Construction Of Prokaryotic And Eukaryotic Expression Vectors Of PRV NSP4 Gene And Study On Their Expression

Rotavirus(RV) are the major cause of diarrhea in young pigs.The young pigs infected RV are induced diarrhea quickly and always died because of dehydration,thus it is a severe hazard to graziery.Because the serotype of RV is numerouS and the crossing of its immunity is weak,to investigate new generation vaccine have become hot.Recent researchs show that NSP4 is associated with RV immune response and may aid in development of an efficacious rotavirus vaccine.Set about initial research of property NSP4 in this study.The main contents as follow:1.Cloning and identification of PRV NSP4 gene.Designed a pair of primer contained restricted enzyme site XhoI and EcoRI according to the sequence ofprocine rotavirus strain OSU has published in genbank,and successfully amplified ORF ofNSP4 gene,which was 547bp,by RT-PCR from viral genome.Then it was inserted in PMD-19Tsemple vecter to be checked by PCR and restricted enzyme.The sequence analysis showed that rotavirus strain OSU was mutated largely via cell culture.The homology of nucleotide was 81% compared with the reported rotavirus strain OSU,and the homology of amino acid was 83 %.The main regions of variability were localized in C-terminuS.This strain was more clined to human rotavirus,because the homology of nucleotide was 93%and the homology of amino acid was 95%compared with human rotavirus strainG 10.2.Construction of prokaryotic expression vectors of NSP4c gene and study on their expression.Designed a pair of primer again and amplificated NsP4 gene 300bp about which abscised the hydrophobic region at the beginning of the protein and inserted it into pMD-19Tsemple vector.NSP4c gene fragment was moved into pet32a(+) vector by digested with XhoI and EcoRI and the recombinant plasmid pet32a-NSP4c was obtained which can expressed C-terminal of NSP4(86-174aa).NSP4c was:expressed and purified, and immunized mice to obtain positive control,then constructed indirect ELISA for detecting NSP4's antibody.3.Construction of eukaryotic expression vectors of NSP4 gene and study on their expression.NSP4 gene fragment was moved into pEGFP-N1 vecter by digested with XhoI and EcoRI and the recombinant plasmid pEGFP-N1-NSP4 was obtained.According to the method of LipofectamineTM2000 recombinant eukaryotic expression plasmid pEGFP-N1-NSP4 was transfected into COS7 cells.The expression results exhibited directly by a fluorescen inverted microscope because of the ptasmids' enhance green fluorescent protein(EGFP).Divided the mouse into 3 group randomly.Group 1 and 2 were blank and empty vecter,injecting Sodium Chloride(100μl per mice) and empty vecter pEGFP-N1(100μg per mice) respectively.Group 3 was experimental group injected recombinant plasmid pEGFP-N1-NSP4(100μg per mice).Every two weeks immunifaction one time.All progressed three times immunifaction.Get the blood serum of mice at 0d, 14d,21d,28d,35d and 42d for the immunifaction then detected the level of antibody by ELISA.At 2 Id the positive antibodys of NSP4 were detected.At 28d and 35d the antibody level of it was increased and at 42d was inclined to descend.The experimental results showed that recombinant eukaryotic expression plasmid pEGFP-N1-NSP4 was expressed in cells at the time of animal immunifaction and the expressed products induced humoral immune reaction and generated antibody.This trial laid materials and technique foundation for the structural and functional research of NSP4 and the research of RV DNA vaccine.