Kadokura H, Yoda K, Imai M, Yamasaki M
Department of Agricultural Chemistry, University of Tokyo, Japan.
Appl Environ Microbiol. 1990 Sep;56(9):2742-7. doi: 10.1128/aem.56.9.2742-2747.1990.
The diagnostically important surface antigen pre-S2 of hepatitis B virus was produced in large amounts in the periplasmic space of Escherichia coli. The DNA fragments (pre-S2) coding the pre-S2 antigen were tandemly duplicated or triplicated and ligated in the same reading frame to a fragment containing the promoter and the signal sequence of the alkaline phosphatase-coding gene (phoA) of E. coli. Further, a DNA fragment (bla) coding mature beta-lactamase was joined to the region coding the C terminus of the pre-S2 repeat to stabilize the gene product. Upon induction of the phoA-(pre-S2)3-bla fusion gene, the fusion protein was produced at up to 30% of the total cellular protein. Fractionation of the cellular components and trypsin accessibility of the product showed that the antigen was secreted in the periplasm and formed inclusion bodies there. The signal sequence of alkaline phosphatase was found to be correctly processed in E. coli.
乙型肝炎病毒具有诊断重要性的表面抗原前S2在大肠杆菌的周质空间中大量产生。编码前S2抗原的DNA片段（前S2）被串联重复或三倍重复，并在相同阅读框中与包含大肠杆菌碱性磷酸酶编码基因（phoA）启动子和信号序列的片段连接。此外，编码成熟β-内酰胺酶的DNA片段（bla）与编码前S2重复序列C末端的区域连接，以稳定基因产物。诱导phoA-（前S2）3-bla融合基因后，融合蛋白的产量高达细胞总蛋白的30%。细胞成分分级分离及产物的胰蛋白酶可及性表明，该抗原分泌到周质中并在那里形成包涵体。发现碱性磷酸酶的信号序列在大肠杆菌中得到了正确加工。
