The Construction Of Eukaryotic Expression Vector Of The Vp6 Gene Of Porcine Group A Rotavirus And The Comparison Of Immunodetection

The total RNA of porcine rotavirus as template, VP6 gene was cloned by the reverse transcription-polymerase chain reaction (RT-PCR). The length of target gene was 1172bp. The products of RT-PCR were ligated with Vector pMD-18—T, and the ligation product was used to transform E.coli JM109. By the analyses of restriction endonuclease, sequencing and PCR, it showed that the inserted gene had been a positive reading frame.Cloning plasmid pMD-18T-VP6 and pVAXI were digested by Bam HI and KpnI double restriction enzymes,the Vp6 gene was purified and subcloned into the expression vector pVAXI, and the recombinant eukaryotic expression vector pVAXI-Vp6 was construted. The recombinant expression plasmid was transfected into MA-104 cells by liposome-mediated gene transfer method and observed through Fluorescence microscopy, RT-PCR was used to demonstrate successful transcription.Result, A recombinant eukaryotic expression plasmid pVAX1-VP6 was successfully constructed Green fluorescent protein could be detected in the transfected MA-104 cells 72 hours post transfected and the expression of pVAX1 mRNA was confirmed using RT-PCR.The BABL/c mice used for nucleic acid immunization were divided into A, B, C three groups, 25 individuals/group. Group A was injected with 100 μg/100μL eukaryotic expression plasmid pVAX1;Group B was injected with 100μg/100μL of the recombinant plasmid pVAX1-VP4, pVAX1-VP7; Group C was injected with 100μg/100 μL of the pVAX1-VP6, pVAX1-VP4, pVAX1-VP7respectively, and the mice in each group were in injected three times totally. After primary injection, all mice received booster injection two times with the same dosage and vin the same route of primary injection at interval of two weeks. Blood samples collected from mice at Od before primary injection and those collected from mice of each group at 14, 28, 42d, were used for detecting antibody levels by ELISA and the differences in numher of CD3~+, CD4~+, CD8~+, CD4~+/CD8~+, CD19~+, CD21~+T cells in peripheral blood lymphocytes, and also with the lymphocyte transformation experiment (LT).