变形链球菌CH43变链素Ⅰ基因片段的克隆及表达

Cloning of mutA and expression of its coding protein

目的:获得编码变形链球菌CH43变链素Ⅰ前体肽基因片段,并诱导其在不杀伤工程菌的前提下高效表达。方法:用PCR技术从变形链球菌CH43基因库中扩增出编码变链素Ⅰ成熟肽mutA基因片段相应大小的DNA片段,将片段与pMD18 T载体连接后测序。将测序正确的mutA按照BamHⅠ和HindⅢ酶切位点克隆入原核表达载体pProEX,将连接产物转化入E.coliDH5α,挑出阳性克隆,IPTG诱导表达重组的6×His融合蛋白。以IPTG浓度、A600值、诱导时间各梯度优化表达。结果:PCR获得的mutA序列与GenBank报道的一致(为147bp)。优化了诱导条件使重组载体在E.coliDH5α中高效表达,融合蛋白经SDS PAGE,在相对分子质量为5. 7×103处有特异的蛋白条带,在A600为1. 666时,加入IPTG至终浓度为1. 0mmol/L,诱导6h,目的蛋白表达量最高,占菌体总蛋白量的20%左右。结论:成功克隆变形链球菌CH43变链素ⅠmutA基因片段,并在E.coliDH5α中高效表达。

Objective:To obtain mutA gene of Strepto c occus mutans (Ms),and to express it in E.coli DH5α.Methods: mutA gene was amplified by PCR with specific primers from genome of Ms CH43 strain. After sequencing, the gene segment was inserted into vector pProEX and expressed in E.coli DH5α.The protein expression was induced by ITPG an d the protein products were examined by 180 ml/L SDSPAGE electrophorosis. Results:The length of PCR product was 147 bp and was identical to mu tA gene reported by GenBank.The mutA gene product was expressed in E.col i DH5α with Mr of 5.7×10 3.The maximum mutA protein product amount (20% of the total bacterial protein) was obtained when the A 600 value of DH5α was 1.666,IPTG concerntration 1.0 mmol/L and induction time 6 h.Conclus ion:mutA of Ms CH43 can be cloned and expressed in E.coli DH 5α.