中国人MBL基因编码区的克隆与原核表达

Cloning and Prokaryotic Expression of Encoding Region of Chinese MBL Gene

目的克隆中国人MBL基因编码区序列,并在原核细胞中表达。方法提取肝总RNA,RT-PCR扩增MBL编码区序列,并克隆到pGEM-T-easy载体上,再亚克隆到pET-30(a)上。诱导表达重组MBL,并通过SDS-PAGE检测表达情况,表达蛋白经初步纯化后,用ELISA及点杂交试验检测其免疫原性。结果克隆得到长747bp的MBL编码区序列,与GenBank中的MBL序列同源性在99%以上。重组MBL相对分子质量约为36000,以包涵体形式存在,表达量占菌体总蛋白量的20%左右,表达蛋白经初步纯化,纯度可达95%以上。ELISA检测显示重组MBLA490值与阴性对照差异有显著意义(10倍以上),点杂交检测结果为阳性。结论已成功克隆中国人MBL基因的编码区序列,并在大肠杆菌中表达,表达产物与天然MBL具有相似的免疫原性。

Objective To clone the encoding region of Chinese mannan-binding lectin(MBL) gene and exp ress in prokaryotic cells. Methods Extract total RNA from human liver tissue,amplify the encoding region of MBL by RT-PCR,clone into vector pGEM-T-easy,then subclone to plasmid pET-30(a) for expression under induction of IPTG.The expressed protein was identified by SDS- PAGE and primarily purified,then analyzed for immunogenicity by ELISA and Dot bl ot. Results The encoding region,at a length of 747 bp,was cloned and showed a homology of mo re than 99% to that of MBL gene reported in GenBank.The expressed recombinant MB L,with a relative molecular weight of about 36 000 ,existed in a form of incl usion body and contained about 20% of total somatic protein.The purity of expres sed protein after preliminary purification was above 95%.The recombinant MBL was positive in Dot blotting,and its ELISA result (A value) deviated by more th an 10 folds from that of negative control. Conclusion The encoding region of Chinese MBL gene was successfully cloned and expressed in E.coli,and the immunogenicity of expressed product was similar to that of n ature MBL.