| Summary: | 碩士 === 國立中興大學 === 獸醫學系 === 90 === Classical swine fever (CSF) is an important infectious disease of swine, its causative agent is Classical swine fever virus (CSFV). The virion is surrounded by an envelope containing three viral glycoproteins, Erns (E0) , E1 as well as E2 both Erns and E2 have been shown to be capable of inducing neutralizing antibodies in host. The purpose of this study is to produce the CSFV glycoprotein E2 amount in the yeast Pichia pastoris eukaryotic expression system and furtherly to developing an efficient CSFV subunit vaccine. Glycoprotein E2 gene was cloned by polymerase chain reaction (PCR) cloning strateg and then subcloned into the P. pastoris expression vector pGAPZαC to generate the recombinant plasmid pE2 6-2G. After transformation of P. pastoris, the recombinant strains resisting to high concentration of the antibiotic Zeocin were selected. The chromosome DNA of each transformant was extracted and further identified to contain the CSFV E2 gene by PCR analysis. The expressed E2 proteins in culture supernatant and cellular lysates of recombinant P. pastoris were analyzed by SDS-PAGE after 96h incubation, and the major 55 kDa secretary protein was specifically recognized by the monoclonal antibody against the E2 protein. Furthermore, immunostaining of the CSFV-infected cells using mouse serum immuned with the expressed E2 protein in indirect fluorescent antibody (IFA) assay revealed specifically cytoplasmic staining.
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