RECOMBINANT SEQUENCE SPECIFICALLY EXPRESSING HUMAN BETA-GLOBIN IN ERYTHROID CELLS, AND USE THEREOF

Abstract

Disclosed is an erythroid-specific human β-globin gene promoter and a human β-globin-expressing recombinant sequence, and use thereof, which belongs to the technical field of genetic engineering. An erythroid-specific human β-globin gene promoter with a nucleotide sequence set forth in SEQ ID NO: 1 is provided in the present disclosure, which may achieve high-efficiency and erythroid-specific initiation of the expression of a functional gene. A recombinant sequence specifically expressing human β-globin in erythroid cells is also provided in the present disclosure, which is human β-globin locus control region (LCR) HS 3-1+β-globin gene promoter+β-globin gene+β-globin gene enhancer.

Claims

1. A recombinant sequence specifically expressing human β-globin in erythroid cells, wherein the recombinant sequence is human β-globin locus control region (LCR) HS 3-1+human β-globin gene promoter+β-globin gene+β-globin gene enhancer; and the human β-globin gene promoter is an erythroid-specific human β-globin gene promoter with a nucleotide sequence set forth in SEQ ID NO: 1.

2. The recombinant sequence according to claim 1, wherein a nucleotide sequence of the β-globin gene enhancer is set forth in SEQ ID NO: 2.

3. The recombinant sequence according to claim 1, wherein a nucleotide sequence of the β-globin gene is set forth in SEQ ID NO: 3.

4. The recombinant sequence according to claim 1, wherein the β-globin LCR HS 3-1 comprises β-globin LCR HS3, β-globin LCR HS2, and β-globin LCR HS14 in sequence; a nucleotide sequence of the β-globin LCR HS1 is set forth in SEQ ID NO: 4; a nucleotide sequence of the β-globin LCR HS2 is set forth in SEQ ID NO: 5; and a nucleotide sequence of the β-globin LCR HS3 is set forth in SEQ ID NO: 6.

5. The recombinant sequence according to claim 1, wherein the recombinant sequence comprises at least one selected from the group consisting of the following DNA sequences: 1) a DNA sequence with a nucleotide sequence set forth in SEQ ID NO: 7; and 2) a DNA sequence encoding a recombinant protein with an amino acid sequence set forth in SEQ ID NO: 8.

6. The recombinant sequence according to claim 2, wherein the recombinant sequence comprises at least one selected from the group consisting of the following DNA sequences: 1) a DNA sequence with a nucleotide sequence set forth in SEQ ID NO: 7; and 2) a DNA sequence encoding a recombinant protein with an amino acid sequence set forth in SEQ ID NO: 8.

7. The recombinant sequence according to claim 3, wherein the recombinant sequence comprises at least one selected from the group consisting of the following DNA sequences: 1) a DNA sequence with a nucleotide sequence set forth in SEQ ID NO: 7; and 2) a DNA sequence encoding a recombinant protein with an amino acid sequence set forth in SEQ ID NO: 8.

8. The recombinant sequence according to claim 4, wherein the recombinant sequence comprises at least one selected from the group consisting of the following DNA sequences: 1) a DNA sequence with a nucleotide sequence set forth in SEQ ID NO: 7; and 2) a DNA sequence encoding a recombinant protein with an amino acid sequence set forth in SEQ ID NO: 8.

9. A recombinant vector comprising the recombinant sequence according to claim 1, wherein a backbone vector of the recombinant vector comprises a mammalian expression vector and/or a lentiviral vector.

10. Use of the recombinant sequence according to claim 1 in the preparation of a drug for gene therapy of β-thalassemia.

11. The use according to claim 10, wherein the drug comprising the recombinant sequence, or a recombinant vector, and wherein the recombinant vector comprises the recombinant sequence, and a backbone vector of the recombinant vector comprises a mammalian expression vector and/or a lentiviral vector.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIGS. 1A-B show the electrophoresis results of PCR products of promoters with different lengths and ligated plasmids subjected to enzyme digestion, FIG. 1A shows the electrophoresis results of PCR products of promoters with lengths of 152 bp, 266 bp, and 422 bp, corresponding to 8005 plasmid, 8004 plasmid, and 8006 plasmid, respectively; and FIG. 1B shows the double enzyme digestion identification results of the constructed promoter plasmids with different lengths;

[0028] FIG. 2 shows the sequencing and alignment results of the 8004 plasmid;

[0029] FIGS. 3A-B show the viability detection results of K562 cells transfected with promoter plasmids with different lengths, where FIG. 3A shows the 12-h detection results; and FIG. 3B shows the 24-h detection results;

[0030] FIG. 4 shows the specificity detection results (24 h) in different cells transfected with the 8004 promoter plasmid;

[0031] FIGS. 5A-D show the detection results of β-globin expression in K562 cells into which the 8019 plasmid is constructed and transfected, where FIG. 5A is an electropherogram of the 8019 plasmid subjected to double digestion with AseI and NotI; FIG. 5B is a conclusion of sequencing and alignment results of the 8019 plasmid; FIG. 5C shows the detection results of β-globin mRNA in K562 cells electroporated with the 8019 plasmid; and FIG. 5D shows the detection results of β-globin in K562 cells electroporated with the 8019 plasmid;

[0032] FIGS. 6A-C show the construction of reverse β-globin plasmids with different HSs and the comparison results of effectiveness in K562 cells, where FIG. 6A shows the electrophoresis results of the 8023, 8405, and 8406 plasmids subjected to KpnI digestion; FIG. 6B shows the Western blotting (WB) detection results of β-globin in K562 cells electroporated with the 8023, 8405, and 8406 plasmids; and FIG. 6C is a conclusion of WB detection results of β-globin in K562 cells electroporated with the 8023, 8405, and 8406 plasmids;

[0033] FIG. 7 shows the sequencing and alignment results of the 8405 plasmid fragments; and

[0034] FIG. 8 shows the preliminary verification results of specificity of the 8023 recombinant plasmid in different cells.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] The present disclosure provides an erythroid-specific human β-globin gene promoter with a nucleotide sequence set forth in SEQ ID NO: 1

TABLE-US-00001 (ATCGTAAATACACTTGCAAAGGAGGATGTTTTTAGTAGCAATTTGTACTG ATGGTATGGGGCCAAGAGATATATCTTAGAGGGAGGGCTGAGGGTTTGAAG TCCAACTCCTAAGCCAGTGCCAGAAGAGCCAAGGACAGGTACGGCTGTCAT CACTTAGACCTCACCCTGTGGAGCCACACCCTAGGGTTGGCCAATCTACTC CCAGGAGCAGGGAGGGCAGGAGCCAGGGCTGGGCATAAAAGTCAGGGCAGA GCCATCTATTGCTT).

[0036] In the present disclosure, human fi-globin gene promoters with different lengths are predicted by software, and recombinant plasmids containing promoters of three different lengths (152 bp, 266 bp, and 422 bp) are constructed. Results show that the promoter with a length of 266 bp can specifically and efficiently initiate the expression of the target gene in erythroid cells, and the expression levels of the target gene initiated by the other two promoters are low, with a significant difference.

[0037] The present disclosure provides a recombinant sequence specifically expressing human β-globin in erythroid cells, which is human β-globin LCR HS 3-1+human β-globin gene promoter+β-globin gene+β-globin gene enhancer; and the human β-globin gene promoter is the erythroid-specific human β-globin gene promoter. In the present disclosure, a nucleotide sequence of the β-globin gene enhancer is preferably set forth in SEQ ID NO: 2. A nucleotide sequence of the β-globin gene is preferably set forth in SEQ ID NO: 3. The β-globin LCR HS 3-1 may preferably include β-globin LCR HS3, β-globin LCR HS2, and β-globin LCR HS1 in sequence; a nucleotide sequence of the β-globin LCR HS1 is preferably set forth in SEQ ID NO: 4; a nucleotide sequence of the β-globin LCR HS2 is preferably set forth in SEQ ID NO: 5; and a nucleotide sequence of the β-globin LCR HS3 is preferably set forth in SEQ ID NO: 6. The recombinant sequence may include at least one selected from the group consisting of the following DNA sequences: 1) a DNA sequence with a nucleotide sequence set forth in SEQ ID NO: 7; and 2) a DNA sequence encoding a recombinant protein with an amino acid sequence set forth in SEQ ID NO: 8. There is no particular limitation on the source of the recombinant sequence in the present disclosure, and a recombinant sequence source well known in the art will do, such as a synthetic or PCR amplified sequence.

[0038] In the present disclosure, different regulatory regions are selected to express the human β-globin, and results show that, compared with the regulatory region HS 1-4, the regulatory region HS 1-3 without HS 4 enables the highest expression of β-globin by the β-globin plasmid. Therefore, the regulatory region HS 1-3 without HS 4 is selected as a part of the recombinant sequence for efficient regulation of the expression of human β-globin.

[0039] The present disclosure provides a recombinant vector including the recombinant sequence. A backbone vector of the recombinant vector may preferably include a mammalian expression vector and a lentiviral vector. The mammalian expression vector may preferably include pEGFP-N1 and pUC57 vectors. The lentiviral vector may preferably include a pLV-EF1a-OCT4-IRES-eGFP vector. There is no particular limitation on the sources of the mammalian expression vector and the lentiviral vector in the present disclosure, and vector sources well known in the art will do.

[0040] The present disclosure provides use of the erythroid-specific human β-globin gene promoter, the recombinant sequence, or the recombinant vector in the preparation of a drug for gene therapy of β-thalassemia.

[0041] The present disclosure also provides use of the erythroid-specific human β-globin gene promoter, the recombinant sequence, or the recombinant vector in the gene therapy of β-thalassemia.

[0042] In the present disclosure, a method for the gene therapy of β-thalassemia may preferably include the following steps: inserting the recombinant sequence into a mammalian expression vector to obtain a mammalian recombinant expression vector, or inserting the recombinant sequence into a lentiviral vector to obtain a recombinant lentiviral vector, and introducing the mammalian recombinant expression vector or the recombinant lentiviral vector into a patient to realize the recombinant expression of β-globin in the patient, which increases an expression level of β-globin to achieve the purpose of treating β-thalassemia.

[0043] The present disclosure provides a drug for gene therapy of β-thalassemia, including the erythroid-specific human β-globin gene promoter, the recombinant sequence, or the recombinant vector. The drug may preferably further include a pharmaceutically acceptable adjuvant. The drug achieves the purpose of treating β-thalassemia through the recombinant expression of the recombinant sequence in cells of a patient to increase a content of β-globin.

[0044] The recombinant sequence specifically expressing human β-globin in erythroid cells and use thereof provided by the present disclosure are described in detail below with reference to examples, but these examples may not be understood as a limitation to the protection scope of the present disclosure.

Example 1

[0045] Construction of Promoter Plasmids with Different Lengths and Determination of a Promoter with the Optimal Activity

[0046] 1. A promoter core region for β-globin was predicted on the website http://www.genomatix.de/, and in combination with the promoter lengths in different vectors described in queried literatures, the fragments of 152 bp, 266 bp, and 422 bp before exon 1 for β-globin were finally selected.

[0047] 2. A single restriction site was selected upstream and downstream of a promoter of a pEGFP-N1 backbone plasmid, with Vsp I upstream and NheI downstream. A forward primer (FP) and a reverse primer (RP) were designed according to an NCBI gene sequence and a predicted promoter region, and a restriction site and a protective base were added at the 5′ end to synthesize primer sequences. Exon-RP was adopted as a common reverse primer. Details were shown in Table 1.

TABLE-US-00002 TABLE 1 Primers for amplification of the three promoter fragments Primer name Sequence Sequence No. E152 FP CGATTAATAGTGCCAGAAGAGCCAAGGA SEQ ID NO: 9 E266 FP CGATTAATCGTAAATACACTTGCAAAGG SEQ ID NO: 10 AGG E422 FP CGATTAATCTGGAGACGCAGGAAGAGAT SEQ ID NO: 11 Exon-RP ATATTGCTAGCAAGCAATAGATGGCTCT SEQ ID NO: 12 GCC

[0048] 3. Genomic DNA (gDNA) (Item No.: DP304) was extracted from normal human blood, and specific steps were as follows:

[0049] 1) 200 μL of blood was taken and centrifuged at 3,000 rpm for 5 min, and a resulting supernatant was discarded;

[0050] 2) 200 μL of Buffer GA was added, and a resulting mixture was pipetted up and down for mixing;

[0051] 3) 20 μL of Proteinase K was added, and a resulting mixture was thoroughly mixed by inverting a tube up and down;

[0052] 4) 200 μL of Buffer GB was added, a resulting mixture was thoroughly mixed by inverting the tube up and down and then incubated in a 70° C. metal bath for 10 min, and after a clear solution was obtained, the solution was centrifuged briefly;

[0053] 5) 200 μL of absolute ethanol was added, and a resulting mixture was thoroughly shaken for 15 s and then centrifuged briefly;

[0054] 6) the above solution was transferred to a CB3 adsorption column marked in advance, centrifugation was conducted at 12,000 rpm for 1 min, and a resulting filtrate was discarded;

[0055] 7) 500 μL of Buffer GD was added, centrifugation was conducted at 12,000 rpm for 1 min, and a resulting filtrate was discarded;

[0056] 8) 600 μL of Buffer PW was added, centrifugation was conducted at 12,000 rpm for 1 min, and a resulting filtrate was discarded;

[0057] 9) step 8) was repeated, then centrifugation was conducted at 12,000 rpm for 2 min, a resulting filtrate was discarded, and the adsorption column was transferred to a clean 1.5 ml EP tube marked in advance and air-dried for 5 min to 10 min with a cap open;

[0058] 10) 100 μL of purified water was added to a center of the adsorption column, incubation was conducted at 55° C. for 5 min, centrifugation was conducted at 12,000 rpm for 2 min, and a resulting liquid in the EP tube was collected.

[0059] 4. The promoter fragment was subjected to PCR amplification (Q5 enzyme, Item No.: M0491S), and specific steps were as follows:

[0060] 1) Forward and reverse primers were designed and synthesized according to the target fragment to be amplified, and each diluted to 10 mM for use;

[0061] 2) An ice box was prepared, gloves were worn during the experimental operation, a 0.2 ml PCR tube was prepared, and different pipette tips were used for different reagents. Sample addition was conducted according to the following principle: The samples were added from large-amount to small-amount; the enzyme was added last, which was taken out just before addition; be marked in advance.

[0062] 3) PCR reaction system and parameters were shown in Table 2 (the system could be multiplied according to the experimental purpose).

TABLE-US-00003 TABLE 2 Reaction system Component Volume 5 × Q5 reaction Buffer 10 μL 5 × Q5 GC enhancer 10 μL 10 mM dNTP 1 μL 10 mM forward primer 2.5 μL 10 mM reverse primer 2.5 μL DNA template 0.1-2 μg Q5 enzyme 0.5 μL Making up with ddH.sub.2O to 50 μL

[0063] PCR Amplification Procedure:

[0064] Step 1 pre-denaturation: 95° C. for 3 min;

[0065] Step 2 denaturation: 95° C. for 30 s;

[0066] Step 3 annealing: 60° C. for 30 s;

[0067] Step 4 extension: 72° C. for 30 s, 40 cycles;

[0068] Step 5 re-extension: 72° C. for 5 min.

[0069] 5. Purification of PCR product (Item No.: DP204-02):

[0070] 1) column equilibration: 500 μL of buffer BL was added to a CB3 adsorption column, centrifugation was conducted at 12,000 rpm for 1 min, and a resulting filtrate was discarded;

[0071] 2) 5 folds of buffer PB was added to the PCR product, and a resulting mixture was thoroughly mixed;

[0072] 3) the mixture obtained in step 2) was transferred to the equilibrated CB3 (which needed to be marked in advance), centrifugation was conducted at 12,000 rpm for 1 min, and a resulting filtrate was discarded;

[0073] 4) 600 μL of PW was added, the column was allowed to stand for 2 min to 5 min at room temperature, centrifugation was conducted at 12,000 rpm for 1 min, and a resulting filtrate was discarded;

[0074] 5) step 4) was repeated;

[0075] 6) centrifugation was conducted at 12,000 rpm for 2 min, a resulting filtrate was discarded, and the column was allowed to stand at room temperature for few minutes to be air-dried; and

[0076] 7) CB3 was transferred to a new 1.5 mL EP tube marked in advance, 50 μL or more of an eluent was added dropwise in a suspended manner to a center of an adsorption membrane, centrifugation was conducted at 12,000 rpm for 2 min, and a resulting filtrate was collected for concentration determination.

[0077] 6. VspI and NheI double enzyme digestion backbone and target fragment (Item No.: VspI: FD0914, and NheI: FD0947). An enzyme digestion system was shown in Table 3 (which could be proportionally decreased or increased).

TABLE-US-00004 TABLE 3 Enzyme digestion reaction system Component Volume Backbone or target fragment <2 μg 10 × Green buffer 3 μL Vsp I 1 μL Hind III 1 μL Making up with ddH.sub.2O to 30 μL

[0078] 7. A target band obtained after enzyme digestion was recovered by subjecting the PCR product to gel extraction, and a long EGFP-containing fragment among enzyme digestion products of the backbone plasmid was recovered (gel extraction kit, Item No.: K0691). Specific steps were as follows:

[0079] 1) an electrophoresis tank and the like were cleaned, and 1% agarose gel was prepared;

[0080] 2) sample loading: 5 μL of Mark was added, and all digestion products were added to sample wells every other well;

[0081] 3) electrophoresis was conducted at 120 V for 30 min, and a 1.5 mL EP tube was taken, marked, and weighed;

[0082] 4) gel with the target band was cut and collected in the EP tube;

[0083] 5) a total weight of the gel and the tube was measured and subtracted by a weight of the tube to obtain a weight of the gel;

[0084] 6) binding buffer was added to the gel, and a resulting mixture was incubated in a 55° C. metal bath for 10 min until the gel was completely dissolved, where a ratio of the weight of gel to a volume of binding buffer was 1:1;

[0085] 7) isopropyl alcohol (IPA) was added, and a resulting mixture was pipetted up and down for mixing, where a ratio of the weight of gel to a volume of IPA was 1:1;

[0086] 8) the mixture was transferred to an adsorption column marked in advance, centrifugation was conducted at 12,000 rpm for 1 min, and a resulting filtrate was discarded; and then 100 μL of binding buffer was added, centrifugation was conducted at 12,000 rpm for 2 min, and a resulting filtrate was discarded;

[0087] 9) 700 μL of wash buffer was added, centrifugation was conducted at 12,000 rpm for 2 min, and a resulting filtrate was discarded; centrifugation was conducted at 12,000 rpm for 2 min, and a resulting filtrate was discarded; and the column was transferred to a new EP tube marked in advance, and then air-dried for 5 min; and

[0088] 10) 30 μL of pure water was added, and the mixture was incubated in a 55° C. metal bath for 5 min; and centrifugation was conducted at 12,000 rpm for 2 min, a resulting filtrate was collected, and 2 μL of the filtrate was taken for concentration determination.

[0089] 8. Ligation (T4 DNA ligase, Item No.: EL0011). Specific steps were as follows:

[0090] Fragment:backbone=3:1 (mass ratio), and thus fragment (ng)=backbone (ng)×3×fragment length/backbone fragment length. A specific ligation system was shown in Table 4. After samples were added, the ligation was conducted at room temperature for 2 h or overnight.

TABLE-US-00005 TABLE 4 Ligation reaction system Component Volume Backbone 50-100 ng Target fragment 300 ng × fragment length/backbone length T4 DNA Ligase buffer 1 μL T4 DNA Ligase 1 μL ddH.sub.2O Making up to 20 μL

[0091] 9. Transformation (Item No.: CB101-02):

[0092] 1) the plasmid was taken out and put on ice, and competent cells were taken out and thawed on ice;

[0093] 2) 5 μL of the plasmid was added to 50 μL of the competent cells, and a resulting mixture was allowed to stand on ice for 30 min;

[0094] 3) heat shock: the heat shock was conducted at 42° C. for 90 s;

[0095] 4) resulting cells were immediately allowed to stand on ice for 5 min;

[0096] 5) recovery: 150 μL of a resistance-free liquid medium was added, and the cells were cultivated for 1 h at 37° C. and 160 rpm;

[0097] 6) coating: a recovered bacterial solution was taken and spread on an LB resistant solid medium by sterilized beads; and

[0098] 7) cultivation was conducted overnight at 37° C. and the colony growth was observed.

[0099] 10. Amplification of monoclonal colonies:

[0100] a 15 mL centrifuge tube was taken, 5 mL of a resistance-free liquid medium was added, kana was added to make its final concentration 50 μg/mL, and single colonies were picked and added to the centrifuge tube with a high-pressure pipette tip, and the colonies were cultivated overnight at 37° C. and 160 rpm.

[0101] 11. Extraction of amplified plasmid (Item No.: AP-MN-P-50): Specific steps were as follows:

[0102] 1) 1 ml to 4 ml of a bacterial culture was taken and centrifuged at 12,000 rpm for 2 min, and a resulting supernatant was discarded (the bacterial culture could be centrifuged multiple times, and a resulting bacterial precipitate was collected in a centrifuge tube).

[0103] 2) 250 μL of Buffer S1 (if it was used for the first time, RNaseA was added before use) was added to the centrifuge tube with the bacterial precipitate, and a resulting mixture was pipetted up and down and resuspended until there was no small bacterial mass;

[0104] 3) 250 μL of Buffer S2 was added to the centrifuge tube, and the centrifuge tube was gently inverted up and down for 6 to 8 times to fully lyse the bacteria (notes: the mixing must be conducted gently to avoid contamination to the bacterial gDNA, such that a resulting bacterial solution became clear and viscous; and a time of the mixing should not exceed 5 min, such as to avoid damage to the plasmid);

[0105] 4) 350 μl of Buffer S3 was added to the centrifuge tube, and the centrifuge tube was immediately gently inverted up and down for 6 to 8 times until a white flocculent precipitate appeared; a resulting mixture was centrifuged at 12,000 rpm for 10 min, and a resulting supernatant was carefully transferred to an adsorption column marked in advance (the adsorption column was added to the collection tube) and placed at room temperature for 2 min; and centrifugation was conducted at 12,000 rpm for 1 min, a resulting filtrate was discarded, and the adsorption column was put back to the collection tube;

[0106] 5) 500 μL of a washing solution W1 was added to the adsorption column, centrifugation was conducted at 12,000 rpm for 1 min, a resulting filtrate was discarded, and the adsorption column was put back to the collection tube;

[0107] 6) 700 μL of a washing solution W2 was added to the adsorption column (it was checked whether absolute ethanol had been added before the addition of the washing solution), centrifugation was conducted at 12,000 rpm for 1 min, a resulting filtrate was discarded, and the adsorption column was put back to the collection tube;

[0108] 7) step 6) was repeated;

[0109] 8) centrifugation was conducted at 12,000 rpm for 2 min, the adsorption column was transferred to a new EP tube marked in advance, and the tube was placed at room temperature for few minutes with a cap open to remove the residual washing solution in the adsorption column; and

[0110] 9) 60 μL to 80 μL of ddH.sub.2O was added dropwise in a suspended manner to a center of an adsorption membrane, incubation was conducted at 55° C. for 8 min, centrifugation was conducted at 12,000 rpm for 2 min, and a resulting filtrate was collected, which was the extracted plasmid DNA.

[0111] 12. Verify of whether the recombinant plasmid was correctly ligated. The recombinant plasmid pEGFP-βp (Exon152 bp) was numbered 8005; the recombinant plasmid pEGFP-βp (Exon266 bp) was numbered 8004; and the recombinant plasmid pEGFP-βp (Exon422 bp) was numbered 8006.

[0112] 1) Electrophoresis verification after double digestion: An enzyme digestion system was the same as above, and a small system of 10 μL or 5 μL could be used. A band size was verified by electrophoresis after enzyme digestion, as shown in FIG. 1B. The band sizes of the plasmids 8005, 8004, and 8006 after enzyme digestion were 4,148 bp+152 bp, 4,148 bp+266 bp, and 4,148 bp+422 bp, respectively.

[0113] 2) Verification by sequencing: The extracted plasmids were sent to a company for sequencing the inserted fragments, such as to further verify whether the plasmids were constructed successfully, and it was determined that the plasmid fragments 8005, 8004, and 8006 were correct. A sequencing result of the plasmid 8004 was shown in FIG. 2.

[0114] 13. Activity detection and specificity verification for recombinant promoter plasmids:

[0115] 1) Activity detection for recombinant promoter plasmids with different lengths:

[0116] Cell transfection: K562 cells (Item No.: L3000015) were transfected with a liposome. Specific steps were as follows:

[0117] Culture plates were prepared. Cells were collected by centrifugation, washed once with PBS, inoculated into a 6-well plate or a 12-well plate, and the plate was put back to an incubator for continuous culture. Then a transfection system was added: tube A: diluted liposome: 250 μL opti-MEM+7.5 μL lipofection; and tube B: diluted DNA: 250 μL opti-MEM+5 μL P3000+1 μg recombinant plasmid DNA+1 μg mCherry plasmid with red fluorescence. A and B were thoroughly mixed, incubated at room temperature for 15 min, and the incubation solution was added to K562 cell wells, and the cells were cultivated in the incubator. At 12 h and 24 h of the cultivation, the number of cells with green fluorescence in the red fluorescence was observed, and the promoter activity was detected. The activity detection results were shown in FIGS. 3A-B, and it can be seen that, either at 12 h (FIG. 3A) or at 24 h (FIG. 3B), the recombinant plasmid 8005 (that is, when the promoter length was 266 bp) showed the optimal promoter activity.

[0118] 2) Specificity detection of the recombinant promoter plasmid 8004:

[0119] Cell transfection: a liposome was used to transfect different cell lines (Item No.: L3000015), and specific steps were as follows:

[0120] Culture plates were prepared. Cells were collected by centrifugation, washed once with PBS, inoculated into a 6-well plate or a 12-well plate, and the plate was put back to an incubator for continuous culture. Then a transfection system was added: tube A: diluted liposome: 250 μL opti-MEM+7.5 μL lipofection; and tube B: diluted DNA: 250 μL opti-MEM+5 μL P3000+1 μg recombinant plasmid DNA+1 μg mCherry plasmid with red fluorescence. A and B were thoroughly mixed, incubated at room temperature for 15 min, and the incubation solution was added to K562, Huvec, 293T, 293, and HL60 cell wells, and the cells were cultivated in the incubator. At 12 h and 24 h of the cultivation, the number of cells with green fluorescence in the red fluorescence was observed, and the promoter specificity was detected. The specificity detection results were shown in FIG. 4, and it can be seen that an expression level in K562 cells was significantly higher than expression levels in other groups, and the expression levels in the other groups were extremely low.

Example 2

[0121] Construction of a Plasmid without β-Globin LCR (HS) Using the Selected Promoter Length

[0122] 1. An enhancer core region of β-globin was predicted on the website http://www.genomatix.de/, and in combination with the promoter lengths in different vectors described in queried literatures, a β-globin LCR (HS)-free fragment from the promoter with the optimal activity (266 bp before exon 1) to the β-globin enhancer was amplified, which was named pβp (Exon266 bp)-βglobin-βE-713 and numbered 8019.

[0123] 2. A single restriction site was selected on the backbone plasmid, with VspI upstream and NotI downstream; and forward and reverse primers were designed according to the NCBI gene sequence and the predicted promoter region, and the restriction site and a protective base were added at the 5′ end.

[0124] Table 5 Sequence information of primers for amplification of the promoter protected by the present disclosure

TABLE-US-00006 TABLE 5 Sequence information of primers for amplification of the promoter protected by the present disclosure Primer name Sequence Sequence No. E266FP CGATTAATCGTAAATACACTTGCAA SEQ ID NO: 10 AGGAGG enhancer-R ATGCGGCCGCTGGTAACACTATGCT SEQ ID NO: 13 AATAA

[0125] 3. gDNA was extracted from normal human blood (Item No.: DP304), and specific steps were the same as in Example 1.

[0126] 4. The β-globin fragment (Phusion enzyme, Item No.: F530S) without the β-globin LCR (HS) was subjected to PCR amplification. A PCR amplification system was shown in Table 6.

TABLE-US-00007 TABLE 6 PCR amplification reaction system for β-globin gene fragment Component Volume 5 × HF Buffer 20 μL 10 mM dNTP 2 μL 10 mM E266FP 5 μL 10 mM enhancer-R 5 μL DNA template 0.1-2 μg Phusion enzyme 1 μL Making up with ddH.sub.2O to 100 μL

[0127] PCR Amplification Procedure:

[0128] Step 1 pre-denaturation: 98° C. for 2 min;

[0129] Step 2 denaturation: 98° C. for 10 s;

[0130] Step 3 annealing: 63° C. for 30 s;

[0131] Step 4 extension: 72° C. for 30 s to 5 min, depending on an amplification length, 40 cycles;

[0132] Step 5 re-extension: 72° C. for 5 min;

[0133] Step 6 temporary storage: about 30 min at 4° C., depending on a specific time demand.

[0134] 5. Purification of PCR amplification product (common DNA product purification kit, Item No.: DP-204-02), and specific steps were the same as in Example 1.

[0135] 6. VspI and NheI double enzyme digestion backbone and target fragment (Item No.: VspI: FD0914, and NotI: FD0594). An enzyme digestion system was shown in Table 7 (which could be proportionally decreased or increased).

TABLE-US-00008 TABLE 7 VspI and NheI double enzyme digestion reaction system Component Volume Backbone or target fragment 2 μg 10 × Green buffer 3 μL VspI 1 μL NotI 1 μL Making up with ddH.sub.2O to 30 μL

[0136] 7. The target fragment and 713 plasmid backbone were recovered by gel (gel extraction kit, Item No.: K0691), and specific steps were the same as in Example 1.

[0137] 8. Ligation (T4 DNA ligase, Item No.: EL0011): Specific steps were the same as in Example 1:

[0138] 9. Transformation (Item No.: CB101-02): Specific steps were the same as in Example 1.

[0139] 10. Amplification of monoclonal colonies: Specific steps were the same as in Example 1.

[0140] 11. Extraction of amplified plasmid (Item No.: AP-MN-P-50): Specific steps were the same as in Example 1.

[0141] 12. Verify of whether the LCR-free β-globin recombinant plasmid was correctly ligated. The recombinant plasmid was named pβp(Exon266 bp)-βglobin-βE-713 and numbered 8019.

[0142] 1) Electrophoresis verification after double digestion: An enzyme digestion system was the same as above, and a small system of 10 μL or 5 μL could be used. A band size was verified by electrophoresis after enzyme digestion, as shown in FIG. 5A. The band sizes of the plasmid 8019 after enzyme digestion were 2,718 bp and 3,339 bp.

[0143] 2) Verification by sequencing: The extracted plasmid was sent to a company for sequencing the inserted fragment, such as to further verify whether the plasmid was constructed successfully. As shown in FIG. 5B, the sequencing result showed that there were 8 sites inconsistent with the NCBI sequence, but these sites were all outside the open reading frame (ORF).

Example 3

[0144] The effectiveness test of the β-globin recombinant plasmid without LCR, K562 cells were electrotransfected with the recombinant plasmid, and untransfected cells and blank transfected cells were set as control groups. A relative expression level of β-globin mRNA was detected by qPCR, and an expression level of β-globin was detected by WB. Results were shown in FIG. 5C and FIG. 5D. Compared with the control groups, the expression of β-globin was not significantly changed with reference to the blank group at either the mRNA level or the protein level, without statistically-significant difference.

[0145] (1) K562 cells were electroporated (electrode tube set, Item No.: No. 1207) by the following specific steps:

[0146] A culture plate was prepared, a complete medium was added to the culture plate, and the culture plate was incubated in an incubator. Cells were collected by centrifugation and washed once with PBS, 20 μL of an electroporation solution and 4 μg of the recombinant plasmid were added to the cells, and a resulting mixture was pipetted up and down for mixing and then transferred to an electroporation cuvette; then electroporation was conducted according to different electroporation conditions of different cells, and a system obtained after the electroporation was added to a corresponding well of the culture plate incubated in the incubator; and the culture plate was incubated in the incubator for 48 h, then the RNA and protein were extracted, and a content of β-globin was determined.

[0147] (2) A specific method for the detection of the relative expression level of β-globin mRNA by qPCR was as follows:

[0148] 1) RNA extraction (RNA extraction kit, Item No.: R1058): Specific steps were as follows:

[0149] A: cells were resuspended and washed once with PBS;

[0150] B: cell counting was conducted;

[0151] C: a cell precipitate obtained after the washing was collected, 300 μL of a cell lysis solution was added, and a resulting mixture was pipetted up and down for thorough mixing;

[0152] D: lysed cells were transferred to a yellow adsorption column, centrifugation was conducted at 12,000 rpm for 2 min, a resulting filtrate was retained, and the adsorption column was discarded;

[0153] E: 150 μL of absolute ethanol was added to the filtrate, a resulting mixture was thoroughly mixed and transferred to a green adsorption column, centrifugation was conducted at 12,000 rpm for 2 min, and a resulting filtrate was discarded;

[0154] F: 400 μL of wash buffer was added to the adsorption column, centrifugation was conducted, and a resulting filtrate was discarded;

[0155] G: an enzyme-free EP tube was taken, 75 μL of DNA digestion buffer and 5 μL of DNase were thoroughly mixed and added to the adsorption column, and then the adsorption column was incubated at room temperature for 15 min;

[0156] H: after the incubation was completed, 400 μL of RNAprep buffer was added to the column, centrifugation was conducted at 12,000 rpm for 2 min, and a resulting filtrate was discarded;

[0157] I: 700 μL of RNA WASH BUFFER was added to the column, centrifugation was conducted at 12,000 rpm for 2 min, and a resulting filtrate was discarded; and 400 μL of RNA WASH BUFFER was added, centrifugation was conducted at 12,000 rpm for 3 min, and a resulting filtrate was discarded; and

[0158] J: an enzyme-free EP tube was taken, the green adsorption column was transferred to the EP tube, 100 μL of DNase/RNase Free Water was added to a center of the column, and centrifugation was conducted at 12,000 rpm for 2 min; an appropriate amount of the obtained RNA was taken out and dispensed for concentration determination and electrophoresis, which was intended to reduce the repeated freezing and thawing of RNA; and the RNA was stored at −80° C. (RNA electrophoresis: an electrophoresis solution was replaced in advance, 100 ng to 500 ng of RNA was taken, thoroughly mixed with loading and enzyme-free water, and then loaded, and electrophoresis was conducted at 10 V/cm)

[0159] 2) cDNA was synthesized by reverse transcription (reverse transcription kit: Thermo: RevertAid First Strand cDNA Synthesis Kit, Item No. K1621), and specific steps were as follows:

[0160] A: a required amount of RNA was calculated. 1 μg of RNA was generally used for a 20 μL system. The system could be appropriately enlarged or reduced (0.1 ng to 5 μg of RNA could be used);

[0161] B: reagents other than the enzyme in the reverse transcription kit were taken out and placed on ice;

[0162] C: a 0.2 ml enzyme-free EP tube was taken, marked, and placed on ice;

[0163] D: reaction preparation (12 μL): RNA 1 μg+Random Primer 1 μL+enzyme-free water (up to 12 μL)

[0164] E: the reaction was incubated in a 65° C. metal bath for 5 min, and then put back on ice; and

[0165] F: the following reagents were added in sequence: 8 μL in total;

TABLE-US-00009 TABLE 8 Reverse transcription reaction Component Volume 5 × Reation Buffer 4 μL RiboLock RNase Inhibito 1 μL 10 mM dNTP Mix 2 μL RevertAid M-MμLV RT (200 U/μL)(enzyme) 1 μL

[0166] G: brief centrifugation was conducted, and incubation was conducted in a 42° C. metal bath for 60 min;

[0167] H: pre-synthesis was conducted at 25° C. for 5 min;

[0168] I: a heat treatment was conducted at 70° C. for 5 min to terminate the reaction; and

[0169] J: a product was stored at −80° C. or used for subsequent experiments.

[0170] 3) q-PCR detection of globin level:

[0171] q-PCR system: Experimental group: enzyme-free water 6.8 μL+2×SYBR 10 μL+cDNA 1.6 μL+forward primer 0.8 μL+reverse primer 0.8 μL (forward primer: mRNA-F-2: CTGAGGAGAAGTCTGCCGTTA, SEQ ID NO: 14, reverse primer: mRNA-R-2: GAGGTTGTCCAGGTGAGCCA, SEQ ID NO: 15). Blank control group: enzyme-free water 8.4 μL+2×SYBR 10 μL+forward primer 0.8 μL+reverse primer 0.8 μL (3 replicate wells were required for each group, and thus water and cDNA could be mixed in advance and then evenly dispensed to each well).

[0172] q-PCR procedure: Details can be seen in the instrument manual. However, an annealing temperature of the primers should be known in advance, an annealing temperature of the primers for the internal reference should not be too different from an annealing temperature of the primers for the experimental group, and an average value is generally taken.

[0173] (3) Detection of β-globin level by WB (primary and secondary antibodies for β-globin, Item No.: SC-21757 and 62-6520): Specific steps were as follows:

[0174] 1) cell collection: cells were collected by centrifugation, washed once with PBS, and counted by a cell counter;

[0175] 2) total cellular protein (TCP) extraction: a lysis solution (strong lysis solution+PMSF=100:1) was added at an amount enabling 50 μL to 100 μL of lysis solution for 3 to 5×10.sup.6 cells, and a resulting mixture was shaken on ice for 1 h;

[0176] 3) protein denaturation: 5×protein loading buffer was added to a protein sample at a final concentration of 1×, and a sample tube was wrapped with a sealing tape and then incubated in a 100° C. metal bath for 10 min;

[0177] 4) a centrifuge was pre-cooled, then centrifugation was conducted at 12,000 rpm and 4° C. for 20 min, and a resulting supernatant was collected, which was the protein sample;

[0178] 5) gel preparation;

[0179] 6) the supernatant obtained after the centrifugation was taken and loaded for electrophoresis;

[0180] 7) transfer and blocking;

[0181] 8) primary antibody incubation: the membrane was incubated for 1 h at room temperature or overnight at 4° C., and then washed with PBST three times, each time for 10 min;

[0182] 9) secondary antibody incubation: the membrane was incubated for 1 h at room temperature or overnight at 4° C., and then washed with PBST three times, each time for 10 min; and

[0183] 10) an exposure solution was prepared according to a ratio of 1:1 and then added dropwise onto the washed membrane, and exposure was conducted with a Bio-rad exposure machine.

Example 4

[0184] With the screened promoter length, reverse β-globin plasmids with different β-globin LCRs (HS) were constructed, the effectiveness and specificity of which were tested, and an efficient and specific recombinant sequence carrying human β-globin gene was finally obtained.

[0185] 1. Synthesis of reverse β-globin plasmid with HS 1-4: The LCR region of β-globin was predicted on the website http://www.genomatix.de/. According to results of related literatures, HS 1-4 can increase the expression of β-globin, and in most literatures, β-globin is inserted into a recombinant plasmid reversely, which may enhance the expression of β-globin. Therefore, HS 1-4 was ligated to the β-globin sequence in Example 2. Because the recombinant plasmid 8019 constructed in Example 2 included a mutated sequence, a recombinant β-globin gene sequence that had no mutation and included the β-globin LCR HS 4-1, β-globin gene promoter, β-globin coding sequence, and β-globin gene enhancer sequences was designed, which was specifically shown as follows. The sequence was synthesized by Sangon and ligated to a pUC57 vector, and a resulting plasmid was named pHS(4-1)-βp(Exon266 bp)-βglobin-βE-pUC57 and numbered 8023. The plasmid was digested and sequenced. Digestion results of 8023 were shown in FIG. 6A. According to alignment results, the sequence was correct.

[0186] 2. A recombinant reverse β-globin plasmid without HS 4 was constructed with 8023 as a template, and the constructed plasmid was named pHS(3-1)-βp(Exon266 bp)-βglobin-βE-pUC57 and numbered 8405. A specific construction method was as follows: a forward primer was designed upstream of HS3, and a KpnI restriction site was added, where the forward primer was HS3-pUC57-F: CTTggtac caagactgagctcagaaga (SEQ ID NO: 16); a reverse primer was designed downstream of the enhancer, and a SalI restriction site was added, where the reverse primer was lv-R-2: GCCGTCGACtggtaacactatgctaataac (SEQ ID NO: 17); PCR was conducted with 8023 as a template, and a product was purified; the PCR product was digested with DpnI, kpnI, and SaII, and the backbone was digested with kpnI and SaII (DpnI Item No.: FD1703, kpnI Item No.: FD 0524, and SaII Item No.: FD0644); and target fragments were recovered by gel extraction, then ligated by T4 DNA ligase, and transformed for amplification. Enzyme digestion and sequencing verification were conducted, and according to enzyme digestion and sequencing verification results, the fragment sequence was correct. The enzyme digestion results were shown in FIG. 6A, the sequencing results were shown in FIG. 7, and detailed experimental methods involved in the construction were the same as above.

[0187] 3. A recombinant reverse β-globin plasmid without HS 1 was constructed with 8023 as a template, and the constructed plasmid was named pHS(4-2)-βp(Exon266 bp)-βglobin-βE-pUC57 and numbered 8406. A specific construction method was as follows: a forward primer was designed downstream of HS 1, a reverse primer was designed downstream of HS 2, and a BgIII restriction site was added at 5′ end of each of the forward and reverse primers, where the primers were as follows: BgIII-E266-F: GCGagatctATCGTAAATACACTTGC (SEQ ID NO: 18), and BgIII-HS2-R: GCGagatctTTCAGGAAATAATATATTC (SEQ ID NO: 19); PCR was conducted with 8023 as a template, and a product was purified; enzyme digestion was conducted overnight with DpnI and BgIII (BgIII Item No.: FD0084); and target fragments were recovered by gel extraction, then ligated by T4 DNA ligase, and transformed for amplification. KpnI enzyme digestion and sequencing verification were conducted, and according to enzyme digestion and sequencing verification results, the fragment sequence was correct. The enzyme digestion results were shown in FIG. 6A, and detailed experimental methods involved in the construction were the same as above.

[0188] 4. K562 and 293T cell lines were each electrotransfected with 4 μg of the plasmid 8023 to verify the specificity of the recombinant plasmid, and a specific method was the same as in Example 2. Results were shown in FIG. 8, and it can be seen that, after transfection of the same β-globin, an expression level of β-globin in 293T cells was still not significantly increased, indicating that the sequence is relatively specific. According to the specificity detection results of the promoter, it is considered that the constructed plasmid has some erythroid specificity.

[0189] 5. The effectiveness of the above three reverse β-globin sequences with different β-globin LCRs (HS) was detected, and a specific method was as follows:

[0190] 1) K562 cells were electroporated (electrode tube set, Item No.: No. 1207), and specific steps were the same as in Example 2.

[0191] 2) Detection of a relative expression level of β-globin mRNA by qPCR: Specific steps were the same as in Example 2.

[0192] 3) Detection of β-globin level by WB (primary and secondary antibodies for β-globin, Item No.: SC-21757 and 62-6520): Specific steps were the same as in Example 2.

[0193] 4) Detection results by WB were shown in FIG. 6B and FIG. 6C, and it can be seen that the recombinant plasmid 8405 led to the highest expression level of β-globin, that is, the reverse β-globin plasmid without HS 4 (HS(3-1)-βp(Exon266 bp)-βglobin-βE-puc57) led to the highest expression level of β-globin, indicating that the HS3-1-βp(Exon266 bp)-βglobin-βE sequence can efficiently express β-globin.

[0194] It can be seen from the above examples that, in the present disclosure, three human β-globin gene promoter plasmids (promoter-EGFP plasmids) with different lengths were first constructed and optimized, and a promoter region with high activity, strong specificity, and a length of 266 bp was screened out by using the co-transfection method with the Mcherry plasmid. In the present disclosure, with the promoter length screened out, a plasmid without β-globin LCR (HS) is constructed and the effectiveness thereof is tested; then with the promoter length screened out, reverse β-globin plasmids with different β-globin LCRs (HS) are constructed and the effectiveness and specificity thereof are tested; and finally a recombinant sequence with the regulatory region of HS3-1 and the 266 bp promoter and enhancer of the human β-globin gene in sequence is obtained, and the recombinant sequence can efficiently and specifically express the human β-globin, which provides a basis for subsequent gene therapy of thalassemia.

[0195] The above descriptions are merely preferred implementations of the present disclosure. It should be noted that those skilled in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.