Study On High-level Expression Of Recombinant Hepatitis B Surface Antigen In CHO Cell

At present, there are 3.5-4 hundred million chronic hepatitis Bvirus carriers in the world, among them ninety million people developinto liver diseases. About 1-2 million patients die of HBV infectionand its following diseases every year. Our country is highly endemicregion of HBV infection, about 1.7 hundred million people are infectedwith HBV and 170 million people are suffering from chronic hepatitis.About 500 thousand people die of liver cancer or cirrhosis caused byHBV every year. Inoculating HBV vaccine is an important means for preventinghepatitis. Currently, there are two expression systems of yeast andCHO cell that express HBsAg, of which the HBsAg expressed in CHO cellhas the advantage of powerful immunogenicity and high efficiency ofpositive transformation, but low yield is the main problem. The objective of our research was to construct recombinant CHOengineered cell that highly expressed HBsAg. Firstly, the genome ofC28 cell that currently was used to produce HBsAg was extracted, andthe S gene in C28 genome was amplified , then the three rare codonsin S gene were changed into the preferred codons of mammal that were213TTA CTG;216TTA CTG;224GTA GTG.The result of sequencingproved that we succeeded in mutating S gene. constructing P1 and P4 vectors we considered the selection ofregulatory elements containing promoter and enhancer, polyadenylationsignal, IVS, selectable marker, multiple cloning region and antibiotic iv吉林大学硕士学位论文 重组 CHO 乙型肝炎疫苗的高效表达resistance gene. The two fragments from pCIneo and pMD-T-Sm vector digested withrestriction enzyme were ligated and transformed into E.coliJM109(pCIneo-Sm). The SV +DHFR gene fragment in which the attenuationenhancer of SV40 promoter was deleted was amplified,and thenincorporated into pCIneo-Sm plasmid. The P1 expression vector wasconstructed successfully. The P1 vector was identified by digestingwith restriction enzyme and PCR amplification . The result ofidentification was in accordance with our expectation. The digested fragments from pMD-T-Sm and pCI vectors digestedwith NheⅠ and NotⅠ were ligated and transformed into E.coliJM109(pCI-Sm). . DHFR gene was amplified and incorporated into thedownstream of S gene pCI-Sm vector so that DHFR and S gene formed adiscistronic transcription unit. So the pCI-Sm-DHFR vector wasconstructed .With the primers envisaged according to NEO gene sequence,N5 and N3 fragments were amplified and ligated to intron by turns. theligated product was transformed into E.coli JM109, the pMD18-T-N+itransformant was acquired. The big fragment that derived from digestedpCIneo vector was blunted by Klenow and ligated. The ligated productis transformed into E.coli JM109. The recombinant plasmid andpMD-18-T-N+I were digested, ligated and transformed. with the tworestriction site of BglⅡ and NotⅠin intron, the Sm-DHFR gene fragmentwith hCMV promoter was incorporated into NEO gene .the P4 vector wasidentified by digesting with restriction enzyme and PCR v吉林大学硕士学位论文 重组 CHO 乙型肝炎疫苗的高效表达amplification . The result of identification was in accordance withour expectation. There were hCMV promoter regions, poly(A) signal chimeric intron,DHFR gene and S gene without 5′ and 3′ UTR in P1 and P4 vectorsconstructed. The enhancer of the SV40 promoter in the front of DHFRgene in P1 vector was deleted so that the transcription efficient ofDHFR gene was weakened. The long DNA fragment was inserted into NEOgene in P4 vector so that the post-transcription process of NEO genewas influenced and the expression of NEO gene was weakened. At the sametime, the expression of DHFR gene in P4 vector was also weakened becauseDHFR gene was at the downstream of S gene and th...