COMPOSITION FOR TARGETING CANCER CELL, COMPRISING STRAIN EXPRESSING MONOMERIC STREPTAVIDIN, AND BIOTINYLATED COMPOUND

Abstract

The present invention relates to host cells expressing monomeric streptavidin. The host cells according to the present invention may express streptavidin in vivo, making it possible to visualize and monitor in real time the biodistribution of cancer tissue, pre-targeted by the host cell, with a biotinylated diagnostic agent, as well as to increase the cancer-targeting efficiency of biotinylated anticancer drugs.

Claims

1-59. (canceled)

60. A composition comprising host cells transformed by introduction of a gene encoding biotin-binding protein thereinto.

61. The composition of claim 60, wherein the composition is for an in vivo cell tracking platform.

62. The composition of claim 60, wherein the composition is for a cancer pretargeting platform.

63. The composition of claim 60, wherein the biotin-binding protein is monomeric streptavidin (mSA).

64. The composition of claim 60, wherein a gene encoding fusion partners for improving solubility and expression of recombinant proteins has been introduced into the host cells.

65. The composition of claim 60, wherein a regulatory gene that regulates expression of the gene encoding monomeric streptavidin has been introduced into the host cells.

66. The composition of claim 65, wherein the regulatory gene is at least one selected from the group consisting of a ribosome binding site (RBS), a 5-untranslated region (5-UTR), a transcription factor binding site, and an inducible promoter.

67. The composition of claim 65, wherein the regulatory gene causes the monomeric streptavidin to be expressed in a periplasm of the host cell when a recombinant vector comprising the regulatory gene is transformed into the host cell.

68. The composition of claim 65, wherein the regulatory gene has a total Gibbs free energy change (G.sub.total) of 0 or less.

69. The composition of claim 65, wherein the regulatory gene has a translation initiation rate (TIR) controlled within a predetermined range.

70. The composition of claim 65, wherein the regulatory gene has a sequence length of to 39 bp.

71. The composition of claim 65, wherein the regulatory gene comprises a gene sequence represented by any one of SEQ ID NOs: 5 to 7.

72. The composition of claim 71, wherein a spacing between a 3 end of the gene sequence represented by any one of SEQ ID NOs: 5 to 7 in the regulatory gene and an initiation codon of the gene encoding monomeric streptavidin is 6 to 13 bp.

73. The composition of claim 60, wherein the host cells are of any one or more types selected from the group consisting of bacteria, yeast, fungal cells, plant cells, insect cells, and animal cells.

74. The composition of claim 60 wherein the composition further comprises a biotinylated compound.

75. A method for visualizing host cell biodistribution with imaging techniques, the method comprising a step of identifying monomeric streptavidin-expressing host cells after the host cells have been administered into a subject of interest.

76. The method of claim 75, wherein a biotinylated compound has been further administered to the subject of interest.

77. The method of claim 75, wherein the identification of host cell is continuously visualized through multiple injection of biotinylated imaging agents after the repeated expression of monomeric streptavidin in subjects or cancer tissues.

78. The method of claim 75, wherein the method is for pretargeting and visualizing cancer tissues in the subject of interest.

79. A method for preventing or treating cancer, the method comprising administering to a subject in need thereof a composition comprising host cells transformed by introduction of a gene encoding monomeric streptavidin (mSA) thereinto.

80. The method of claim 79, further comprising administering to the subject a biotinylated compound.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0143] FIG. 1 shows the results of analyzing the expression of plasmids transduced with mSA gene alone in Experimental Example 1.

[0144] FIG. 2 shows the results of analyzing the expression of MBP-mSA gene in Experimental Example 2.

[0145] FIG. 3 shows the results of Western blotting performed to determine the expression and activity of MBP-mSA gene in Experimental Example 2.

[0146] FIG. 4 is a graph showing the results of analyzing biotin binding to recombinant strains in Experimental Example 2.

[0147] FIG. 5 depicts confocal microscope images showing biotin binding to recombinant strains in Experimental Example 2.

[0148] FIG. 6 depicts confocal microscope images showing biotin binding to recombinant strains in Experimental Example 2.

[0149] FIG. 7 shows the results of analyzing the expression of MBP-mSA gene in Experimental Example 3.

[0150] FIG. 8 shows the results of Western blotting performed to determine the expression and activity of MBP-mSA gene in Experimental Example 3.

[0151] FIG. 9 shows the results of Western blotting performed to determine the expression of MBP-mSA gene in Experimental Example 4.

[0152] FIG. 10 shows the results of Western blotting performed to compare the expression of MBP-mSA gene in Experimental Example 4.

[0153] FIG. 11 is a graph showing the results of analyzing biotin binding to recombinant strains in Experimental Example 4.

[0154] FIG. 12 depicts confocal microscope images showing biotin binding to a recombinant strain in Experimental Example 4.

[0155] FIG. 13 depicts confocal microscope images showing biotin binding to recombinant strains in Experimental Example 4.

[0156] FIG. 14A is a graph showing the results of analyzing the specificity of biotin binding to recombinant strains in Experimental Example 5.

[0157] FIG. 14B is a graph showing the results of analyzing the specificity of biotin binding to recombinant strains in Experimental Example 5.

[0158] FIG. 15A is a graph showing the results of analyzing the specificity of biotin binding to recombinant strains in Experimental Example 5.

[0159] FIG. 15B is a graph showing the results of analyzing the specificity of biotin binding to recombinant strains in Experimental Example 5.

[0160] FIG. 16 depicts images showing biotin binding to a recombinant strain injected intramuscularly into mice in Experimental Example 5.

[0161] FIG. 17 shows the CFU count of a recombinant strain injected intramuscularly into mice in Experimental Example 5.

[0162] FIG. 18 depicts images showing biotin binding to a recombinant strain injected intraperitoneally into mice in Experimental Example 5.

[0163] FIG. 19 shows the CFU count of a recombinant strain in the bowels of mice into which the recombinant strain has been injected intraperitoneally in Experimental Example 5.

[0164] FIG. 20 depicts images showing biotin binding to a recombinant strain injected intravenously into mice in Experimental Example 5.

[0165] FIG. 21 shows the CFU count of a recombinant strain in the livers of mice into which the recombinant strain has been injected intravenously in Experimental Example 5.

[0166] FIG. 22 depicts images showing biotin binding to a recombinant strain administered orally to mice in Experimental Example 5.

[0167] FIG. 23 shows the CFU count of a recombinant strain in the bowel of mice into which the recombinant strain has been administered orally in Experimental Example 5.

[0168] FIG. 24 depicts images showing biotin binding to recombinant strains in tumor animal models in Experimental Example 5.

[0169] FIG. 25 depicts images showing biotin binding to recombinant strains in tumor animal models in Experimental Example 5.

[0170] FIG. 26 depicts images showing biotin binding to recombinant strains in tumor animal models in Experimental Example 5.

[0171] FIG. 27 depicts images showing biotin binding to recombinant strains in harvested tumors in Experimental Example 5.

[0172] FIG. 28 depicts images showing biotin binding to a recombinant strain in tumor animal models in Experimental Example 5.

MODE FOR INVENTION

[0173] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention according to the subject matter of the present invention is not limited by these examples.

[Example 1] Construction of mSA Expression Plasmids

[0174] [1-1] Construction of mSA Gene-Inserted Plasmids

[0175] In order to construct a plasmid for mSA expression in a recombinant strain, the monomeric streptavidin (mSA) gene represented by SEQ ID NO: 2 was synthesized (Macrogen, Korea), amplified, digested with restriction enzymes EcoRI and SalI, and purified to obtain a gene amplification product which was then cloned into a pBAD24 plasmid digested with the same restriction enzymes, thus constructing a pBAD-mSA (B-mSA) plasmid.

[0176] Additionally, in order to increase the expression of the mSA gene, BBa_B0032, BBa_B0030, and BBa_B0034, which are the ribosome binding sites (RBSs) shown in Table 1 below, were each inserted downstream of the promoter, thereby constructing pBAD_RBS 0.3-mSA (B_R0.3-mSA), pBAD_RBS 0.6-mSA (B_R0.6-mSA), and pBAD_RBS 1.0-mSA (B_R1.0-mSA) plasmids.

[0177] In addition, in order to increase the expression and solubility of the gene, the mSA gene was amplified using the pBAD-mSA plasmid as a template, and then digested with restriction enzymes EcoRI and HindIII and purified to obtain a gene amplification product which was then cloned into each of pMA1_p2x and pMA1_c2x plasmids digested with the same restriction enzymes, thereby constructing pMA1_p2x-mSA (M_p-mSA) and pMA1_c2x-mSA (M_c-mSA) plasmids.

[0178] [1-2] Construction of MBP-mSA-Expressing Plasmids

[0179] Next, for use in animal experiments, the maltose binding protein (MBP)-encoding gene represented by SEQ ID NO: 4, the mSA gene represented by SEQ ID NO: 2, and the BBa_B0034 sequence were each cloned into a pBAD24 plasmid, thereby constructing pBAD_p2x-mSA (B_p-mSA), pBAD_c2x-mSA (B c-mSA), pBAD_RBS1-p2x-mSA (B_R1.0-p-mSA), and pBAD_RBS1-c2x-mSA (B_R1.0-c-mSA) plasmids.

[0180] In addition, the mSA gene was amplified using the pBAD-mSA plasmid as a template, and then digested with restriction enzymes EcoRI and HindIII, and purified to obtain a gene amplification product which was then cloned into each of pMA1_p2x and pMA1_c2x plasmids digested with the same restriction enzymes, thereby constructing pMA1_p2x-mSA (M_p-mSA) and pMA1_c2x-mSA (M_c-mSA) plasmids.

[0181] [1-3] Construction of RBS-Substituted Plasmids

[0182] In order to increase the expression level and functionality of mSA, gene constructs in which the existing RBS was substituted with a new regulatory gene were additionally constructed (Table 1 below). First, a sequence library was prepared by analyzing the RBS sequence of the plasmid. Next, the translation initiation rate (TIR) of the B_p-mSA plasmid was analyzed using the RBS calculator (Penn State University) program, and then a regulatory gene library having a translation initiation rate value ranging from 3.97 to 42,889 as calculated by the RBS library calculator was constructed. The regulatory gene constructed according to the library was cloned to substitute for the RBS sequence of the B_p-mSA plasmid, and then the resulting colonies were selected, thereby constructing the final plasmids pBAD R01-p2x-mSA (B_R01-p-mSA), pBAD R02-p2x-mSA (B_R02-p-mSA), pBAD R1-p2x-mSA (B_R1-p-mSA), pBAD R11-p2x-mSA (B_R11-p-mSA), pBAD R12-p2x-mSA (B_R12-p-mSA), pBAD R13-p2x-mSA (B_R13-p-mSA), pBAD R2-p2x-mSA (B_R2-p-mSA), and pBAD R21-p2x-mSA (B_R21-p-mSA) (see Table 1 below).

[0183] The name, abbreviation, backbone plasmid and entire sequence of each gene construct obtained in Examples 1-1 to 1-3 are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Gene construct Name Backbone Entire (abbreviation) plasmid mSA MBP RBS sequence pBAD-mSA pBAD24 SEQ ID NO: Not added Unsubstituted SEQ ID NO: (B-mSA) 2 8 pBAD_RBS 0.3-mSA pBAD24 SEQ ID NO: Not added SEQ ID NO: SEQ ID NO: (B_R0.3-mSA) 2 26 9 pBAD_RBS 0.6-mSA pBAD24 SEQ ID NO: Not added SEQ ID NO: SEQ ID NO: (B_R0.6-mSA) 2 27 10 pBAD_RBS 1.0-mSA pBAD24 SEQ ID NO: Not added SEQ ID NO: SEQ ID NO: (B_R1.0-mSA) 2 28 11 pMAl_p2x- pMAl_p2x SEQ ID NO: SEQ ID NO: Unsubstituted SEQ ID NO: mSA(M_p-mSA) 2 4 12 pMAl_c2x-mSA pMAl_c2x SEQ ID NO: SEQ ID NO: Unsubstituted SEQ ID NO: (M_c-mSA) 2 4 13 pBAD_p2x-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (B_p-mSA) 2 4 29 14 pBAD_c2x- pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: mSA(B_c-mSA) 2 4 29 15 pBAD_RBS1-p2x- pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: mSA(B_R1.0-p-mSA) 2 4 28 16 pBAD_RBS1-c2x- pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: mSA(B_R1.0-c-mSA) 2 4 28 17 pBAD_R01-p2x- pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: mSA(B_R01-p-mSA) 2 4 30 18 pBAD_R02-p2x-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (B_R02-p-mSA) 2 4 31 19 pBAD_R1-p2x-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (B_R1-p-mSA) 2 4 32 20 pBAD_R11-p2x-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (B_R11-p-mSA) 2 4 33 21 pBAD_R12-p2x-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (B_R12-p-mSA) 2 4 34 22 pBAD_R13-p2x-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (B_R13-p-mSA) 2 4 35 23 pBAD_R2-p2x-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (B_R2-p-mSA) 2 4 36 24 pBAD_R21-p2x-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (B_R21-p-mSA) 2 4 37 25 pBAD R-lib-1-1-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-1-1-mSA) 2 4 66 39 pBAD R-lib-1-5-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-1-5-mSA) 2 4 67 40 pBAD R-lib-1-7-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-1-7-mSA) 2 4 68 41 pBAD R-lib-1-10-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-1-10-mSA) 2 4 69 42 pBAD R-lib-1-11-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-1-11-mSA) 2 4 70 43 pBAD R-lib-1-12-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-1-12-mSA) 2 4 71 44 pBAD R-lib-1-13-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-1-13-mSA) 2 4 72 45 pBAD R-lib-1-14-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-1-14-mSA) 2 4 73 46 pBAD R-lib-1-16-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-1-16-mSA) 2 4 74 47 pBAD R-lib-1-17-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-1-17-mSA) 2 4 75 48 pBAD R-lib-1-18-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-1-18-mSA) 2 4 76 49 pBAD R-lib-2-2-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-2-2-mSA) 2 4 77 50 pBAD R-lib-2-3-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-2-3-mSA) 2 4 78 51 pBAD R-lib-2-4-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-2-4-mSA) 2 4 79 52 pBAD R-lib-2-5-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-2-5-mSA) 2 4 80 53 pBAD R-lib-2-6-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-2-6-mSA) 2 4 81 54 pBAD R-lib-2-7-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-2-7-mSA) 2 4 82 55 pBAD R-lib-2-8-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-2-8-mSA) 2 4 83 56 pBAD R-lib-2-14-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-2-14-mSA 2 4 84 57 pBAD R-lib-2-16-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-2-16-mSA 2 4 85 58 pBAD R-lib-2-17-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-2-17-mSA 2 4 86 59 pBAD R-lib-3-4-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-3-4-mSA 2 4 87 60 pBAD R-lib-3-5-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-3-5-mSA 2 4 88 61 pBAD R-lib-3-11-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-3-11-mSA 2 4 89 62 pBAD R-lib-3-13-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-3-13-mSA 2 4 90 63 pBAD R-lib-3-18-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-3-18-mSA 2 4 91 64 pBAD R-lib-3-20-mSA pBAD24 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: (R-lib-3-20-mSA 2 4 92 65

[0184] [1-4] Calculation of Total Gibbs Free Energy Changes of Regulatory Gene Transcripts

[0185] For the sequence from the promoter to the initiation codon of the ribosome binding site (RBS) constructed in Example 1-3, in order to confirm the mSA expression ability of the gene construct depending on the total Gibbs free energy change (G.sub.total), the total Gibbs free energy change (G.sub.total) was calculated by calculating the following parameters, and the results are shown in Table 2 below: G.sub.mRNA-rRNA which is the Gibbs free energy change when a reaction that forms a complex of the mRNA of the regulatory gene and the 30S ribosomal subunit occurs; G.sub.spacing which is a Gibbs free energy penalty that occurs when the spacing between the sequence forming the 30S ribosomal subunit complex and the initiation codon in the mRNA transcript of the regulatory gene is not optimized; G.sub.stacking which is the Gibbs free energy change of nucleotides stacked in the region of the spacing; G.sub.standby which is the Gibbs free energy penalty when a binding reaction between the standby site of the mRNA transcript of the regulatory gene and a ribosome occurs; G.sub.start which is the Gibbs free energy change when a reaction that forms an mRNA-tRNA complex occurs; and G.sub.mRNA which is the Gibbs free energy change when the mRNA transcript of the regulatory gene forms a folded complex structure. Here, the total Gibbs free energy change (G.sub.total) was calculated using Equations 1 and 2 below.

G.sub.total(G.sub.final)(G.sub.initial)[Equation 1]

(G.sub.final)(G.sub.initial)=[(G.sub.mRNA-rRNA)+(G.sub.spacing)+(G.sub.stacking)+(G.sub.standby)+(G.sub.start)](G.sub.mRNA)[Equation 2]

TABLE-US-00002 TABLE 2 RBS SEQ ID Unit: kcal/mol NO G.sub.mRNA-rRNA G.sub.spacing G.sub.stacking G.sub.standby G.sub.start G.sub.mRNA G.sub.total 29 14.4914 4.992 0 2.41 2.76 15.01 4.944348676 30 6.84135 1.525 0 1.5336 2.76 7.76 1.328848924 31 1.158649 0.005326 0 0.4314 0.42 3.4 4.641874373 32 7.05135 0 0 1.0898 2.76 5.79 3.555751381 33 1.37135 0 0 0.0786 2.76 4.17 0.1498488 34 0.24135 0 0 0.29 2.76 6.12 3.239348447 35 5.39135 0.672 0 4.0728 2.76 7.41 3.862748471 36 9.37135 0.288 0 4.0728 2.76 4.61 3.458051381 37 7.44135 0 0 4.8986 2.76 8.12 2.647948438 66 7.44135 0 0 4.8986 2.76 10.25 4.777948552 67 13.8614 0.288 0 4.0728 2.76 4.62 7.9380514 68 7.77135 0.288 0 4.0728 2.76 5.98 0.488051352 69 5.39135 0.288 0 4.0728 2.76 5.98 1.891948643 70 2.68135 0.288 0 3.3234 2.76 4.61 2.482548676 71 5.39135 0.288 0 4.0728 2.76 4.61 0.521948757 72 2.68135 0.288 0 4.0728 2.76 5.37 3.991948428 73 2.56135 0 0 3.3234 2.76 4.61 2.4427488 74 3.96135 0.288 0 4.8986 2.76 6.51 4.67774886 75 6.95135 0.288 0 4.8986 2.76 6.45 1.627748371 76 7.49135 0.288 0 4.0728 2.76 6.78 0.59194881 77 9.37135 0 0 2.6504 2.76 5.68 4.216651686 78 7.27135 0 0 2.6504 2.76 4.59 3.283051219 79 7.27135 0 0 2.6504 2.76 4.59 2.966951219 80 10.9714 0 0 2.6504 2.76 3.99 7.41975141 81 8.87135 0 0 2.6504 2.76 2.9 6.510651419 82 10.9714 0 0 2.6504 2.76 3.58 7.860951495 83 9.37135 0 0 2.6504 2.76 4.59 5.403751362 84 8.87135 0 0 2.6504 2.76 3.82 5.729451581 85 9.37135 0 0 2.6504 2.76 2.44 7.456651457 86 8.87135 0 0 2.6504 2.76 2.31 7.100651572 87 7.58135 1.525 0 0.0168 2.76 4.78 4.109751104 88 5.27135 0 0 0.4314 2.76 4.46 3.219451333 89 5.27135 0 0 0.4314 2.76 4.27 3.76635139 90 5.27135 0 0 0.4314 2.76 5.47 2.476551581 91 5.06135 0.005326 0 0.2168 2.76 4.1 3.800725876 92 7.75135 1.525 0 0.4314 2.76 4.95 3.695151507

[0186] [1-5] Calculation of Translational Initiation Rates of Regulatory Genes

[0187] In order to confirm the mSA expression ability of the plasmid depending on the translation initiation rate (TIR) of the regulatory gene, the translation initiation rate of each regulatory gene sequence constructed as described above was calculated, and the results are shown in Table 3.

TABLE-US-00003 TABLE 3 Regulatory gene Translation initiation rate (AU) Unsubstituted 1 SEQ ID NO: 29 133.266441 SEQ ID NO: 30 678.2310709 SEQ ID NO: 31 152.7003477 SEQ ID NO: 32 6110.586323 SEQ ID NO: 33 1152.963841 SEQ ID NO: 34 287.0559877 SEQ ID NO: 35 216.8312084 SEQ ID NO: 36 5847.72872 SEQ ID NO: 37 374.5906748 SEQ ID NO: 66 143.6296318 SEQ ID NO: 67 43914.95671 SEQ ID NO: 68 1536.365645 SEQ ID NO: 69 526.4033937 SEQ ID NO: 70 403.5382313 SEQ ID NO: 71 975.1871797 SEQ ID NO: 72 204.582929 SEQ ID NO: 73 410.8314232 SEQ ID NO: 74 150.2547754 SEQ ID NO: 75 592.8668512 SEQ ID NO: 76 944.9445588 SEQ ID NO: 77 8227.297678 SEQ ID NO: 78 5404.842895 SEQ ID NO: 79 4688.141139 SEQ ID NO: 80 34778.40472 SEQ ID NO: 81 23100.64943 SEQ ID NO: 82 42417.31004 SEQ ID NO: 83 14037.12899 SEQ ID NO: 84 16253.13229 SEQ ID NO: 85 35360.78091 SEQ ID NO: 86 30125.96282 SEQ ID NO: 87 7840.852282 SEQ ID NO: 88 5252.334127 SEQ ID NO: 89 6718.078741 SEQ ID NO: 90 3759.681518 SEQ ID NO: 91 6822.815917 SEQ ID NO: 92 6506.222717

[0188] As shown in Table 3, it was confirmed that, among the regulatory genes of the constructed plasmids, the regulatory genes of SEQ ID NOs: 29 to 37 and 65 to 92 had translation initiation rates in the range of 50 to 45,000 AU, and thereamong, the regulatory genes of SEQ ID NOs: 32 and 36 had translation initiation rates in the range of 900 to 9,000 AU.

[0189] [1-6] Sequence Analysis of Regulatory Genes

[0190] In order to examine the mSA expression ability of the plasmid depending on whether not the regulatory gene sequence comprises the AGG, TAGG or ATAGG sequence and on the spacing between the 3 end of the AGG sequence and the initiation codon, the regulatory gene sequence of each plasmid and the spacing (unit: bp) between the 3 end of the AGG sequence and the initiation codon were analyzed, and the results are shown in Table 4 below.

TABLE-US-00004 TABLE4 Spacing Regulatorygenesequence (bp) SEQIDNO:26 TCACACAGGA 4 AAG SEQIDNO:27 ATTAAAGAGG 5 AGAAA SEQIDNO:28 AAAGAGGAGA 5 AA SEQIDNO:29 ACCCGTTTTT 14 TGGGCTAACA GGAGGAAGCT AGCGCT AGC SEQIDNO:30 TAGCACTCGT TGACATACGG ACGTCAC SEQIDNO:31 ACTACTGAGG 5 CTACT SEQIDNO:32 TGGAACAGCT 6 CACGCAAAAA TAGGTTTCTT SEQIDNO:33 CGCTTTTTAT CGCAACTCTC TACTGTTTCT CCAT SEQIDNO:34 TCTGAGAAAG ACACGATCTT ACTAG SEQIDNO:35 TCTAGAGAAA GAGCGGATCC TACCTAG SEQIDNO:36 TCTAGAGAAA 10 GATAGGAGAA TACTAG SEQIDNO:37 TCTAGAGAAA 11 GAGGCGACGG TACTAG SEQIDNO:66 TCTAGAGAAA 12 GAGGCGAGTG TACTAG SEQIDNO:67 TCTAGAGAAA 10 GATAGGAGGT TACTAG SEQIDNO:68 TCTAGAGAAA 12 GAGGGGACAC TACTAG SEQIDNO:69 TCTAGAGAAA GAGCGGAAAC TACTAG SEQIDNO:70 TTCTAGAGAA AGATTTGAAT ATACTAG SEQIDNO:71 TCTAGAGAAA GAACGGACAT TACTAG SEQIDNO:72 TCTAGAGAAA GACATGACTA TACTAG SEQIDNO:73 TCTAGAGAAA GAACTGAAGA TACTAG SEQIDNO:74 TCTAGAGAAA 12 GAGGCGATCC TACTAG SEQIDNO:75 TCTAGAGAAA GAAGAGAGCC TACTAG SEQIDNO:76 TCTAGAGAAA 7 GACTTGAGGC TACTAG SEQIDNO:77 GAACCCTAAT 9 ACATTAGGAG ATCTTCT SEQIDNO:78 GAACCCTAAT 9 ACATTAGGAC ATATTCT SEQIDNO:79 GAACCCTAAT 9 ACATTAGGAC ATCATCT SEQIDNO:80 GAACCCTAAT 9 ACATAAGGAG ATCATAT SEQIDNO:81 GAACCCTAAT 9 ACATAAGGAC ATAATAT SEQIDNO:82 GAACCCTAAT 9 ACATAAGGAG ATTATCT SEQIDNO:83 GAACCCTAAT 9 ACATTAGGAG ATTATAT SEQIDNO:84 GAACCCTAAT 9 ACATAAGGAC ATCTTAT SEQIDNO:85 GAACACTAAT 9 ACATTAGGAG ATCTTCT SEQIDNO:86 GAACACTAAT 9 ACATAAGGAC ATAATAT SEQIDNO:87 TTAAGTAGTT 6 AAACAGGGTA TATAGGGGAA GA SEQIDNO:88 TTAAGTAGTT 6 AAACAGGGTA TATAGGACGA GA SEQIDNO:89 TTAAGTAGTT 6 AAACAGGGTA TATAGGGCTA TA SEQIDNO:90 TTAAGTAGTT 6 AAACAGGGTA TATAGGAGGA TA SEQIDNO:91 TTAAGTAGTT 6 AAACAGGGTA TATAGGGCGA TA SEQIDNO:92 TTAAGTAATT 6 AAACAGGGTA TATAGGGGAA GA

[0191] As shown in Table 4, it was confirmed that, among the regulatory genes of the constructed plasmids, the regulatory genes of SEQ ID NOs: 26, 27, 28, 29, 31, 32, 36, 37, 66, 67, 68, 74, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 and 92 contained the AGG sequence, and in particular, the spacing between the 3 end of the AGG sequence in the regulatory genes of SEQ ID NOs: 32 and 36 and the initiation codon was 6 to 13 bp. In addition, it was confirmed that, among the regulatory genes of the constructed plasmids, the regulatory genes of SEQ ID NOs: 32, 36, 67, 77, 78, 79, 83, 85, 87, 88, 89, 90, 91 and 92 contained the TAGG or ATAGG sequence.

[Example 2] Transformation and Culture of Host Cells

[0192] After each of the plasmids constructed in Example 1 was transformed into a bacterial strain, each of the transformed strains was cultured overnight using an LB solid medium containing ampicillin. Then, the resulting colonies were diluted at a ratio of 1:100 using an LB liquid medium containing antibiotics, and when the OD.sub.600 value reached 0.5 to 0.7 during additional culture, arabinose was added to the culture at a final concentration of 0.1%, followed by culturing in a shaking incubator under conditions of 200 rpm and 37 C.

[Experimental Example 1] Analysis of mSA Expression Level of Recombinant mSA Plasmid

[0193] In order to analyze the expression level of the plasmid into which the mSA gene was inserted alone, recombinant E. coli colonies containing each of the plasmids B-mSA, B_R0.3-mSA, B_R0.6-mSA and B_R1.0-mSA constructed in Example 1 were transformed and cultured as described in Example 2. Next, the cultured recombinant E. coli was added to SDS-PAGE sample buffer based on OD4, boiled at 95 C. for 10 minutes, and then loaded on SDS-PAGE to determine the expression level of the protein, and the results are shown in FIG. 1.

[0194] As shown in FIG. 1, it could be confirmed that the strains containing the plasmid in which the RBS sequence known as BBa_B0032, BBa_B0030 or BBa_B0034 was inserted upstream of the mSA gene sequence to improve protein expression did not express the mSA protein on SDS-PAGE. Thereby, it could be seen that the addition of the RBS sequence to the pBAD expression system did not significantly affect the overexpression of the mSA gene alone.

[Experimental Example 2] Analysis of mSA Expression Level and Activity of MBP-mSA Plasmid

[0195] [2-1] SDS-Page

[0196] The present inventors examined the mSA expression level of a strain transformed with an MBP-mSA plasmid in which the MBP gene was fused with mSA in order to increase the expression and solubility of mSA. Specifically, M_p-mSA and M_c-mSA plasmids obtained by fusion with the MBP gene were constructed as described in Example 1, and transformation and culture were performed as described in Example 2. When the OD.sub.600 value reached 0.5 to 0.7 during culture, isopropyl beta-D-1-thiogalactopyranoside (IPTG) was added to the culture at a final concentration of 0.1 mM, followed by culturing in a shaking incubator under conditions of 200 rpm and 37 C. The cultured recombinant E. coli was added to SDS-PAGE sample buffer based on OD4, boiled at 95 C. for 10 minutes, and then loaded on SDS-PAGE to confirm the expression level of the protein, and the results are shown in FIG. 2.

[0197] As a result, as shown in FIG. 2, it was confirmed that the mSA protein fused with MBP was overexpressed on the gel, and that the mSA gene fused with the MBP gene without the secretion sequence was more overexpressed than the mSA gene fused with the MBP gene with the secretion sequence.

[0198] [2-2] Western Blot Analysis

[0199] Western blot analysis was performed to analyze the mSA expression level and biotin binding activity of the recombinant strain transformed with the MBP-mSA plasmid. Specifically, the culture of the strain of Example 2 was diluted with PBS to 410 CFU/ml, and the pellet was collected by centrifugation at 13,000 rpm for 5 minutes. The pellet fraction was washed with PBS and mixed with SDS sample buffer containing 0.2% beta-mercaptoethanol (catalog number: EBA-1052, ELPIS BIOTECH) to obtain a strain lysate. Then, the strain lysate was electrophoresed on 12% SDS-PAGE gel, and the protein was transferred from the gel to a nitrocellulose membrane, followed by blocking with 5% skim milk at room temperature. Then, the expression level of mSA was confirmed using his tag antibody, and the biotin-binding activity of mSA was confirmed using biotinylated peroxidase. The results are shown in FIG. 3.

[0200] As shown in FIG. 3, it was confirmed that the expression level of the MBP-fused mSA protein of each of the M_c-mSA and Mp-mSA plasmids into which both the MBP gene and the mSA gene were inserted was higher than the expression level of the non-MBP-fused mSA protein of the B-mSA plasmid into which only the mSA gene was inserted.

[0201] In addition, it could be confirmed that, although the expression level of the protein expressed from the M_c-mSA plasmid was higher than that from M_p-mSA, the biotin binding activity of MBP-fused mSA with the secretion sequence was higher.

[0202] [2-3] Biotin Uptake Assay

[0203] In order to analyze the biotin binding activity of the recombinant strain transformed with the MBP-mSA plasmid, biotin uptake assay was performed, and the results are shown in FIG. 4. Specifically, biotinylated fluorescent dye (biotin-flamma 675 dye, BioActs) was added to and reacted with the cultured strain, followed by washing with PBS to remove biotinylated fluorescent dye not bound to the strain. Then, the fluorescence value of the fluorescent dye absorbed by the recombinant strain was measured using a fluorescence measurement reader (Infinite m200, Tecan).

[0204] As a result, as shown in FIG. 4, it was confirmed that the increase in the biotin binding signal (biotin activity) before/after the addition of arabinose was higher in the strain containing the control B-mSA plasmid (344%) than in the strains containing pBAD (151%), M_c-mSA (141%), and M_p-mSA(158%), indicating that the biotin binding ability of the recombinant strain expressing MBP-mSA was not improved.

[0205] [2-4] Confocal Microscopic Observation

[0206] In order to actually image the binding of the biotinylated fluorescent dye to the recombinant strain, the recombinant strains cultured as described in Example 2 were fixed to slides and observed with a confocal microscope, and the results are shown in FIGS. 5 and 6.

[0207] As shown in the biotin uptake assay results in FIGS. 5 and 6, the number of biotinylated fluorescent dye particles bound to the strain was very small regardless of MBP fusion. Thereby, it could be confirmed that mSA expression was improved through MBP gene fusion, but changes in gene expression and solubility through MBP could not improve biotin binding activity.

[Experimental Example 3] Analysis of mSA Expression Level and Activity of RBS-Added Plasmid

[0208] [3-1] SDS-Page

[0209] SDS-PAGE was performed to examine the mSA expression and activity of the recombinant strain transformed with the RBS-added plasmid. Specifically, SDS-PAGE was performed on recombinant strains transformed with each of Bp-mSA and B c-mSA plasmids obtained by cloning the MBP-mSA gene into the pBAD plasmid, and B_R1.0-p-mSA and B_R1.0-c-mSA plasmids obtained by adding the BBa_B0034 sequence to improve the expression of the plasmids, and the results are shown in FIG. 7.

[0210] As shown in FIG. 7, as a result of loading the recombinant strains on SDS-PAGE and examining the expression level of the protein, it was confirmed that the MBP-fused mSA protein was overexpressed even from the pBAD plasmid.

[0211] [3-2] Western Blot Analysis

[0212] Western blot analysis was performed to examine the mSA expression and activity of the recombinant strain transformed with the RBS-added plasmid. Western blot analysis was performed on B_p-mSA and B c-mSA plasmids obtained by cloning the MBP-mSA gene into the pBAD plasmid, and B_R1.0-p-mSA and B_R1.0-c-mSA plasmids obtained by adding the BBa_B0034 sequence to improve the expression of the plasmids, in the same manner as in Experimental Example 2-2, and the results are shown in FIG. 8.

[0213] As shown in FIG. 8, as a result of performing Western blot analysis, it was confirmed that the B_p-mSA, B c-mSA, B_R1.0-p-mSA and B_R1.0-c-mSA plasmids obtained by inserting both the MBP gene and the mSA gene overexpressed the MBP-fused mSA protein, and the expression level of the MBP-fused mSA protein was higher than that of the non-MBP-fused mSA protein.

[Experimental Example 4] Analysis of mSA Expression Level and Activity of RBS-Substituted Plasmid

[0214] [4-1] Western Blot Analysis (1)

[0215] The present inventors analyzed the RBS sequence of the B_p-mSA plasmid to induce increased functional expression of the gene in the recombinant strain, and constructed B_R01-p-mSA, B_R02-p-mSA, B_R1-p-mSA, B_R11-p-mSA, B_R12-p-mSA, B_R13-p-mSA B_R2-p-mSA and B_R21-p-mSA plasmids as described in Example 1. A strain was transformed with each of the constructed plasmids and cultured. In order to examine the protein expression level of each of the recombinant strains, Western blot analysis was performed in the same manner as in Experimental Example 2-2, and the results are shown in FIG. 9.

[0216] As shown in FIG. 9, it was confirmed that, among the mSA-expressing strains, the recombinant strains transformed with each of the B_R1-p-mSA and B_R2-p-mSA plasmids showed higher mSA expression levels than the other strains.

[0217] [4-2] Western Blot Analysis (2)

[0218] Additional experiments were performed on the two selected strains transformed with each of the B_R1-p-mSA and B_R2-p-mSA plasmids having high mSA expression levels, and the results are shown in FIG. 10.

[0219] As shown in FIG. 10, it was confirmed that, in the control group, the expression and secretion levels of mSA were higher in the recombinant strain containing the Mp-mSA plasmid than in the recombinant strain containing the BAD-mSA plasmid. In addition, it was confirmed that, even in the experimental group, the expression and secretion levels of mSA were higher in the recombinant strain containing the Mp-mSA plasmid than in the recombinant strains containing each of the B_R1-p-mSA and B_R2-p-mSA plasmids.

[0220] In addition, it was shown that the secretion level versus expression level of the protein was lower in the recombinant strains containing each of the B_R1-p-mSA and B_R2-p-mSA plasmids than in the recombinant strain containing the Mp-mSA plasmid, indicating that mSA expressed from each of the B_R1-p-mSA and B_R2-p-mSA plasmids remained in the periplasm of the strain. The biotin binding activity was higher in the order of the recombinant strains containing the BAD-mSA, B_R1-p-mSA, Mp-mSA and B_R2-p-mSA plasmids, respectively, and the secreted protein binding activity was higher in the order of the recombinant strains containing the Mp-mSA, BAD-mSA, B_R1-p-mSA, B_R2-p-mSA plasmids, respectively.

[0221] [4-3] Biotin Uptake Assay

[0222] In addition, in order to analyze the biotin binding activity of the recombinant strain with improved expression, biotin uptake assay was performed in the same manner as in Experimental Example 2, and the results are shown in FIG. 11.

[0223] As shown in FIG. 11, it was confirmed that the recombinant strain containing each of the B_R1-p-mSA and B_R2-p-mSA plasmids had significantly higher biotin binding activity than the recombinant strain containing each of the pBAD and B-mSA plasmid as a control, indicating that mSA expressed from each of the B_R1-p-mSA and B_R2-p-mSA plasmids had a significant biotin binding activity effect compared to mSA expressed from the other plasmids. In addition, it was confirmed that the biotin-binding activity was not proportional to the protein expression level, indicating that the biotin-binding activity effect could not be predicted simply by the protein expression level alone.

[0224] [4-4] Confocal Microscopic Observation

[0225] In order to actually image the binding of the biotinylated fluorescent dye to the recombinant strain, the cultured strains were fixed to slides and observed with a confocal microscope, and the results are shown in FIGS. 12 and 13.

[0226] As shown in FIG. 12, it could be confirmed that the biotinylated fluorescent dye more strongly bound to the recombinant strain containing each of the B_R1-p-mSA and B_R2-p-mSA plasmids than to the control B-mSA and BAD-mSA shown in FIG. 13, and in particular, mSA expression by the B_R2-p-mSA plasmid was optimal for binding to the biotinylated fluorescent dye. Thereby, it could be seen that even the strain in which the expression of the mSA gene was improved through MBP gene fusion did not sufficiently bind to external biotin, but in the case in which the MBP gene and the RBS gene were fused with the mSA gene, the expression of the mSA gene was functionally improved, and thus the ability to bind to external biotin was significantly improved.

[Experimental Example 5] Confirmation of Tracking Function for mSA-Expressing Recombinant Strain

[0227] [5-1] Biotin Uptake Assay

[0228] In order to confirm whether the mSA gene expressed in the constructed recombinant strain of the present invention is specific to biotin, as described in Experimental Example 1, each of the pBAD, B-mSA, Bp-mSA, B_R1-p-mSA and B_R2-p-mSA plasmids was transformed into each of E. coli and Salmonella strains which were then cultured. Next, biotin uptake assay was performed in the same manner as in Experimental Example 2, and the results are shown in FIGS. 14A, 14B, 15A and 15B.

[0229] As shown in FIG. 14A or 14B, it was confirmed that, when the recombinant strains were treated only with the biotinylated fluorescent dye, the recombinant E. coli and Salmonella strains containing the B_R2-p-mSA plasmid had the highest biotin uptake. On the other hand, as shown in FIG. 15A or 15B, it was confirmed that, when biotin without the fluorescent dye was added to the recombinant strains which were then treated with the biotinylated fluorescent dye, the uptake of the biotinylated fluorescent dye by the recombinant strains decreased. Through the difference between FIGS. 14A, 14B, 15A and 15B, it was confirmed that the binding of the biotinylated fluorescent dye to the recombinant strain could be inhibited when 200 nM of biotin without the fluorescent dye was added prior to addition of the biotinylated fluorescent dye, suggesting that the recombinant strain of the present invention binds specifically to biotin.

[0230] [5-2] Tracking of Strain in Muscle Tissue (1)

[0231] In order to confirm the strain tracking effect in muscle tissue according to the present invention, an in vivo imaging system (IVIS) imaging was performed. Specifically, 110.sup.9 CFUs of the recombinant strain transformed with B_R2-p-mSA was injected intramuscularly (IM) into the right thigh of each mouse (BALB/C). Then, arabinose was injected to express mSA in the recombinant strain, and arabinose was not injected into the control group. Subsequently, biotin-dye was injected into each of the experimental group and the control group, the signal was examined, and the results are shown in FIG. 16.

[0232] As shown in FIG. 16, it was confirmed that, in in the right thigh of the mouse (right) injected with the recombinant strain and arabinose, a strong signal caused by biotin staining appeared for more than 6 hours, but in the control mouse into which arabinose was not injected (left), the signal caused by biotin staining did not appear. This suggests that, when biotin staining is used after mSA is expressed by injecting the recombinant strain and arabinose, it is possible to track the distribution of the recombinant strain in muscle tissue.

[0233] [5-3] Tracking of Strain in Muscle Tissue (2)

[0234] In order to confirm whether the recombinant strain of the present invention is actually distributed in muscle tissue, the present inventors harvested the right thigh tissue, into which the strain was injected in Experimental Example 5-2, and counted the CFUs of the strain. Specifically, the present inventors harvested the right thigh tissue from each of the mouse (Induction) showing the signal of the recombinant strain, caused by biotin staining, and the control mouse (Non-induction), which were used in Experimental Example 5-2, and counted the CFUs of the strain remaining therein. The results are shown in FIG. 17.

[0235] As shown in FIG. 17, it was confirmed that the recombinant strain of the present invention was present throughout the thigh muscle tissue regardless of the presence or absence of arabinose treatment, indicating that the distribution of the recombinant strain in muscle tissue can be tracked by mSA expressed in the recombinant strain by arabinose treatment.

[0236] [5-4] Tracking of Strain Administered Intraperitoneally (1)

[0237] In order to confirm the strain tracking effect according to the present invention, in vivo imaging system (IVIS) imaging was performed on the strain injected intraperitoneally. Specifically, 510.sup.9 CFU of the recombinant strain transformed with B_R2-p-mSA were injected intraperitoneally (IP) into each mouse (BALB/C). Then, arabinose was injected to express mSA in the recombinant strain, and arabinose was not injected into the control group. Subsequently, biotin-dye was injected into each of the experimental group and the control group, the signal was examined, and the results are shown in FIG. 18.

[0238] As shown in FIG. 18, it was confirmed that, in the abdominal organ of each of the mouse injected with the recombinant strain and arabinose (right), a strong signal caused by biotin staining appeared for more than 6 hours, but in the control group into which the recombinant strain was not injected (left) or the control mouse into which arabinose was not injected (middle), the signal caused by biotin staining disappeared after 6 hours. In addition, as a result of harvesting the bowel and examining the signal, it was confirmed that the signal appeared only in the mouse into which the recombinant strain and arabinose were injected (right). This suggests that, when biotin staining is used after mSA is expressed by injecting the recombinant strain and arabinose, it is possible to track the distribution of the recombinant strain injected intraperitoneally.

[0239] [5-5] Tracking of Strain Administered Intraperitoneally (2)

[0240] In order to confirm whether the recombinant strain of the present invention is actually distributed in the abdominal cavity and intestinal tract, the CFUs of the strain in the bowel harvested in Experimental Example 5-4 above were counted. Specifically, the present inventors harvested the bowel from each of the mouse (Induction) showing the signal of the recombinant strain, caused by biotin stain, and the control mouse (Non-induction), which were used in Experimental Example [5-4], and counted the CFUs of the strain remaining therein. The results are shown in FIG. 19.

[0241] As shown in FIG. 19, it was confirmed that the recombinant strain of the present invention was present in the bowel regardless of the presence or absence of arabinose treatment, indicating that the distribution of the recombinant strain in the abdominal cavity and intestinal tract can be tracked by mSA expressed in the recombinant strain by arabinose treatment.

[0242] [5-6] Tracking of Strain Administered Intravenously (1)

[0243] In order to confirm the strain tracking effect according to the present invention, in vivo imaging system (IVIS) imaging was performed on the strain injected intravenously. Specifically, 110.sup.9 CFU of the recombinant strain transformed with B_R2-p-mSA were injected intravenously (IV) into each mouse (BALB/C). Then, arabinose was injected to express mSA in the recombinant strain, and arabinose was not injected into the control group. Subsequently, biotin-dye was injected into each of the experimental group and the control group, the signal was examined, and the results are shown in FIG. 20.

[0244] As shown in FIG. 20, it was confirmed that, in the mouse injected with the recombinant strain and arabinose (right), a strong signal caused by biotin staining appeared for more than 6 hours, but in the control group into which the recombinant strain was not injected (left) or the control mouse into which arabinose was not injected (middle), the signal caused by biotin staining disappeared after 6 hours. In addition, as a result of harvesting the liver and spleen and examining the signal, it was confirmed that the signal appeared only in the mouse into which the recombinant strain and arabinose were injected (right). This suggests that, when biotin staining is used after mSA is expressed by injecting the recombinant strain and arabinose, it is possible to track the distribution of the recombinant strain in organs.

[0245] [5-7] Tracking of Strain Administered Intravenously (2)

[0246] In order to confirm whether the recombinant strain of the present invention is actually distributed in organs, the present inventors counted the CFUs of the strain in the liver harvested in Experimental Example 5-6. Specifically, the present inventors harvested the liver and spleen from each of the mouse (Induction) showing the signal of the recombinant strain, caused by biotin stain, and the control mouse (Non-induction), which were used in Experimental Example 5-6, and counted the CFUs of the strain remaining therein. The results are shown in FIG. 21.

[0247] As shown in FIG. 21, it was confirmed that the recombinant strain of the present invention was present in the liver regardless of the presence or absence of arabinose treatment, indicating that the biodistribution of the recombinant strain can be tracked by mSA expressed in the recombinant strain by arabinose treatment.

[0248] [5-8] Tracking of Strain Administered Orally (1)

[0249] In order to confirm the strain tracking effect according to the present invention, in vivo imaging system (IVIS) imaging was performed on the strain administered orally. Specifically, 110.sup.9 CFU of the recombinant strain transformed with B_R2-p-mSA were orally administered to each mouse (BALB/C). Then, arabinose was injected to express mSA in the recombinant strain, and arabinose was not injected into the control group. Subsequently, biotin-dye was injected into each of the experimental group and the control group, the signal was examined, and the results are shown in FIG. 22.

[0250] As shown in FIG. 22, it was confirmed that, in the mouse injected with the recombinant strain and arabinose (right), a strong signal caused by biotin staining appeared in the intestinal tract for more than 6 hours, but in the control group into which the recombinant strain was not injected (left) or the control mouse into which arabinose was not injected (middle), the signal caused by biotin staining disappeared after 6 hours. In addition, as a result of harvesting the bowel and examining the signal, it was confirmed that the signal appeared only in the mouse into which the recombinant strain and arabinose were injected (right). This suggests that, when biotin staining is used after mSA is expressed by injecting the recombinant strain and arabinose, it is possible to track the distribution of the recombinant strain in the intestinal tract.

[0251] [5-9] Tracking of Strain Administered Orally (2)

[0252] In order to confirm whether the recombinant strain of the present invention is actually distributed in the intestinal tract, the present inventors counted the CFUs of the strain in the bowel harvested in Experimental Example 5-8 above. Specifically, the present inventors harvested the bowel from each of the mouse (Induction) showing the signal of the recombinant strain, caused by biotin stain, and the control mouse (Non-induction), which were used in Experimental Example 5-8, and counted the CFUs of the strain remaining therein. The results are shown in FIG. 23.

[0253] As shown in FIG. 23, it was confirmed that the recombinant strain of the present invention was present in the intestinal tract regardless of the presence or absence of arabinose treatment, indicating that the distribution of the recombinant strain in the intestinal tract can be tracked by mSA expressed in the recombinant strain by arabinose treatment.

[0254] Specifically, through the above experiments, it was confirmed that, when the recombinant vector or construct according to the present invention, especially the regulatory gene according to the present invention, is included, the monomeric streptavidin (mSA) expressed has excellent stability and can strongly bind to external biotin, and this is effective even in vivo, and treatment with the biotinylated fluorescent dye may be performed multiple times or at adjusted time intervals.

[0255] [5-10] Tumor Imaging Assay (1)

[0256] In order to confirm the biotin binding activity of the recombinant strain of the present invention, in vivo imaging system (IVIS) imaging was performed. Specifically, first, the CT26 cell line was subcutaneously injected into the flanks of Balb/c mice to construct tumor animal models. Each recombinant strain was injected into the tumor animal model, and as a control, only dye was injected into the tumor animal model. The recombinant strains were those transformed with B-mSA, 13p-mSA, B_R2-p-mSA (non-induction) and B_R2-p-mSA, respectively. After 3 days, biotinylated fluorescent dye was injected into each mouse. The results of IVIS imaging performed 6 hours after biotinylated fluorescent dye injection are shown in FIG. 24, the results of IVIS imaging performed 9 hours after biotinylated fluorescent dye injection are shown in FIG. 25, and the results of IVIS imaging performed 24 hours after biotinylated fluorescent dye injection are shown in FIG. 26.

[0257] As shown in FIGS. 24 to 26, it could be seen that the biotinylated fluorescent dye injected into each of the tumor animal model injected only with dye (only dye) as a control and the tumor animal models injected with the recombinant strains transformed with B-mSA, B_p-mSA, and B_R2-p-mSA (non-induction) plasmids, respectively, was gradually eliminated in vivo over time after injection, suggesting that there was no tumor specificity. On the other hand, it was confirmed that the tumor animal model injected with the recombinant strain containing the B_R2-p-mSA plasmid showed a stronger signal in the tumor tissue than the control group after injection of the biotinylated fluorescent dye, and the signal was still strongly maintained even after 24 hours after injection of the biotinylated fluorescent dye. Thereby, it was confirmed that the biotinylated fluorescent dye strongly bound only to the recombinant strain of the present invention in small animals. In particular, it could be seen that, when the recombinant strain with tumor specificity is used, the biotinylated fluorescent dye can bind specifically to the tumor through the recombinant strain, suggesting that the signal generated from the biotinylated fluorescent dye can be detected by an imaging means, enabling real-time tumor imaging.

[0258] [5-11] Tumor Imaging Assay (2)

[0259] In addition, in order to confirm the biotin binding activity of the recombinant strain of the present invention, cancer tissue was harvested from the tumor animal model and imaged with an in vivo imaging system (IVIS). Specifically, 24 hours after the biotinylated fluorescent dye was injected into the tumor animal model, the tumor was harvested from each group and imaged with an IVIS to detect the signal of the biotinylated fluorescent dye, and the results are shown in FIG. 27.

[0260] As shown in FIG. 27, it was confirmed that the group treated with the recombinant strain containing the B_R2-p-mSA plasmid maintained strong fluorescence activity compared to the other groups. Thereby, it was confirmed that the biotinylated fluorescent dye strongly bound only to the recombinant strain of the present invention in small animals, and in particular, it could be seen that real-time tumor imaging using the recombinant strain having tumor specificity is possible.

[0261] [5-12] Tumor Imaging Assay (3)

[0262] In order to confirm the multiple-biotin-binding activity of the recombinant strain of the present invention, in vivo imaging system (IVIS) imaging was performed. Specifically, first, the CT26 cell line was subcutaneously injected into the flanks of Balb/c mice to construct tumor animal models. The recombinant strain was injected into the tumor animal models. Three days after injecting the recombinant strain into the tumor animal models, the biotinylated fluorescent dye was injected (first injection). Two days later, the biotinylated fluorescent dye was injected into the same tumor animal models (second injection). IVIS imaging was performed before, 6 hours after, and 9 hours after the first injection of the fluorescent dye, and then IVIS imaging was performed before, 6 hours after, and 9 hours after the second injection of the fluorescent dye, and the results are shown in FIG. 28.

[0263] As shown in FIG. 28, it was confirmed that the signal of the biotinylated fluorescent dye after first injection was strongly maintained in the cancer tissue over time by the recombinant strain of the present invention, and after the biotinylated fluorescent dye was eliminated in vivo, the signal of the biotinylated fluorescent dye after second injection was strongly maintained in the cancer tissue over time by the recombinant strain of the present invention. This means that, by regulating mSA expression of the recombinant strain of the present invention, it is possible to continuously acquire tumor images even when multiple treatments with the biotinylated fluorescent dye are performed, and that treatment with the biotinylated conjugate may be performed at adjusted time intervals.

[0264] Specifically, through the above experiments, it was confirmed that, when the recombinant vector or construct according to the present invention, especially the regulatory gene according to the present invention, is included, the monomeric streptavidin (mSA) expressed has excellent stability and can strongly bind to external biotin, and this is effective even in vivo, and treatment with the biotinylated fluorescent dye may be performed multiple times or at adjusted time intervals.

[0265] Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only description of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereto.