Expression Of Porcine Rotavirus VP7 Gene In Schizosaccharomyces Pombe And Identification

Porcine Rotavirus(PRV) is a member of Rotavirus of Reoviridae family, a kind of no capsule membrane, double helix RNA virus. This virus is a cause and major pathogens of infant, child and many kinds of young animals died of acute diarrhoea. In pig production, A group PRV virus is one of the main cause and virulence pathogens of diarrhoea. After young pigs infected by PRV, the organisms immunity sharp drop cause by diarrhoea, and easily affect by many kinds of bacterial infections, leading to young pig living rate declined, and survivors grow slowly or stagnation, bring tremendous economic losses to the production of pig fanning industry. PRV containing 11 sections of the genome, its coding nine protein, structural protein VP1, VP2, VP3, VP4, VP6, VP7 and non-structural protein NS34, NS35, NS28. VP7 protein is a major component of the virus envelope, not only a basis to the virus serotype division, but also a main antigen which lure organisms produce idiosyncrasy antibodies. Therefore cloning VP7 genes and the certification of its transformation, expression, identification in Schizosaccharomyces pombe have great significance for further study of VP7 protein expression in plants and the development of nuclear PRV genetic engineering vaccines.This experiment used RG-2 plasmids as templates, according VP7 complete sequence designed the primer, progressing PCR acquired 1000bp length VP7 gene products. Connected it to the pMD18-T vector, through filtration white fleck training in added IPTG, X-gal and Amp LB media, acquired the pMD18-T-VP7 recombinant plasmids, by PCR and double digest identified that the VP7 gene have been cloning in pMD18-T-VP7 successfully.Digest VP7 PCR product with Nhe I and Bgl II, then using Alkaline Phosphatase (Calf intestine) enzyme digest product of digest with double enzyme to phosphoric acid, to prevent VP7 segment from coherence, after purification the product and under the effect of T4 DNA enzymes, directional linking it with the pESP-2 plasmids products of digest with Nhe I and Bgl II, constructions a recombinant plasmids yeast vector pESP-2-VP7. Using heat, electric shocks and LiAC conversion method forced recombinant plasmids pESP-2-VP7 into the S.pombe yeast SP-Q01, basis on weather it can growth in the EMM+VB1 media which without leucine to identify the pESP-2-VP7 positive plasmids, using the PCR, double digest and SDS-PAGE electrophoresis methods to identification the recombinant plasmids in DH5a and SP-Q01. PCR testing proved recombinant plasmids containing purpose size PCR fragments and processing the sequence measurement, comparison with the VP7 complete sequence, degree of similarity is 98.8%; the recombinant plasmids after double enzyme acquired a small segment as the size of VP7 gene and a large segment as the size of pESP-2 plasmids; SDS-PAGE electrophoresis also showed a purpose size of fusion proteins as 61kda. Confirmed that the recombinant plasmids containing VP7 sequences, and the successful Conversion and expression of the recombinant plasmids in S.pombe yeast.