CSFV E2Recombinant Baculovirus Construction And Expression Of Recombinant E2Protein Of CSF In Silkworm Chrysalis

Hog cholera also known as classical swine fever, or European swine fever,is composedof classical swine fever virus infection in swine induced by a highly contagious febrile infectiousdiseases, which is mainly caused by porcine parenchymal organ hemorrhage and sepsis, and thepig is the only natural hosts, which including domesticated pigs and wild boars, of any age, breed,sex pigs and wild boars onset can be. Its high infection and disease rate that has caused the pigindustry serious economic loss in the world.This disease is classified as A class disease by theOIE.Classical swine fever virus is a single positive strand RNA virus, and there is a largedevelopment reading frame (ORF), the open reading frame encoding an approximately3898amino acids of the polyglutamine proteins. The multimeric protein in virus infection within thehost cell is cleaved into12kinds of virus-specific proteins,4of which the viral structural protein,8kinds of non structural protein, E0/Erns, E1, E2is immune properties of3glycoproteins.Wensvoot (1990) study confirmed, E2protein induces immune responses to protect against CSFVinfection by immunization of pigs.Silkworm breeding in China is a long history of traditional industries, and the development ofnew biological pharmaceutical industry in China is one of the goals of the new stage, which usingsilkworm Bombyx mori as reactor expression of CSFV E2protein to the advantage industry of ourcountry and the continuous development of the molecular biology techniques together, not onlyfor the development of broad-spectrum swine fever subunit vaccine. The foundation, but also canbe explored using silkworm as the bioreactor to express protein cost savings, which reduce thedifficulty of pharmaceutical technology.This study aims to construct the recombinant E2gene of classical swine fever virus bybaculovirus in insect cells and expression of recombinant E2protein of classical swine fever insilkworm chrysalis. According to the GenBank registration of classical swine fever lapinized attenuated strain (HM175885) sequence design primer, amplified by RT-PCR CSFV glycoproteinE2gene, PCR products in sequencing primers, which, on the EHU/EHD amplification productnamed PCR-E2, and C6EU/C6ED primers for amplification product named PCR-C6E2, PCRproducts by enzyme digestion and connected to a donor plasmid pFastBacHBM-TOPO andpFastBacI vector, respectively named pFastHBM-E2and pFastBac-C6E2. The identification ofthe correct recombination plasmid pFastHBM-E2and pFastBac-C6E2were transformed intoDH10Bac competent cells, after reorganization and training, screening recombinant white colony.The recombinant baculovirus recombinant plasmid named Bacmid-E2and Bacmid-C6E2wastransfected into Sf9cells of insects, and the recombinant virus respectively named VBac-E2andVBac-C6E2.The use of ELISA, SDS-PAGE and Westem blotting method for the identification of twokinds of recombinant virus in insect cells are capable of secreting expression. Then, therecombinant VBac-E2and VBac-C6E2were injected with silkworm chrysalis, in a certain periodof time to harvest silkworm hemolymph, by ELISA test, the results show that two kinds ofrecombinant virus can also be in chrysalis in vivo expression of recombinant E2protein ofclassical swine fever virus. According to the commercial kits and classical swine fever virus E2protein standard established a recombinant E2protein quantitative method for detection ofclassical swine fever, for the follow-up development of diagnostic reagent kit and subunit vaccinesduring antigen quantitative basis.