CN114478714A - Method for analyzing expression and immunogenicity of recombinant human rotavirus VP7 protein - Google Patents
Method for analyzing expression and immunogenicity of recombinant human rotavirus VP7 protein Download PDFInfo
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Abstract
The invention belongs to the technical field of biological engineering, and particularly relates to an analysis method for expression and immunogenicity of a recombinant human rotavirus VP7 protein. Firstly, designing a primer according to the published VP7 complete sequence (NC _ 007571.1) in GeneBank, extracting the pathogenic nucleic acid, carrying out reverse transcription on cDNA as a template, cloning VP7 gene, constructing an expression vector pESP-2-VP7, and transforming Schizosaccharomyces pombe. VP7 is induced by 1% IPTG, and is separated and purified to obtain VP7 protein, and VP7 protein is used for immunizing mice in different immunization modes to evaluate the immune protection effect. The results show that: the design of the primer is consistent with the prediction result, the length of the cloned VP7 gene is 1050bp, and the SDS-PAGE and Western Blot identification result shows that the protein size is 63 kDa; after a mouse is immunized by the VP7 protein, the IgG, SIgA and neutralizing antibody results show that the immunization effect of 50 mu g/nose drop is better than that of 20 mu g/nose drop and injection groups (50 and 20 mu g/nose drop); and the nasal drop immunity has higher toxic attack protection rate of 100 percent.
Description
Technical Field
The invention belongs to the technical field of biological engineering, and particularly relates to an analysis method for expression and immunogenicity of a recombinant human rotavirus VP7 protein. The expression of rotavirus VP7 protein in Schizosaccharomyces pombe is explored, and the immunoprotection effect of the rotavirus VP7 protein is identified and evaluated.
Background
In the prior art, Rotavirus (Rotavirus, RV) Rotavirus belongs to the genus Rotavirus of reoviridae, and is an enterovirus. Is the main cause of acute gastroenteritis of infants, about 87 million infants die from diarrhea caused by rotavirus every year, is one of the main pathogens causing diarrhea and causing death, and is susceptible to children under the age of 5. According to statistics, about 2000 ten thousand children under 5 years of age see a doctor and 200 ten thousand children are hospitalized all year round. In the area where the RV vaccine is not widely inoculated, 40% of children are hospitalized due to acute gastroenteritis caused by rotavirus, while the RV vaccine is widely inoculated, the rate of children hospitalized due to diarrhea caused by rotavirus is reduced by 38%, and the vaccine development is expected to reduce the burden of virus pathogens all over the world, so that the RV vaccine has important public health significance for deep research on the RV vaccine. However, the rotavirus vaccine currently produced is more demanding on production and storage conditions and therefore more costly, which limits the vaccination of rotavirus vaccines for children in relatively poor regions. How to produce a rotavirus vaccine which is low in cost and easy to store becomes a problem which needs to be solved urgently at present.
Recently, it has been reported that a novel DS-1-like G1P human rotavirus appears and spreads rapidly in Japan. In thailand, such intergenic reassortant strains were found, which means that unusual rotavirus strains are continuously spreading in asia and multiple recombinations occur. Meanwhile, rotavirus antagonizes innate immune response through multiple ways, phylogenetic analysis reveals that a plurality of substitutions are identified in the epitope of the Lebanon specimen, and VP4 and VP7 genes have high genetic variability, so that the research and development of a safe and stable rotavirus vaccine with low cost are necessary to avoid the phenomenon that part of vaccines are broken down due to various existing problems. At present, multiple enterprises in China are developing the research and development work of multivalent rotavirus vaccines, the use of the rotavirus vaccines is being expanded in sub-saharan africa abroad, and the development of the vaccines is expected to reduce the burden of virus pathogens all over the world.
The glycoprotein on the outer layer surface of the rotavirus is VP7 structural protein coded by a structural gene VP7, is also structural neutralizing antigen of the rotavirus, can induce and generate IgG antibody and IgA antibody, is related to protective immunity 4 and 5, and therefore, the glycoprotein is taken as a target protein and has important significance for developing healthy and safe RV vaccines. At present, more and more researches on genetic engineering vaccine production by using a yeast expression system are carried out, and compared with other expression systems, the yeast expression system has the advantages of low cost, easiness in genetic operation, stable genetic character, high protein expression amount, post-translational modification and the like. Schizosaccharomyces pombe, the sixth eukaryotic model organism discovered by the present people, is widely used as a model system for molecular genetics and cell biology research to research the cell division mechanism 6.
Since there is no specific drug for treating Rotavirus diarrhea at present, the development of RV (Rotavirus) vaccine is a major task for preventing RV diarrhea. Rotaviruses are double-stranded RNA viruses, having two forms: infectious virions have a double capsid; non-infectious virions have no outer capsid. The RV genome consists of 11 discrete dsRNA, of which 6 gene segments encode the viral structural protein VP1-VP7 and 5 gene segments encode the non-structural protein NSP1-/NSP 6. The VP7 and VP4 proteins determine the G-and R-type of RV and its virulence. The VP7 protein is a conjugated Ca2+The glycoprotein can stimulate the human body to generate humoral immunity and induce the body to generate specific neutralizing antibodies.
The application utilizes the fission yeast schizosaccharomyces pombe expression system to express the rotavirus VP7 protein, and carries out an immune experiment to evaluate the immune protection effect, so as to lay a foundation for further developing an economic and safe RV transgenic vaccine.
Disclosure of Invention
The invention aims to provide an analysis method for expression and immunogenicity of recombinant human rotavirus VP7 protein, and the obtained monoclonal antibody of rotavirus VP7 gene has the characteristics of simplicity and good specificity compared with the existing human rotavirus vaccine detection method and equipment.
The technical solution of the invention is as follows: an analysis method of expression and immunogenicity of group human rotavirus VP7 protein is characterized in that a monoclonal antibody of human rotavirus VP7 yeast protein is prepared, a target fragment is amplified by RT-PCR to construct recombinant yeast engineering bacteria of a fusion expression vector pESP-2-VP7, and expression protein is induced by IPTG; the method comprises the following steps: firstly, searching a rotavirus VP7 nucleotide sequence obtained from Genebank, designing a specific primer through PrimerPremier5.0, connecting a VP7 gene gram to an 18T vector to obtain a cloned VP7 plasmid, amplifying, determining to successfully obtain a target gene VP7 through PCR analysis, introducing the target gene into a schizosaccharomyces pombe pESP-2 vector to perform prokaryotic expression to obtain a pESP-2-VP7 plasmid, and successfully obtaining schizosaccharomyces pombe positive bacteria for expressing VP7 protein through a series of genetic engineering methods; purified VP7 protein was obtained by SDS-PAGE and Western Blot analysis. The BCA detection results in the concentration of the VP7 protein being 0.58 mg/mL.
The mouse immunization experiment result shows that the VP7 protein obtained by Schizosaccharomyces pombe can induce the mouse to generate high-level IgG, SIgA and neutralizing antibody, and the VP7 protein retains a better immunization prototype. In addition, the mouse challenge test result shows that the diarrhea rate and the death rate of the mouse immunized by the VP7 protein are obviously reduced, the immune protection is very high for the mouse, the protection rate of a nose drop group reaches 100 percent and is obviously higher than that of an injection immune group, the immune mode possibly transmitted with a feces mouth is mainly mucosal immune, the injection immune group mainly stimulates humoral and cellular immunity, and the nose drop immune just can well stimulate the mucosa to generate better immunity. In addition, the specific immunoprotection time and immune mechanisms require further investigation. The application provides a very useful basis for developing novel vaccines for preventing rotavirus infection.
The invention has the advantages that: 1. the method for constructing the recombinant saccharomyces pombe by utilizing the molecular biology means constructs novel engineering bacteria, the schizosaccharomyces pombe is an important host bacteria for synthesizing natural products, the genetic stability is good, the gene expression is controllable, the equipment is simple, a large amount of medicinal protein can be produced, and the green and efficient RV genetic engineering vaccine is formed.
2. Proves the phenomenon and effect that the VP7 protein immunization can induce the body to enhance the immune response.
3. Compared with other expression systems, the yeast expression system has the advantages of low cost, easy genetic operation, stable genetic character, high protein expression amount, post-translational modification and the like.
Drawings
FIG. 1 amplifies the VP7 fragment.
FIG. 2 shows the double restriction enzyme identification of pESP-2-VP 7.
FIG. 3 SDS-PAGE analysis of recombinant VP7 protein.
FIG. 4 Western Blotting analysis of recombinant VP7 protein.
Detailed Description
The expression and immunogenicity analysis method of recombinant human rotavirus VP7 protein includes the following steps:
1 Material Process
1.1 sources of bacterial and viral species
Escherichia coli pMD18-T and Schizosaccharomyces pombe expression vector pESP-2 were purchased from Takala organisms, Schizosaccharomyces pombe strain SPQ-01 (Schizosaccharomyces pombe) and stored in the university of Jilin agriculture bioreactor laboratory. Rotavirus pathogenic material is preserved at-70 deg.C by hospital infectious department in the center of the general market.
Primary reagent
The reverse transcription kit, TaqDNApolymerase and DNAmarker are purchased from Takala biological company; the PCR product recovery and purification kit is purchased from Kangrun organisms; the mouse anti-rotavirus VP7 monoclonal antibody is purchased from Zhuhai Bomei Biotech limited; horseradish peroxidase-labeled goat anti-human IgG was purchased from Invitrogen; isopropyl-beta-D-thiogalactoside (IPTG) detection reagent, IgG, SIgA, neutralizing antibody and IFN-gamma detection kit are purchased from Nanjing Biotechnology Ltd.
Culture medium
The culture medium used in the present application includes: LB solid medium, LB liquid medium, YPD solid medium, YPD liquid medium, BMGY liquid medium, YES liquid medium.
Gene cloning and vector construction
According to the published rotavirus VP7 gene sequence information (NC-007571.1) of Genebank, primer premier5.0 is used for primer design, a pair of specific primers is designed, protective bases and enzyme cutting sites (enzyme cutting sites are underlined, NdeI/XhoI) are added to the primers, and the length of a target fragment is 1050 bp.
VP7-FP:5’-GGGAATTCCATATGATGGTTTGTACAACATTGTACACTG-3’
VP7-RP:5’-CGGCCGCTCGAGTTACGCATACCTCAACATCCAC-3’
The method comprises the following steps of (1) carrying out nucleic acid extraction on a pathological material (feces): adding 50 μ L feces and 500 μ L PBS into 1.5mL EP tube, shaking and mixing, mixing well, centrifuging for 10min at 5000r/min, sucking 100 μ L feces mixed solution, and extracting nucleic acid with ABIPRISMTM 6700; reverse transcription was performed according to the Takala reverse transcription kit instructions and cDNA was assayed by NanoDrop2000(Thermo scientific, USA) followed by VP7 gene cloning, the PCR procedure was as follows: CR reaction (25. mu.L): PCR Premix 22. mu.L, upstream and downstream primers 1. mu.L, and template 1. mu.L were added to each tube in sequence. And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 1min, to complete 25 cycles; extending for 8min at 72 ℃; storing at 4 ℃. The PCR product was detected by electrophoresis on a 1% agarose gel.
Construction of recombinant VP7 Schizosaccharomyces pombe vector
And adding NdeI and XhoI double enzyme digestion into the amplified target fragment and a schizosaccharomyces pombe expression vector pESP-2, incubating overnight at 16 ℃, transforming the obtained product into an E.coli JM109 strain, selecting a single colony on an LB solid culture medium containing Kan, extracting a plasmid, and identifying the connection condition of the target fragment through PCR and enzyme digestion to obtain a recombinant plasmid named pESP-2-VP 7. Transfecting the schizosaccharomyces pombe into schizosaccharomyces pombe by an electrotransformation mode, selecting single colonies on an Edinburgh basal Medium + vitamin B1(Edinburgh Minimal Medium + VB1, EMM + VB1) screening Medium plate, inoculating the single colonies on a liquid Medium containing EMM + VB1, culturing at 30 ℃ for 12h, extracting schizosaccharomyces pombe DNA, and carrying out enzyme digestion and PCR identification. The PCR reaction system was the same as 1.4.
Rotavirus VP7 protein expression purification
Selecting a schizosaccharomyces pombe single colony to culture in 10 mL of YES liquid culture medium (30 ℃, 250 r/min), measuring the OD600 value of the schizosaccharomyces pombe during the culture, adding IPTG (isopropyl-beta-thiogalactoside) for induction when the OD600 is about 0.53, continuing to culture for 36h, taking a sample to place in a centrifuge tube, carrying out resuspension precipitation at 4 ℃, 5000r/min and 10min, collecting precipitated thalli, adding 50mL of TE buffer solution for resuspension precipitation, centrifuging again, removing a supernatant, collecting fusion protein by using a GST kit, and measuring. After the determination, the protein is purified by a nickel column to obtain the recombinant VP7 protein, and the concentration of the target protein is 0.58 mg/mL by BCA protein concentration check. 50 μ L of recombinant protein was taken and analyzed by SDS-PAGE and Western Blot, and the remaining proteins were stored at-20 ℃ for further use.
Recombinant VP7 animal immunization and protection rate test
144 SPF mice were selected and randomly divided into 6 groups of 24 mice, each group was immunized with nasal drip (IN) and intramuscular Injection (IM) at 0, 20. mu.g and 50. mu.g, respectively, and divided into 6 groups of injection control group (IMCK), injection 20. mu.g (IM 20), injection 50. mu.g (IM 50), nasal drip control group (INCK), nasal drip group (IN 20) and nasal drip group 0. mu.g (IN 50) for 0, 2 and 4 weeks, in2, 4 and 6 weeks, 3 mice are selected from each group for orbital blood collection, the mouse SIgA, serum antibody IgG and neutralizing antibody titer are detected, and detecting serum IFN-gamma, immediately performing challenge on the immunized mice after immunization, performing intraperitoneal injection of 0.1mL of rotavirus culture solution containing 102TCID50, and performing statistics on diarrhea occurrence and death within 7 days after challenge.
Data analysis
The experimental data were processed using Excel2010, analyzed for variance using SPSS20.0 statistical software, and multiple comparisons were performed using the Duncan method, and the experimental results were expressed as "mean ± standard deviation". P <0.05 indicates significant difference, and P <0.01 indicates very significant difference.
Results
2.1 cloning of the rotavirus VP7 Gene
DNA DL10000 Marker is used as molecular mass standard, and amplification product of RT-PCR is identified according to agarose gel electrophoresis, so that 1050bp amplification band is obtained, which is consistent with the expected size, as shown in figure 1 (M: DNA molecular standard 10000; 1: VP7 mesh fragment).
PCR, amplification and enzyme digestion identification of recombinant expression plasmid
The positive recombinant clone plasmid was selected, and the VP7 target fragment was recovered by double digestion with XbaI and NotI, ligated to expression vector pESP-2, transferred to SPQ-01 Schizosaccharomyces pombe, and extracted recombinant Schizosaccharomyces pombe plasmid PCR and double digestion, see FIG. 2 (M: DNA molecular Standard 10000; 1: pESP-2-VP7 (XbaI + NotI)).
Purification and characterization of proteins
Selecting pESP-2-VP 7/SPQ-01 control bacterial colonies to be used from the bacterial colonies, further utilizing IPTG to continuously induce for 48h, centrifuging for 10min at 5000r/min to obtain bacterial precipitates, adding 60ml TE buffer solution to resuspend the bacterial precipitates, centrifuging for 10min at 4 ℃ at 5000r/min, and then carrying out GST kit affinity purification on fusion protein and detecting the protein concentration. A30. mu.L sample of the purified protein was subjected to SDS-PAGE. As can be seen from FIG. 3 (M: protein molecule Marker; 1: recombinant fused VP7 protein before IPTG induction; 2: recombinant fused VP7 protein after IPTG induction; 3-6: recombinant VP7 protein after purification), the expression level of the recombinant Schizosaccharomyces pombe protein after IPTG induction is significantly increased compared with that after IPTG non-induction.
Expression of fusion proteins
And (3) inducing and purifying by using IPTG, expressing positive recombinant bacteria, and analyzing the VP7 protein by using a Western Blotting test. FIG. 4 shows (M: protein molecule Marker; 1-2: purified recombinant VP7 protein), a colored strip appears at 63 kD, which further proves that VP7 protein is secreted and expressed by positive recombinant Schizosaccharomyces pombe and has good immunogenicity.
Results of serum IgG, SIgA and neutralizing antibody titers in immunized mice
As shown IN Table 1, the mice induced by VP7 have specific serum IgG antibody detected by ELISA, and after two weeks of enhanced immunization, the average IgG antibody titers of the mice IN the IM group reach the levels of 500+ and 600+, respectively, and the average IgG antibody titers of the mice IN the IN group reach the levels of 500+ and 700 +; while the titer values of the control mice were all below 50. The IgG antibody titers of the mice IN the IM group were not significantly different from those of the mice IN the IN group. IN addition, after boosting, SIgA antibody titers IN the sera of IN group mice reached 35.94 and 57.94, respectively, whereas no significant SIgA was produced IN the sera of IM and CK group mice. IN the result of the neutralizing antibody IN the serum, the neutralizing antibody titer of the mice IN the IM group and the IN group is obviously higher than that of the control group after the mice IN the IM group and the IN group are immunized by VP7, and the neutralizing antibody titer of the mice IN the IN group is obviously higher than that of the mice IN the IM group after the mice are immunized by the same dose of VP 7.
2.6 VP7 immunization mice diarrhea statistics and immune protection rate results
As can be seen from Table 2, after immunization with VP7, the incidence of diarrhea caused by RV challenge IN mice is significantly reduced IN both IM and IN, the immunoprotection rates of mice with IM20 μ g and 50 μ gVP7 are 93.33% and 100%, respectively, and the immunoprotection rates of mice with IN20 μ g and 50 μ gVP7 are 100%.
The above description is only a detailed description of the present invention, and various illustrations are not intended to limit the essence of the present invention.
Claims (2)
1. An analysis method for expression and immunogenicity of recombinant human rotavirus VP7 protein is characterized in that a monoclonal antibody of human rotavirus VP7 yeast protein is prepared, a target fragment is amplified by RT-PCR to construct recombinant yeast engineering bacteria of a fusion expression vector pESP-2-VP7, and expression protein is induced by IPTG; firstly, searching a rotavirus VP7 nucleotide sequence obtained from Genebank, designing a specific primer through PrimerPremier5.0, connecting a VP7 gene gram to an 18T vector to obtain a cloned VP7 plasmid, amplifying, determining to successfully obtain a target gene VP7 through PCR analysis, introducing the target gene into a schizosaccharomyces pombe pESP-2 vector to perform prokaryotic expression to obtain a pESP-2-VP7 plasmid, and successfully obtaining schizosaccharomyces pombe positive bacteria for expressing VP7 protein through a series of genetic engineering methods; purified VP7 protein was obtained by SDS-PAGE and Western Blot analysis.
2. The method for analyzing the expression and immunogenicity of the recombinant human rotavirus VP7 protein according to claim 1, wherein the concentration of VP7 protein obtained by BCA detection is 0.58 mg/mL.
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