Construction Of Human Parvovirus B19-VP2 Gene In Eucaryotic Expression Of Plasmid

Objective: Human Parvovirus B19 is only kind of pathogen causing human disease, which may bring about such critical diseases as infectious erythema, aplastic anemia and so on, and yet no specific vaccine against the virus has been found out. .preventing and treating the infection of Human parvovirus B19 is urgent problem need to be solved. Recently recombination of B19 capsid protein as vaccine antigen is processing in fremdness. The objective of the project is using eukaryotic expression of plasmid pcDNA3.l as vector, selecting the gene fragment of abundance antigen epitope and recombinating it to vector, Constructing pcDNA3.l-VP2 eucaryotic expression of vector. Finally we injected the recombination plasmid directly into an experimental animal to observe its immune reaction in order to explore the feasibility of Human parvovirus B19-VP2 gene as genie vaccine. The subject lay a solid foundation for finally establishing specific utility vaccine of the virus.Met hods: The gene sequence of B19-VP2 can be obtained from gene sequence and its structural analysis of Human parvovirus B19. The B19-VP2 target gene primer was designed on the basis of analysis of the epitope of B19-VP2 with software ANTHEWIN and DNASIS. In order to facilitate cloning and ensure the exactness of insert direction. We designedly joined Hindin and BamHI inscribed enzyme point into primer ;Gene sequence of VP2 was ampliticated from plasmid of DNA sequence of B19 virus with PCR method and was linked to pGEM-T plasmid, which was transformed into JM109. The primer designed with T7/sp6 sequence of pGEM-T plasmid was applied to identify the linking correctness. Furthermore; AMP fastness was also used to select the positive clone and to identify the sequence of DNA for the purpose of the correctness of objective fragment cloned. After the recombinated plasmid pGEM-T-VP2 and pcDNA 3.1 underwent enzyme cutting with both HindTTI and BamHI, and then with electrophoretic purification, they were linked and transformed intobacteria. Once again the work was carried out on selecting and identifying positive clone and DNA sequence. Finally the specific antibody was identified with ELISA method after a series of working processes such as mass culturing, extracting and purifying pcDNA 3.1-VP2 and injecting it into a rabbit in tramuscularly and collecting its blood dynamically.Resu 11: l.Epitope analysis: nine epitopes were primarily identified through analysis onISHGQTTYGNAEDKEYQ (5) GRFPNEKEQ (6) MHTYFPNKGTQQYTD (7) FLKILPQSG (8) FKLGPRKATGRWN(9)LYDPTATDAKQHHRHGYEK2. Amplification of target gene : 800 base pairs were found out by way of whole gene plasmid of B19 virus as template and target gene (4168-4968) in gene VP2 amplificated by PCR.3.Result of recombination and identification of clone and subclone: the positive clone of 960bp was obtained through linking between target gene and pGEM-T plasmid, and then amplificating transformed plasmid by primer T7/sp6.that proved B19-VP2 target gene insert pGEM-T successfully, and we gained recombined pGEM-T-VP2 plasmid. The positive clone 966bp was obtained by means of Linking recombinated plasmid pGEM-T-VP2 and pcDNA3.l after undergoing enzyme cutting with both Hindm and Bam HI. And by means of amplification of plasmid transformed by primer t7/sp6. .that proved B19-VP2 target gene insert pcDNA3.l successfully and we gained recombined pcDNA3.l-VP2 plasmid.4. .Result of sequence analysis of clone and subclone: The identification of DNA in positive clone pGEM-T-VP2 and pcDNA3.l-VP2 demonstrated that the base pains of our cloned or subcloned VP2 gene fragments were identical with the VP2 sequence published in the world .We Constructed Eucaryotic Expression of vector pcDNA3. 1-VP2 successfully.5.Result of ELISA: Highly concentration IgG against B19 virus was generated in the rabbit.Conclusion: This subject make use of molecular biology software analyzing the abundance antigen epitope region of B19-VP2, nine epitopes were concentrated primarily on No.294-541 of vp2.. with molecular biological...