Construction Of Eukaryotic Expression Vector Of HBsAg And The Study Of Its Expression In CHO Cell

Hepatitis B is an infectious disease caused by hepatitis B through blood and body liquid . HBV is responsible to injured liver .At present,there are about 3.5 million hepatitis B virus carriers in the world,among them ninety million people developed to liver diseases.More than 1 million patients die of HBV infection and its following disease every year. Our country is a highly epidemic region of HBV infection, about 1.2 hundred million people were infected with HBV and 120 million people are suffering from chronic hepatitis. Every year about 350 thousand people die of liver cancer or cirrhosis caused by HBV.Inoculating HBV vaccine is an important means for preventing hepatitis,which was integarated into immunization programs system in our country. ten years late, incidence of disease of Hepatitis B is decreased becaused of a large scale of inoculating HBV vaccine which was thinked commonly as safety and effective means.Currently, there are two expression systems of yeast and CHO cell that express HBsAg, of which the HBsAg expressed in CHO cell has the advantage of powerful immunogenicity and high efficiency of positive transformation, but low yield is the main problem.Objective: According to the principle of eukaryotic gene expression, CHO/dhfr-was selected as expression system . we consider the engineering CHO cell construction scheme from expression vector and objective gene.The objective of our research was to construct recombinant CHO engineered cell that highly expressed HBsAg .Methods: We studied carefully the characters of C28 cell strain used in Changchun Institute of Biological Products. On the basis of large quantities of references, we put forward a new designing scheme of expression vectors, including replacing promoter, introducing intron, getting rid of 5′and 3′UTR of objective gene, attenuating antibiotic resistance gene and replacing rare codons in objective gene. Through above methods, four new expression vectors that express HBsAg were constructed.1,clone S gene and gene mutated the S gene in C28 genome was amplified by primers in which there are no 5ˊU TR and 3ˊUTR but there is KOZAK sequence in 5ˊ primer. The primers designing are favorable to the mRNA stability and translation initiation. The three key rare codons in S gene were changed to the preferred codons of mammal that were 213 ,216 fromTTA to CTG; 224 fromGTA toGTG and S^m was obtained.2,Constructed four new expression vectors on the basis of plasmids pCI and pCIneo, NO.1, NO.2, NO.3 and NO.4 expression vector were constructed by recombinant DNA technique. Both pCI and pCIneo carry hCMV promoter ,which is 5 times stronger than SV40 early promoter . There is an intron sequence following hCMV in pCI and pCIneo, which is beneficial for the maturation and stability of objective gene mRNA.The common characters among four new expression vectors are strong promoter and dhfr gene which can amplified greatly under the selection pressure . The attenuation of dhfr could increase the copy of S^m gene and then heighten expression level of HBsAg. There was hEF-1αpromoter regions of NO.2, and others were hCMV promoter regions.3,Constructed engineered cell strain and detectedFour series of expression vectors were extracted in large scale , purified by CsCl2 density gradient centrifugation and high-purified supercoil DNA were obtained. According to LipofectamineTM Reagent instructions, CHO/dhfr- cell was transfected .C1 and C2 cell (cells transfected by NO.1 and NO.2 expression vectors) was screened by different concentrations MTX that were 2x10^-8mol/l, 1x10^-7mol/l and 1x10^-6mol/l MTX .C3 and C4 cell (cells transfected by NO.3 and NO.4 expression vectors ) were firstly cultured in 200μg /ml G418 culture medium, and then were screened as same as C1 and C2 cells. The quantities of expression of HBsAg were detected by ELISA and reversed passive hemagglutination after each screening.Results: S gene was cloned from C28 cell strain and was mutated. Four vectors all express HBsAg and high expression cell strains were selected of which the quantities of HBsAg were stable among 512～1024/two days. The expression level is as twice as expression cell strains before .1,clone S gene and gene mutated S gene was isolated from C28 by PCR and the rare codons at amino acid No. 213, 216 and 224 of S gene were replaced with the preferred codons in mammalian by overlap-PCR technique . There is KOZAK sequence at 5ˊ region of S gene and S^m gene was gained.2,Constructed four new expression vectors The deletion of enhancer of SV40 promoter in expression vector NO.1 attenuated dhfr gene expression. In expression vectors NO.2,NO.3 or NO.4 the dhfr gene and its downstream S^m gene were transcripted into one dicistron, which attenuated expression of dhfr. the deletion of enhancer of SV40 promoter in expression vector NO.1 attenuated dhfr gene expression. In expression vectors NO.2,NO.3 or NO.4 the dhfr gene and its downstream S^m gene were transcripted into one dicistron, which attenuated expression of dhfr.In expression vector NO.3 the promoter of neo^r gene was attenuated. In expression vector NO.4 an intron into which an S^m gene transcription unit was inserted was introduced into neo^r gene that greatly affected the posttranscriptional process of neo^r gene and thereby attenuated the expression of neor. The attenuation of neor gene was benefit to incorporating Sm gene into the hot spot of cell chromosome that was active transcription region.The all of above characters of NO.1, NO.2,NO.3 and NO.4 vectors were favorable for the expression of Sm gene. These four vectors are identified by PCR and digesting with restriction enzymes.3,Constructed engineered cell strain and detected 5μg Lipofectamine is suitable for transfection. The results of detection showed that the quantities of the expression of HBsAg were increased highly after three screenings.The quantities of expression of HBsAg were detected by ELISA and reversed passive hemagglutination after each screening.The monoclones were screened from C1 resistant cells in 1x10-6mol/l MTX, and three high expression cell strains were selected of which the quantities of HBsAg were stable among 512～1024/two days in excretion kinetics of single layer cell culture for 40 days. The results proved it was valuable to do further research. The screening of high-level production strains from C1,C2,C3 and C4 is on the way now.Conclusion:We recombined four new CHO cell strains sucessfully and selected high-level production strains which are two times higher than before. The study provide solid foundation for increasing production of hepatitis B vaccine.