HBcAg表达菌株的筛选、初步鉴定与DNA顺序分析

为提高HBcAg(乙型肝炎核心抗原)在大肠杆菌体内表达水平,将有HBcAg的基因片段用核酸外切酶从两端消化,插入载体质粒pUR222的EcoRI酶切位点,转化Ecoli BMH71—18,用菌落原位酶联免疫法由275个转化子筛选到7株阳性克隆,用ELISA比较了它们的表达水平,表达水平最高者为M2066菌株,P/N=2.0时菌体裂解液的稀泽度为1:33000’比表达最低者M2098高8,000倍,比第一代菌株M206高15,000倍,DNA顺序分析结果表明与mRNA起始密码上游的发卡结构去除有关。用SDS-PAGE和聚乙烯簿膜复印法检测菌体裂解液中HBcAg的分子量为21000,42000及63000,呈单体和聚合物形式存在,比由病人肝脏和血液中提取的HBcAg(19000)分子量大,为融合蛋白。经琼脂免疫双扩散与ELISA阻断试验未发现与β半乳糖苷酶有免疫学交叉反应。用ELIDA法,M2066-HBcAg与肝-HBcAg同时检测40份血清标本的抗-HBc,二者符合率95％。

SCREEING OF E.coli CLONES EXPRESSING HBcAg AND CHARACTERIZATION OF THE HBcAg

To increase the level of expression of HBcAg in E.coli,fragments earring the gnes for core antigen which were digested by BAL-31 were inserted into EcoR1 restriction site in plasmid pUR222,A simple in situ colony enzymoimmunoassay was Used for screening 275 transformants,and 7 of them were found positive for HBcAg.The levels of HBcAg gene expression for these clones have been tested by ELISA.It has been shown that the clone M2066 synthesized HBcAg in the highest level (lysate dilution 1:33000),which was about 15,000 times as much as M206 in bacterial lysates.Three transformants in E.coli showing differet levels of HBcAg gene expression were analyzed for their nucleotide sequences in the junction region by Maxam and Gilbert's method.The results of sequence analysis revealed a hairpin structure lying immediatly before the initiator codon of the core antigen mRNA.HBcAg was dramaticly increased in E.coli by deleting this structure.The M2066-HBcAg molecular weights were examined by Western blot.Their molecular weights were 21,000,42,000 and 63,000 respectively.The molecular weight 21,000 is larger than the native HBcAg (19,000) from serum or liver.Because it is a fusion peptide containing the first 5 amino acids for β galactosidase.No cross-reacting antigen between E.coli HBcAg and βgalactosidase was found by immunodiffusion and the antibody blocking test.Results of parallel ELISA of 40 serum samples from individuals of different anti-HBc status using E.coli HBcAg and liver HBcAg respectively showed a 95% agreement for the two antigens.It may,therefore,be concluded that the E.cli HBcAg can be used to replace liver HBcAg in assays for anti-HBc.