化學合成B型肝炎病毒前表面抗原基因及利用T7RNA聚合?在大腸桿菌中選擇性地表現B型肝炎表面抗原基因

Abstract

中文摘要 本實驗室曾構築了一酵母菌分泌性載體系統，將B型肝炎病毒HBV preS2-sAg基因接到α因數?動子--前導序列下游，雖能表現，但不能分泌。為減少分子太大對分泌功能的影響及期望提高產量，本研究擬合成一段只含246對鹼基對的基因。它含有pre S2的55個胺基酸的順序及酵母菌trp 1基因的轉錄終結子，並將pre S2中密碼子均修改成在酵母菌中最佳的型式。但接合的過程遭遇困難，未能成功。於是利用含有T7 RNA聚合?基因的大腸桿菌溶原菌株BL21(DE3)及SA 2665來表現pre S2-sAg。pAR 2106是一含有T7?動子的質體，pre S2-sAg即是接在T7?動子的下游。結果不論在BL21(DE3)或SA 2665中，放射免疫測試(radioimmunoassay)及免疫吸漬測驗(immunoblot)的結果都沒有表現。但轉形株在IPTG誘導三小時後仍有95％保有質體，而且存活率未下降。並由離體轉錄試驗得到期望的轉錄產物。所以認為，HBV之表面抗原在大腸桿菌中無法穩定存在可能是其在大腸桿菌系統一直表現不理想的主要原因。

Abstract A yeast secretion vector system had been developed previously in our lab. A hepatitis B virus gene coding for preS2—sAg was introduced downstream of the yeast α—factor—promoter—leader sequence but failed to be secreted although it is expressed. In order to minimize the affection of molecular size in secretion and get high yield of expression, we plan to synthesize a gene with lower molecular weight containing 246 base pairs, encoding 55 amino acids of preS2 with optimal codons for yeast and a transcription terminator of yeast trpl gene. The synthesis of the gene involved enzymatic joining of 16 synthetic oligonucleotides, 20—35 nucleotides long, to form DNA duplexes. Because of some experimental limitation, we could not succeed in this line of our work. Another part of this thesis is to use T7 RNA polymerase to direct selective expression of HBV surface antigen gene. HBV preS2—sAg was cloned downstream of T7 promotor in plasmid pAR2106 and transformed to E. coli strains BL21(DE3) and SA2665, both are λ phage lysogen with T7 gene 1 (RNA polymerase gene) integrated in chromosomal DNA under control of lac operon requlatory gene. Both RIA and immunoblot get negative results. But the viability of transformants and stability of plasmids could be maintained even after 3 hours of IPTG induction. The in vitro transcription product, mRNA, is of expected size. The stabliity of HBV surface antigen is probably the major problem for the gene to express in E. coli.