Cloning Of Surface Antigen Genes Of PRRS Virus And Construction Of Its Expressing Vectors

Porcine reproductive and respiratory syndrome is a newly recongnized severe infectious disease. It break out first in 1980s. It is very dangours to the world piggery. PRRSV was first isolated in our country in 1996.It is a enveloped single strand RNA virus and the genomic RNA is 15Kb, which consist of 8 open reading frames (ORFs). The traditional vaccine doesn' t work very well to the disease. Therefore, development of safe and effective vaccines with genetic engineering technology would be a promising approach to protection from the disease. The molecular method was used in this research to construct prokaryotic expression vectorand eucaryotic expression vector. It will provide molecular basis for vaccine research.Based on the nucleotide sequence of E gene published, two primers which have restrict enzyme sites of EcoRI and BamHI were designed and synthesized. Then the E gene fragment of PRRS strain BJ-4 has been amplified by reverse transcription and polymerase chain reaction (RT-PCR) from the culture of PRRS BJ-4. The PCR products were checked by agarose gel electrophoresis and purified. The purified products were cloned into PBV221, an prokaryote expressing vector. The recombinant PRRS virus E protein expression vector (pBV221-E) was successfully constructed. Then the vector pBV221-E was transformed into E.coli strain JM109. The recombinant expressing plasmids were identified by PCR and restriction endo-nuclease analysis. The result indicated that the fragment was correctly inserted into the pBV221-expressing vector. A fusion proteinAbstractwas induced and expressed in 42. ELISA showed that it could react with the antibody to PRRSV. The result indicated that E protein of PRRSV was expressed in prokaryote system.Refer to the published sequences of E gene of PRRSV from Genebank , a different pairs of primers were designed and synthesized. E protein of PRRS virus was modified by PCR technique, the restriction enzyme EcoR I and Not I sites were added to the upstream and downstream separately. After purified and digested by restricted enzymes. E gene of PRRSV was successfully cloned into pPIC9K vector, a eucaryotic expression vector in methylotrophic yeast expression system. The recombinant plasmid was transformed into JM109. The recombinant expressing plasmids were identified by PCR and restriction endo-nuclease analysis. The result indicated the fragment was correctly inserted into the pPIC9K-expressing vector.According to the complete sequence of BJ-4,a pair of primers that have restrict enzyme sites(SacI and BamHI) were designed. N gene fragment was amplified from the cDNA of PRRS by PCR and inserted into the pQE-30. A pQE-30-N recombinant plasimd was constructed and transformed into the JM109.The result indicated that the recombinant vector successfully constructed.The prokaryotic expression vector and eucaryotic expression vector were successfully constructed, which will be used for future gene vaccination.