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Construction And Expression Of The Recombinant Peptide Of Human Cytomegalovirus In The Pichia Pastoris

Posted on:2003-08-26Degree:MasterType:Thesis
Country:ChinaCandidate:Y S ZhengFull Text:PDF
GTID:2120360065457034Subject:Developmental Biology
Abstract/Summary:PDF Full Text Request
In order to express the recombinant peptide of both gp52 and pp150 Oterminal peptides from human cytomegalovirus (HCMV), which seem to show good antigenicity. Recombinant DNA technology was used to construct recombinant plasmid, which was transformed into the Pichia pastoris to express the interesting peptide. The biological function of the interesting peptide has been identified. According to the cDNA sequence of glycoprotein52 (gp52) and phosphoprotein150 (pp150) of human cytomegalovirus, two pairs of primers were designed. The supernate of virus culture was used as templet in overlapping PCR, then the interesting gene was obtained. After digested with EcoRI and NotI, the obtained DNA fragment was linked with pPIC9K by T4DNA ligase and then the recombinant plasmid was transformed into competent DHScc. After positive transformants were sieved out by PCR, digesiting analysis and sequencing were also used to confirm the positive result more. After linerization, the recombinant plasmid was transformed into Pichia pastoris by electroporation, which then were cultured in MD plate free of histidine, from which the positive colones were propagated. Multicopy clones were screened from vary levels of G418 -YPD plate and cultured and induced with methanol to secrete interesting peptide, which was identified by SDS-PAGE and Western blotting.The interesting gene fragment with EcoRI and NotI were amplified by overlapping PCR , which inserted into vector plasmid pPIC9K after degisted by EcoRI and NotI, and the recombinant plasmid was transformed into competent DH5cc. Positive clones were screened by PCR from the LB plate with AMP. Digesting analysis resulte shows that the interesting gene were inserted into the vector pPIC9K with correct direction. Sequencing map also illuminates the interesting DNA fragment of human cytomegaloviruswas cloned into the expression vector correctly. Multicopy integrants were screened with G418 from Pichia pastoris which contains recombinant plasmid, and induced with methanol to secrete interesting peptide. The supernate of Pichia pastoris culture was analysed by SDS-PAGE and Western blotting. A reactive band , which the apparent molecular weight is 36KD , can be detected with sheep anti-HCMV polyclonal antibodies. The result indicates that the recombiant peptide of human cytomegalovirus was expressed in Pichia pastoris successfully.
Keywords/Search Tags:HCMV, recombinant peptide, pPIC9K, Pichia pastoris, overlapping PCR, expression
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