Construction Of Adenovirus Vector For Synthetic Pneumocystis P55 Tandem Antigen Gene And Its Immunogenicity Test

Objective: Pneumocystis pneumonia(PCP),an infectious disease caused by the opportunistic fungus Pneumocystis(Pc),occurs most commonly in the lungs,and can be severely fatal.association with human immunodeficiency viruses(HIV)infection,and in particular PCP was defined in 1981 as signature disease of acquired immune deficiency syndrome(AIDS).In recent years,epidemiological data have been shown that,with the current organ transplantation,malignant tumors,and long-term use of hormones such increase in the number of immunosuppressed patients without HIV infection,the rising number of clinical PCP cases once again aroused great attention of researchers at home and abroad.At present,clinical treatment of PCP is mainly based on compound sulfamethoxazole,although it can achieve a certain degree of efficacy,but due to its toxic side effects,resulting in some patients cannot tolerate,coupled with the emergence of drug resistance and other issues,in the face of such severe situation,many scholars at home and abroad have turned their attention to the development of new PCP prevention strategies and related vaccines.Previous studies have found that P55 protein,one of the components of Pc antigen,plays an important role in anti-PCP.After the body's active immunity to natural P55 protein,the immune response can make the body get a certain degree of protection in the early stages of infection.Therefore,some researchers believe that P55 protein is one of the most promising candidate vaccine against PCP.However,variants of the P55 protein are present and the Pneumocystis is difficult to be cultured in vitro in large quantities,resulting in limited sources of antigen.Therefore,the development of P55 protein vaccine has not achieved effective results.Based on the above reasons,our team constructed the p55 multiepitope tandem gene(p55TAG)including 5 variant effective epitopes of P55 through bioinformatics and molecular biology techniques,and constructed the prokaryotic expression vector and the eukaryotic expression vector proved that p55 TAG can express the target antigen protein effectively,At the same time,it has been verified that the gene has the potential of immunoprotection in immunosuppressed PCP rat model.However, because of Pneumocystis as an opportunistic infectious fungus,mainly infringes upon the immunocompromised patients,it is often difficult to achieve the expected immune effect by using a conventional single molecular vaccine for such people.Therefore,the research group proposed the use of "prime-boost strategy",which is to immunize with two heterogeneous molecular vaccines,so as to improve the anti PCP effect.Based on the above ideas,this study firstly plans to use the adenoviral vector carrying p55 TAG to construct a recombinant adenovirus vector vaccine;secondly,to verify its protein expression effect in eukaryotic cell HEK293;finally,mice are immunized with the packaged recombinant adenoviral vector vaccine and the relevant immune indexes in mice were detected to analyze the immunogenicity of the constructed vaccine.It is expecting to be able provide a "booster" role of the vaccine for "prime-boost strategy",and lay a foundation for the establishment of an effective vaccine against PCP.Method:Part I: Pneumocystis p55 TAG Recombinant Adenovirus Vector Vaccine Construction and Eukaryotic Cells Expression Verification1.Acquisition and amplification of p55 TAG The target gene of p55 TAG on the p MD18-T cloning vector was double digested with Bgl II and Xho I,and the product was sequenced for identification.The correct target gene was identified by PCR amplification,and the product gum was recovered.2.Construction of recombinant adenovirus vector with p55 TAG The adenovirus linkage plasmid p HBAd-EF1-MCS-GFP was digested with Eco RI and connected to p55 TAG target gene,and then transformed into DH5 Escherichia coli.A monoclonal colony by ampicillin resistance screened on the LB medium was amplified by PCR,and the product was sequenced and identified.3.Viral packaging of p55 TAG recombinant adenovirus vector The recombinant adenovirus vector and adenovirus plasmid p BHGlox(delta)E1,3Cre were co-transfected into HEK293 cells,and the expression of GFP and CPE were observed.The recombinant adenovirus after packaging was named p HBAd-p55 TAG.4.Identification of p55 TAG recombinant adenovirus and expression of the target protein in eukaryotic cells The p HBAd-p55 TAG nucleic acid was extracted,and sequenced after PCR amplification.SDS-PAGE and Western blot were used to identify the target protein expression of HEK293 cells infected with recombinant virus.Part II: The Immunogenicity Test of Pneumocystis p55 TAG Adenovirus Vector Vaccine5.Amplification,purification and titer determination of recombinant adenovirus HEK293 cells were repeatedly infected with p HBAd-p55 TAG three times.The last virus solution was collected and purified by Cs Cl gradient centrifugation,and the titer of purified virus was detected by TCID50.6.Recombinant adenovirus vector vaccine for immunization of animals Female(6-8 weeks old)BALB/c mice were divided into three groups according to the principle of randomization: p HBAd-GFP,p HBAd-p55 TAG and PBS,with 10 mice in each group.Single needle immunization was used for intraperitoneal injection,and the injection volume of each mice of each group was 507.Immunogenicity test of recombinant adenovirus vector vaccine Serum levels of IFN--2,IL-4 and IL-17 were detected by MSD at the end of 3d,1w,2w and 3w after immunization.At the end of the third week,serum Ig G,Ig G1 and Ig G2 a antibody subclasses of mice were detected by ELISA assay,and splenocytes from mice were collected and analyzed by flow cytometry for subsets of CD4+T and CD8+T lymphocytes and simultaneous detection changes of m RNA relative expression of four cytokines IFN--2,IL-4 and IL-17 in spleen cells by q PCR.8.Statistical Analysis SPSS19.0 software was used for statistical analysis.Measured data were expressed as mean±standard deviation(±SD).One-way ANOVA was used for comparison between groups and LSD-t test was used for comparison between groups.P<0.05 indicates a statistical difference.Results:Part I: Pneumocystis p55 TAG Recombinant Adenovirus Vector Vaccine Construction and Eukaryotic Cells Expression Verification1.Acquisition and amplification of p55TAG From the vector stored in our lab,it was digested by enzyme,amplified by PCR,and detected by agarose gel electrophoresis.The result showed a specific band around 1,200 bp(full-length p55 TAG gene fragment was 1164 bp).The sequencing result was consistent with the original p55 TAG target gene sequence.2.Construction of recombinant adenovirus vector with p55 TAG After transformation,eight monoclonal colonies were picked from Amp+LB plates and identified by PCR amplification and agarose gel electrophoresis.Six transformants showed specific bands at 1500 bp,and the positive PCR products were sequenced and results consistent with the original p55 TAG target gene sequence,proved that the successful construction of recombinant adenovirus vector.3.Viral packaging of p55 TAG recombinant adenovirus vector The recombinant adenovirus vector was co-transfected with the adenovirus backbone plasmid into HEK293 cells,and the expression of GFP was observed under a fluorescence microscope 48 h after transfection.The results showed that the transfection was successful.After 8 days,the cells began to show the typical CPE phenomenon,indicating that the recombinant adenovirus vector packaging success.4.Identification results of recombinant adenovirus p55 TAG After PCR amplification of p HBAd-p55 TAG,a specific band appeared at 1500 bp after electrophoresis of the product.The sequencing result was consistent with that of the original p55 TAG target gene sequence,demonstrating that the packaged adenovirus contained the p55 TAG target gene.5.Protein expression results of p55 TAG recombinant adenovirus in HEK293 cells Western blot showed a significant band at 60 k Da from the recombinant adenovirus compared with the empty virus vector,which was in line with the expected result.It was proved that the p55 TAG recombinant adenovirus successfully expressed the target protein in HEK293 cells.Part II: The Immunogenicity Test of Pneumocystis p55 TAG Adenovirus Vector Vaccine6.The titer of recombinant adenovirus was amplified and purified The p HBAd-p55 TAG were amplified three generations,and titer of the virus-purified was 1.99 x1010PFU/ml by the TCID50 method,which could be used in the next animal immune experiment.7.Flow cytometric analysis of CD4+T and CD8+T lymphocyte subsets in mice spleen The results of flow cytometry showed that the increase of CD4+T lymphocytes in p HBAdp55 TAG group was statistically significant compared with the other two groups(P<0.05;P<0.01).In the comparison of CD8+T lymphocytes in each group,the elevation of p HBAdp55 TAG group was statistically significant compared with PBS group(P<0.05).8.Analysis mice serum levels of IFN--2,IL-4,IL-17 cytokines The concentrations changes of IFN--2,IL-4 and IL-17 in serum of mice were detected by MSD method.By statistical analysis,the results showed that p HBAd-p55 TAG group of cytokines IFN--17 elevated levels compared with the other two groups showed statistically significant difference(P<0.01),but concentration levels changes of cytokines IL-2 and IL-4 were not statistically significant(P>0.05).9.Analysis of change level of total Ig G,Ig G1 and Ig G2 a antibody subclasses The levels of Ig G and Ig G2 a in serum of p HBAd-p55 TAG group at the end of 3 weeks were significantly higher than those in the other two groups(P<0.01)by ELISA,while the change of Ig G1 subclass in the three groups no difference(P>0.05).After calculating the ratio of Ig G2 a to Ig G1,the ratio of p HBAd-p55 TAG group was statistically different from the other two groups(P<0.05).10.Relative quantitative detection of four cytokines in mice spleen cells by real-time PCR The expression of both p HBAd-p55 TAG group and p HBAd-GFP group compared with PBS group of four kinds of cytokines were increased.Compared with PBS group,p HBAdp55 TAG group had higher levels of the three cytokines besides IL-4(P<0.001).In the statistical comparison of p HBAd-p55 TAG group and p HBAd-GFP group,only the increase level of IL-17 was statistically significant(P<0.001).Conclusion: 1.The p55 TAG recombinant adenovirus vector was successfully constructed in this study.2.The target gene of packaged p55 TAG recombinant adenovirus was successfully expressed in eukaryotic cells HEK293.3.Immunization of BALB/c mice with p55 TAG adenoviral vector vaccine can induce cellular and humoral immune responses and verify that it has certain immunogenicity,which lays the foundation for further study on the new strategy of anti-PCP vaccine.