Expression Of VP1-VP4 Of Entervirus 71 In The Recombinant Adenoviral System And Immunology Evaluation

Enterovirus 71 (EV71) is one of the important etiologic agent for young children, which can cause various kind of diseases. The most common disease caused dy EV71 is HFMD(Hand, Foot, and Mouth Disease) bisease brain stem encephalitis and other nervous system infection to death. In rescent years, EV71 distrubutes throughout the world.Enterovirus is a single-stranded, positive-sense RNA virus whose genome (≈7500 nucleotides) consists of one open reading frame (ORF), and express as large polyprotein that can be divided into 3 regions:P1, P2 and P3. The four structural proteins, VP1,VP2, VP3 and VP4, are encoded within P1 region while the non-structural proteins, such as proteases 3CD.The proteins VP1,VP2 and VP3 are exposed on the surface of virus particles while VP4 locat inside the virus capsid connect with the genomic RNA closely. The VP1, VP2, VP3 and VP4 constitute the area of antigen of EV71 together.Research date showed that mainly antigens decision of the virus located on VP1, but VP2,VP3 and VP4 have combined capabilities of the antigens with antibodies. Existing experimental results showed that a separate VP1 or VP3 just had limited immunogenicity, if we can obtain VP1-VP4, the immunogenicity will be improved obviously.In view of this,to the EV71 virus isolated from Fuyang first we identify it by serological and molecular biology methods, and then cloned the P1 and 3CD genes using RT-PCR method, constructed bicistronic shuttle vector pShuttle-CMV-P1-3CD and single gene shuttle plasmid pShuttle-CMV-P1 and pShuttle-CMV-3CD by means of vector pcDNA3.0BA. By the way homologous recombination in E. coli, the recombinant adenovirus vector rAd-P1-3CD, rAd-P1, rAd-3CD are gained, and transfect them into 293 cells to obtain the corresponding recombinant adenovirus. According to RT-PCR analysis results, all of the recombinant exogenous genes of the three kinds of obtained bicistronic recombinant adenovirus rAd-P1-3CD, rAd-P1 and rAd-3CD have been transcripted mRNA. Immunofluorescence and immunehist-ochemistry results showed that just the bicistronic recombinant adenovirus rAd-P1-3CD could be detected in specific target protein, and the rAd-P1, rAd-3CD failed to detect the expression of specific product. The results indicate that all of three kinds of adenovirus can express EV71 P1 and 3CD genes effectively, the expression products of the recombinant adenovirus containing just P1or 3CD could not be recognized by specific antibodies, but the co-expression products of bicistronic recombinant adenovirus containing P1 gene and 3CD protease gene can be certified having EV71-specific antigen. Promote that P1 protein should be cleaved by 3CD protease and then to show antigenicity. Through the way of the intranasal, intragastric and subcutaneous injection to immune BALB/C mice, the results showed by ELISA assay is that all of the experimental groups serum produced anti-EV71IgG antibody. And the antibody level arose by rAd-P1-3CD is higher than immuned together with rAd-P1/rAd-3CD. Three different immunity means results showed that intragastric is the best inmunity way, intranasal second and subcutaneous injection produce the lowest antibody level. By the limited times of neutralization tests, it is failed detecting EV71 specific protection antibodies of the serum, and the concrete results are being further experimental demonstration.This study cloned containing all of EV71 antigenic role P1 gene and has cleaving effect protease 3CD gene successfully. Results showed the interaction between the proteins of EV71 virus, laid a good foundation for studying the relationship of EV71 structural proteins and non-structural proteins; It is the exploitative experiment for finding the best immunity way in the aspect of interrelated recombinant adenovirus vaccine, and set the stage for genetic engineering vaccine of EV71 research.