| Human parvovirus B19(HPV B19) was one of the smallest single chain DNA virus which confirmed to cause diseases in human. Since 1990 we confirmed that there were B19 virus infection for the first time in our country, more and more researches indicated that B19 virus was contributed to some disesases, such as acute aplastic crisis, acute thrombocytolytic purpura and nonimmune abortion. Especially in immunocompromided patients, the persistent HPV B19 infection could result in chronic anemia due to deficiency of effective therapy, even endanger life. Therefore, it was necessary to establish effective preventing and curing system of B19 virus in our country and even the world.B19 virus genome existed variation and was now divided into three genotypes: type 1 (prototype), type 2 (A6- and LaLi-like), and type 3 (V9-like). There were different distribution of genotype due to regional discrepancy, such as human parvovirus B19 type 1 circulated in European and American , whereas type 2 and 3 encountered infrequently in this area. The type 3 attained commonly in western African. In overall DNA sequence, the three virus types differed by 10%. The most striking DNA dissimilarity, of >20%, was observed within the p6 promoter region. Within the open reading frame(ORF) encoding the VP1/VP2 proteins, the majority of nucleotide substitutions were synonymous: at the nucleotide level, genotypes 2 and 3 differed from the prototype by 9 and 12%, respectively, but at the amino acid level they differed by only 1.1 and 1.4%. We analyzed DNA sequence of the prevalent strain(XA strain) of B19 virus from chinese using the gene cloning and sequencing technology by assistance of the national natural sciences fundation(39870021) and discovered the VP1 unique region of B19-XA strain had the obvious variation in our country. The primary structure differences of its genome could cause alteration of coding amino acids, which might be contributed to the change of viral antigen site and immunal characteristic.The variation of B19 virus gene might result in changes of viral toxicity and pathogenic mechanism. The VP1 protein was closely correlation with viral immunogenicity and generation of protective antibodies after infection. So it was necessary to investigate thoroughly variation of structure and functional position of proteins which were expressed by the region of gene mutation in order to establish the preventing and controlling technology for B19 virus infection in our country. Accordingly, this experiment intended to constructe expressive vectors and express VP1 fusion protein of B19-XA2 strain in procaryon and eukaryon systems, then study change of the protein spatial structure and its functional position, as well as influence to antigenicity so as to reveal biologic activity of the VP1 protein and promote the investigation of B19 virus molecular epidemiology, pathogenic mechanism and preventive vaccine.Objective: Aim of this experiment lies in amplifying the whole VP1 gene of B19-XA2, constructing recombinant procaryotic expressive vector and the shuttle vector (VP1-Bacmid), expressing the fusion protein in E.coli. and Sf9 cells, respectively. In order to investigate immunogenicity of the fusion VP1 protein, rabbits were immunized by the fusion VP1 proteins from procaryon and eukaryotic expressive systems and produced the multiclone antiserum of anti-VP1 protein possessing a certain sensitivity and specificity. In addition, to analyze reactionogenicity of the VP1 fusion protein, the clinical serological detecting were respectively carried out using two kinds of ELISA on the basis of different antigen: one antigen was the VP1 fusion protein from procaryon and eukaryotic expressive systems, the other was parvovirus B19 IgM ELISA Kit of Germany. Above all, this experiment established the substructure for the further investigation of VP1 protein biological function and protection of B19 virus infection.Methods:1. To obtain the interested gene The positive B19 specimen were screened by nested PCR with primers which were independently designed. Then the VP1 whole gene of HPV-B19 (nt2623-nt4968) was amplified from the above positive specimen by conventional PCR assay.2. To express the recombinant protein in procaryon system The VP1 gene was inserted into the multiclone site of the pET28(a) expressive vector, afer that the recombinants were transformed into competent E.coli BL21(DE3). The VP1 fusion protein was expressed by IPTG inducing, analyzed by SDS-PAGE, and identified by Western blot.3. To express the VP1 protein in eukaryon system The VP1 gene and the pFastbac1 donor vector were digestted by Salâ… a nd Xbaâ… restrictive enzyme, connected each other with T4 DNA ligase, and then the recombinant vectors were emerged. Meanwhile the recombinant vectors were transformed into competent E.coli DH10Bac. The positive recombinants--the baculovirus shuttle vectors (VP1-Bacmid) containing the interested gene, were screened by antibiotics and phenotype of Blue-White spot and verified via PCR. In the subseqence testing, the VP1-Bacmid were transfected into Sf9 cells by a cationic lipid(Cellfectin reagent) mediation, then the recombinant baculoviruses were performed and amplified in Sf9 cells. Sf9 transfected cells were freezed repeatly and disintegrated with lysate, and in the end the supernatant containing the VP1 protein was procured by ultracentrifugation.4. Domestic rabbits were respectively immunized by the fusion VP1 proteins from procaryon and eukaryotic expressive systems and produced the multiclone antibody of anti-VP1 protein which valence was detected by ELISA. In the other hand, to analyze reactionogenicity of the VP1 fusion protein, the clinical serological detection were respectively carried out using two kinds of ELISA on the basis of different antigen: one antigen was the VP1 fusion protein from the supernatant of recombinant bacteria disintegrated by ultrasonic wave and the supernatant of transfected Sf9 cells after disintegration by lysate, the other was parvovirus B19 IgM ELISA Kit of Germany.Results:1. The VP1 whole gene of B19 were successfully amplified from one of the positive B19 specimen screened with nested PCR, and be named as B19-XA2 strain.2. The VPl-PET28(a) vectors were digested by Salâ… a nd Xbaâ… restrictive enzyme, and then we observed that the length of fragment matched with anticipative results by agarose gel electrophoresis. Sequencing result confirmed that the sequence of recombinant was entirely accurate. 3. As the recombinant bacteria VP1-PET28(a)-BL21(DE3) were propagated by IPTG induction, the fusion protein were expressed and exactly proved to be the VP1 fusion protein using Western-blot, which molecular weight was approximately 87kDa and could conjugate with 6-His antibody.4. The recombinant baculovirus shuttle vectors (VP1-Bacmid) which were screened by antibiotics and phenotype of Blue-White spot, successfully constructed and verified via PCR. Meanwhile, the VP1-Bacmid were transfected into Sf9 cells by cellfectin reagent, then the recombinant baculovirus were amplified in Sf9 cells. Sf9 cells emerging typical pathological changes were assembled and disintegrated. The 87kDa interested protein which molecular weight was consistent with the anticipated was found by SDS-PAGE.5. As antigen, both of the VP1 fusion proteins from procaryon and eukaryotic expressive systems could make rabbits to produce the multiclone antibodies of anti-VP1 protein. Its valence was detected by ELISA, and the results showed that valence of the former was 1:12800, while the later 1:50000.6. The VP1 protein producing in recombinant baculovirus system could well react to the positive sera of patients after B19 virus infection, moreover its OD value was lower than that of the German ELISA Kit. However, the VP1 protein producing from recombinant E.coli. system didn't exist immunologic reaction with these positive sera.Conclusion:1. We successfully constructed the recombinant E.coli. VP1-PET28(a)- BL21(DE3).2. We successfully constructed the recombinant baculovirus shuttle vectors(VP1-Bacmid), and effectively expressed the VP1 protein in Sf9 insect cells. 3. The fusion VP1 proteins from either procaryon or eukaryotic expressive systems could stimulate animals to generate the specific antibody, so both of them possesed immunogenicity.4. The VP1 protein expressing in Bac-to-Bac system, which bioactivity was much similarly to native protein, could be used to detect specific antibody after B19 virus infection, but also its valence attained the parallel level compared with abroad kit. |
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