Expression Of The Main Antigenic Domains Of CSFV E2 Protein In E.coli And Primary Application

Classical swine fever (CSF), formerly called hog cholera(HC), is a highly contagious and often fatal disease of domestic pigs and wild boars. Outbreaks of CSF usually cause huge economic losses, and impair internal and international trade of pigs and pig products. CSF is classified in List A disease of the Office International des Epizooties (OIE) .It has been successfully eradicated from Canada (1963) and United States (1976), and under effective control with in European Union (EU) in recent years. However, the situation of CSF epidemic is still severe in Asia, Latin America, East Europe and former USSR area . Especially in Asia, the reported cases in 2004 increase nearly 20% than those in 2003. Recently, outbreaks re-emerged in South Africa, which had been free of CSF since 1918 .E2 protein, one important protective antigen of the CSFV, determine virus infectivity. Even if the nucleotide sequences of various genomes among the distinct strains of CSFV are different, the variation of E2 gene is biggest. In present study, on the basis of the gene of E2 protein of CSFV cloned and expressed. Then expressed the E2 protein as a antigen and use Dot-ELISA to diagnosis of antibody against CSFV.The design of primer for RT-PCR was based on the nucleotide sequence of E2 gene of the C- strain in Genbank. The predicted amplified product is 978bp. The recovered target fragment was linked with pGEM-T vector, was transformed Ecoli.DH5αcompetent cell, culture and extract plasmid. The recombinant plasmid was sequenced after the PCR identification and double enzyme-digested. The homology between target gene and other genes of CSFV in Genbank was above 98.9%, which showed CSFV E2 gene was obtained through amplification.E2 gene was subcloned into prokaryoutic high-expressing vector pGEX-6p-1. We used vector's general premier to sequence pGEX-6p-1-E2 recombinant plasmid. The result showed that the sequenced E2 gene was identical with the protosequence and the target gene which was inserted into expressing vector had uniformity, and had correct direction and site. BL21 competent cell was transformed by positive recombinant plasmid and was induced to express by IPTG. Expession product being analysized through SDS-PAGE, target protein were expressed, and a target approximative band of 61kDa. Then we studied the expressing condition of recombinant protein. There are more recombinant proteins expressing with more time and 5h after being induced, recombinant protein reach its maximum. Then used CSFV positive serum by Western Blotting and the E2 protein has immunogenicity. E2 protein expressed product is approximately 61kDa. The study offered necessary experiment material and basis for further study constitutive protein's constitution and function of CSFV .In the present study, the protein was used as envelope antigen to detect experimental sera by Dot-ELISA. The result is correct.