APPLICATION OF PLANT AS HOST IN EXPRESSING VACCINE OF MIDDLE EAST RESPIRATORY SYNDROME

Abstract

Provided is an application of a plant as a host in expressing a vaccine of Middle East respiratory syndrome. In particular, lettuce is utilized to transiently express fusion proteins to prepare a vaccine for Middle East respiratory syndrome.

Claims

1. A method for expressing a vaccine for Middle East respiratory syndrome, comprising using a plant as a host.

2. The method according to claim 1, wherein the plant is selected from the group consisting of lettuce, tobacco, Chinese cabbage, rice, corn, soybean and wheat; and the organ of the plant for use is selected from the group consisting of a leaf, a seed, a rhizome and a whole plant.

3. An expression vector comprising CTB, RBD-Fc and a vector.

4. The expression vector according to claim 3, wherein the codons of CTB and RBD-Fc are plant-preferred codons, and a codon-optimized sequence of CTB and RBD-Fc fusion protein is set forth in SEQ ID NO: 7.

5. The expression vector according to claim 3, wherein the vector is a binary plant vector.

6. The expression vector according to claim 3, wherein the method of constructing the expression vector comprises the steps of: Step 1: linking CTB with RBD-Fc to obtain CTB-5377-588-Fc; Step 2: optimizing the codons of CTB, RBD and Fc to plant-preferred codons respectively, and linking the optimized CTB with the optimized RBD-Fc to obtain a codon-optimized CTB-5377-588-Fc sequence; Step 3: adding KpnI restriction site at the 5 end, and SacI and Pad sites at the 3 end of the codon-optimized CTB-5377-588-Fc sequence and CTB-5377-588-Fc sequence, and then generating pWT-CTB-RBD-Fc vector and pOP-CTB-RBD-Fc vector respectively; and Step 4: producing gene fragments of WT-MersCoV and OP-MersCoV using KpnI/SacI, respectively, and cloning WT-MersCoV and OP-MersCoV into the binary plant expression vector pCam35S to obtain transient expression vectors of p35S-WT-MersCoV and p355-OP-MersCoV respectively.

7. A method for expressing a vaccine for Middle East respiratory syndrome, comprising using the expression vector according to claim 3.

8. The method according to claim 7, comprising transforming the expression vector into agrobacterium, performing agrobacterium-mediated vacuum infiltration on a plant tissue, and extracting and isolating protein to obtain a vaccine for Middle East respiratory syndrome.

9. The method according to claim 8, wherein the agrobacterium-mediated vacuum infiltration comprises the steps of: Step 1: vacuuming for 2545s; Step 2: maintaining under a vacuum of 95 kPa for 3060s; Step 3: releasing pressure to allow a penetrating fluid to penetrate into the plant tissue; and repeating Step 1 to Step 3 for 2 to 3 times, and then keeping in dark for 4 days.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings to be used in the embodiments or the description of the prior art will be briefly described below.

[0041] FIG. 1 shows the constructs of Mers-CoV WT and optimized gene (constructed and synthesized by ThermoFisher).

[0042] FIG. 2 shows a diagram of the construction of the plant binary expression vector p355-OP-MersCoV and p35S-WT-MersCoV. WT-CTB-RBD-Fc and CTB-RBD-Fc fragments of FIG. 1 are generated by restriction endonuclease double digestion (KpnI/SacI), and then the fragments are ligated into the KpnI/SacI sites of pCam35S to generate plant binary expression vectors p35S-OP-MersCoV and p35S-WT-MersCoV.

[0043] FIG. 2 (A) shows p35S-WT-MersCoV and FIG. 2 (B) shows p35S-OP-MersCoV;

[0044] wherein, 35S is the CaMV35S promoter with tobacco mosaic virus (TMV) 5UTR; NPTII is used for the expression of nptII gene for kanamycin resistance; Nos 3 is a terminator; wild type and plant codon-optimized sequences; CTB is cholera toxin B subunit; RBD is MERS-CoV receptor binding domain (S377-588); Fc is Fc fragment of human IgG.

[0045] FIG. 2 (C) shows the enzyme digestion (KpnI/SacI) of p35S-OP-MersCoV and p35S-OP-MersCoV fragments; lane 1 shows WT-CTB-RBD-Fc fragment and lane 2 shows OP-CTB-RBD-Fc fragment.

[0046] FIG. 3 shows SDS-PAGE detection of the affinity reaction between purified CTB-RBD-Fc and DPP4. Lane 1: purified recombinant CTB-RBD-Fc (WT) (5 g); Lane 2: purified recombinant CTB-RBD-Fc (OP) (5 g); Lane 3: DPP4 (5 g); Lane 4: DPP4+CTB-RBD-Fc (WT) (5 g); Lane 5: DPP4+CTB-RBD-Fc (OP) (5 g); Lane 6: sample without vacuum infiltration treatment as negative control.

DETAILED DESCRIPTION OF THE INVENTION

[0047] The invention discloses use of a plant as a host for expressing a vaccine for Middle East respiratory syndrome. Those skilled in the art can learn from the contents of this document and appropriately improve the process parameters. It is specifically to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described by the preferred embodiments, and it is obvious that the methods and applications described herein can be modified or appropriately changed and combined to implement and apply the techniques of the present invention without departing from the scope of the present invention.

[0048] The invention utilizes lettuce as an effective platform for recombinant protein production. The growth time of tobacco plants used for vacuum agrobacterium infiltration is usually 4 to 6 weeks. The invention eliminates the plant growth cycle and greatly saves the time for planting plants in the early stage. The exogenous MERS-CoV vaccine protein was expressed in a lettuce system and successfully isolated under mild conditions, demonstrating that the lettuce expression platform can be used to produce MERS-CoV vaccines against large-scale sudden infection of MERS-CoV.

[0049] The materials and reagents used in the application of the plant as a host for expressing a vaccine for Middle East respiratory syndrome according to the present invention are commercially available.

[0050] The present invention is further illustrated below in conjunction with the examples.

Example 1. Construction of Plant Transient Expression Vector

[0051] To improve expression and translation of proteins in the lettuce system, in the present invention, the codons of CTB-S377-588-Fc were redesigned to preferentially match the codon frequency found in plants (sequences are set forth in SEQ ID NO: 7). The cholera toxin B subunit (CTB) is shown to increase antigen uptake and effectively induce mucosal responses. To improve the immunogenicity of the intranasal vaccine, CTB (Genbank ID: AY475128.1) was fused to RBD (Genbank ID: KM027288.1)-Fc (Genbank ID: BC156864.1). The optimized codons for CTB-5377-588-Fc were designed and synthesized by GeneArt GeneOptimizer (ThermoFisher).

[0052] KpnI restriction site was added at the 5 end and the SacI and Pad sites were added at the 3 end of CTB-5377-588-Fc and the optimized sequence thereof pWT-CTB-RBD-Fc and pOP-CTB-RBD-Fc vectors were generated by ThermoFisher. The gene fragments were obtained by KpnI/SacI digestion and cloned into the binary plant expression vector pCam35S to generate the transient expression vectors p35S-WT-MersCoV and p35S-OP-MersCoV, respectively, which were confirmed to be full size fragment by double enzyme digestion. There two plant expression constructs were transformed into Agrobacterium tumefaciens GV3101 by electroporation using Multiporator (Eppendorf, Hamburg, Germany), respectively. The resulting strains were spread evenly on selective LB plates containing kanamycin antibiotic (50 mg/L). After incubating for 2 days at 28 C. in the dark, single colonies were picked and inoculated into 0.5 LYEB (yeast extract broth, 5 g/L sucrose, 5 g/L tryptone, 6 g/L yeast extract, 0.24 g/LMgSO.sub.4, pH 7.2) supplemented with antibiotic liquid medium (50 mg/L kanamycin). The inoculated culture was incubated at 25 to 28 C. for 72 h in a shaker (220 rpm). The OD 600 value was measured and adjusted to 3.5 to 4.5 by adding YEB medium. The culture medium was then collected and centrifuged (4,500 rpm) for 10 min. The agrobacterium cells were resuspended in penetrating medium (10 mM MES, 10 mM MgSO4) until O.D.600 was 0.5.

[0053] The gene fragments of WT-MersCoV and the optimized fragment OP-MersCoV are shown in FIG. 1. The two binary plant expression vectors p35S-WT-MersCoV and p35S-OP-MersCoV are constructed as shown in FIG. 2A, B. After completion of the construction, digestion was performed with specific restriction enzymes to confirm that the gene fragment was intact (FIG. 2C). Most of the lettuce tissue was submerged during vacuum infiltration. Except for the solid midrib area, the remaining parts turned to a yellowish brown area after 4 days of vacuum infiltration.

Example 2. Agrobacterium-Mediated Vacuum Infiltration

[0054] The prepared agrobacterium culture suspension was added into a 2 L beaker and the beaker was placed in a desiccator. 10% of the top part of the lettuce leaves were removed with a knife, and the rest part was inverted (core up) and gently spun in the bacterial suspension, and the desiccator was then sealed. Vacuum was applied using a vacuum pump (Welch Vacuum, Niles, Ill., USA) for about 25 to 45 s until bubble formation in the leaf space and the penetrating fluid in the leaf tissue were observed. After keeping the lettuce under the pressure for 30-60s, the pressure was released quickly, allowing the penetrating fluid to penetrate into the space inside the tissue. This procedure was repeated 2 to 3 times until the penetrating fluid diffuses significantly in the lettuce tissue. The lettuce tissue was then gently removed from the penetrating fluid and rinsed three times with distilled water and then transferred to a container covered with a plastic film. The treated samples were kept in the dark for 4 days.

Example 3. Protein Extraction and Isolation

[0055] The lettuce sample after agrobacterium vacuum infiltration was stirred with a stirrer and homogenized at a high speed in the extraction buffer (100 mM KPi, pH 7.8; 5 mM EDTA; 10 mM -mercaptoethanol) at 1:1 ratio for 1 to 2 minutes. The homogenate was adjusted to pH 8.0, filtered through gauze, and the filtrate was centrifuged at 10,000 g at 4 C. for 15 min to remove cell debris. The supernatant was collected, mixed with ammonium sulfate (50%), and incubated on ice for 60 min with shaking, and then was again separated by a centrifuge (10,000 g) at 4 C. for 15 min. The resulting supernatant was subjected to a second round of ammonium citrate (70%) precipitation, sit on ice for 60 min with shaking, and again centrifuged at 10,000 g at 4 C. for 15 min. Then, the supernatant was discarded, and the precipitated protein of the treated sample was dissolved in 5 mL of a buffer (20 mM KPi, pH 7.8; 2 mM EDTA; 10 Mm -mercaptoethanol) and stored at 4 C. The purified protein was further purified by the purification of the His-tagged protein. Approximately 200 L of protein extract was mixed with 1 mL of equilibrated Ni-NTA agarose (Qiagen) buffer A (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, pH 8.0), and shaken on a shaker at 4 C. for 1 h. The mixture was then added to a pre-equilibrated 1 mL-polypropylene column with 1 mL Buffer B (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 5 mM imidazole, pH 8.0). Thereafter, the mixture was washed with 10 mL of Buffer A, followed by 5 mL of Wash Buffer B to flow out by gravity. Purified His-tagged protein was eluted with elution buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 1 M imidazole, pH 8.0). The protein concentration was determined by Bradford method. Bradford kit (Bio-rad) was used to quantify the purified recombinant protein.

[0056] Downstream processing of plant-derived recombinant proteins is often difficult and expensive because cellulose cell walls are difficult to lyse and secondary plant metabolites are produced. In the present inventor, a stirrer is used to perform homogenization, which greatly saves the cost and simplifies the process of homogenization. The recombinant MersCoV vaccine protein was separated by SDS-PAGE and a band with an estimated molecular weight of approximately 70 kDa was observed in the lane (FIG. 3, lanes 1, 2), and there is no apparent corresponding bands in the negative control lane. The protein content of the purified sample was determined to be 0.58 mg based on the Bradford assay and the optical density measurement control group. In addition, a band of approximately 150 kDa was also detected in the affinity reaction between DPP4 and CTB-RBD-Fc (FIG. 3, lanes 4, 5), consistent with the predicted size.

Example 4. SDS-PAGE Gel Electrophoresis and Western Blot

[0057] The purified protein extracted from the lettuce after agrobacterium vacuum infiltrated was collected. 5 L sample was heat-denatured (95 C.), mixed with loading buffer (Biorad, Hercules, Calif., USA), and then subjected to electrophoresis on 4 to 12% Bolt Bis-Tris Plus SDS-gel (ThermoFisher Scientific, Waltham, Mass., USA). After the completion of electrophoresis, the gel was stained with Coomassie Blue G250 (Biorad) and photographed again.

Example 5. Binding of DPP4 to Recombinant MERS CoVRBD-Fc In Vitro

[0058] DPP4 is a functional receptor for MERS-CoV and is important in regulating immune responses. The binding affinity of MERS-CoVRBD to DPP4 was analyzed by performing co-immunoprecipitation assay. Recombinant human DPP4 (Sigma-Aldrich, St. Louis, Mo.) was incubated with recombinant MERS-CoV RBD. Samples were separated using SDS-PAGE to detect the size of the resulting complex.

[0059] When recombinant human DPP4 was incubated with recombinant MERS-CoVRBD, a band of approximately 150 kDa was isolated and detected by SDS-PAGE, demonstrating a significant affinity between recombinant MERS-CoV RBD and recombinant human DPP4.

Example 6

[0060] Control group: a vaccine for Middle East respiratory syndrome generated in animals;

[0061] Experimental group 1: a vaccine for Middle East respiratory syndrome generated in the plant provided by the present invention;

[0062] Experimental group 2: a vaccine for Middle East respiratory syndrome generated in tobacco leaves.

TABLE-US-00001 TABLE 1 vaccines for Middle East respiratory syndrome Production cost Production Protein Difficulty in the downstream protein (yuan per gram Group Time (d) Concentration purification of protein) Control 14 0.21 mg/g It is difficult to remove animal cell 5000 group impurities. In particular, animal cells are often contaminated with human viruses and have low safety. Experimental .sup.4**.sup.# 0.56**.sup.# mg/g Relatively easy. The downstream About 1200**.sup.## group 1 homogenization is carried out with a yuan per gram of stirrer, saving time and money, and protein eliminating the need to remove impurities such as nicotine and nicotine. Experimental 7* 0.32* mg/g Relatively difficult. The About 1500* group 1 time-consuming, laborious and yuan per gram of expensive liquid nitrogen grinding protein was required, and special steps were required to remove nicotine, nicotine impurities. *indicates P 0.05 compared with the control group; **indicates P 0.01 compared with the control group; .sup.#indicates P 0.05 compared with the experimental group 2; .sup.##indicates P 0.01 compared with the experimental group 2.

[0063] As can be seen from Table 1, compared with the animal system of the control group, the lettuce system provided by the present invention expresses the vaccine protein for Middle East respiratory syndrome, which reduces the production cycle very significantly (P0.01), improves the protein content very significantly (P0.01), simplifies the purification of the protein and reduces production costs very significantly (P0.01).

[0064] Compared with the tobacco leaf system of experimental group 2, the lettuce system expresses the vaccine protein for Middle East respiratory syndrome, which reduces the production cycle significantly (P0.05), improves the protein content significantly (P0.05), simplifies the purification of the protein and reduces production costs significantly (P0.01).

[0065] Compared with the animal system of the control group, the tobacco leaf system of experimental group 2 transiently expresses the vaccine protein for Middle East respiratory syndrome, which reduces the production cycle significantly (P0.05), improves the protein content significantly (P0.05), simplifies the purification of the protein and reduces production costs significantly (P0.05).

[0066] The above test results show that plant system, especially lettuce system, is a more economical and efficient expression platform for rapid and transient expression of recombinant proteins, which can produce large-scale vaccines of Middle East respiratory syndrome in a short period of time.

[0067] The above application of a plant as a host for expressing a vaccine for Middle East respiratory syndrome according to the present invention is described in detail. The principles and embodiments of the present invention are set forth herein in terms of specific examples, and the description of the above embodiments is only to aid in understanding the method of the present invention and its core concepts. It should be noted that those skilled in the art can make various modifications and changes to the present invention without departing from the spirit and scope of the invention.