| At present, there are 3.5-4 hundred million chronic hepatitis B virus carriers in the world, among them ninety million people developed to liver diseases. About 1-2 million patients die of HBV infection and its following diseases every year. Our country is a highly endemic region of HBV infection, about 1.7 hundred million people were infected with HBV and 170 million people are suffering from chronic hepatitis. About 500 thousand people die of liver cancer or cirrhosis caused by HBV every year.Inoculating HBV vaccine is an important means for preventing hepatitis. The World Health Organization (WHO) recommended to inoculate infants in the country where hepatitis prevailed. At present, our country has integrated hepatitis B vaccination into immunization programs system.In our country, the HBsAg expressed in CHO cell appeared on market in 1992, which was studied by Chinese Academy of Preventive Medicine and Changchun Institute of Biological Products. 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. At present, the cell strain used in Changchun Institute of Biological Products was constructed in the late 1980's, the yield of which is 1-2 mg HBsAg/48 hr/L and the products demanded exceed supplied. In order to enlarge market portion and offer to society more high quality hepatitis vaccines, we plan to construct a new engineered cell strain highly expressing HBsAg.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, two new expression vectors that express HBsAg were constructed.Promoter is one of the key factors that influence expression efficiency of exogenous gene. No. 1 and No. 3 expression vectors constructed were based on pCI and pCIneo plasmids. Both pCI and pCIneo carry hCMV promoter, which is 5 times stronger than SV40 early promoter.It is beneficial for the stability of mRNA to introduce a natural or chimeric intron into expression vector. There is an intron sequence following hCMV in pCI and pCIneo, which is beneficial for the maturation and stability of objective gene mRNA.There maybe form secondary structure in the 5′ UTR of the objective gene which could affect the translation initiation of the objective gene. And maybe there are some regions that could influence mRNA stability and life and further decrease the expression of exogenous gene in the 3′ UTR. So when we construct No.1 and No.3 expression vectors, the 5′ and 3′ UTR in objective gene were gotten rid of.The copies of the co-amplification gene will be greatly increased by attenuating the dhfr gene. By attenuating the neo gene we can screen the transformers in which the exogenous genes are incorporated into the hot spot of chromosome. In vector No.1, the expression of dhfr gene was attenuated by deleting the enhancer of the SV40 early promoter. In vector No.3, the neo gene was attenuated by deleting the enhancer of SV40 early promoter besides attenuating expression of the dhfr gene.Rare codon affects translation in eukaryotic cell. There are two most rare codons, TTA and TCG, in eukaryote. When these rare codons are close to each other, it will seriously hinder ribosome from advancing, lower the efficiency of translation and reduce the level of expression.We analyzed the codons in S gene and found that there were two lowest frequency codons at amino acid No. 213 and 216, which were at a distance of only 6 nucleotides. We estimated that these two codons could greatly affect the translation and reduce the protein expression. So we replaced TTA with the most preferred codon in mammal. Meanwhile, we replaced GTA with GTG at amino acid No. 224. |
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