丝状支原体丝状亚种SC型LppB基因的克隆与表达纯化

目的克隆丝状支原体丝状亚种SC型(MmmSC)LppB蛋白基因,并进行表达及纯化。方法通过PCR,扩增MmmSC标准株PG1的LppB全基因,克隆至pMD18-T载体上,并测序鉴定。同时选LppB基因中两个TGA之间903bp片段,与pET30a载体构建重组质粒,进行诱导表达,表达产物经Ni-NTA-His系统纯化。结果PCR扩增的LppB基因长度片段约1869bp,与预期大小相符。将其与NCBI上发布的Afade株、LC型代表株Y-goat和同属的Bovine7株的LppB基因序列相比较,同源性分别为99·8%、92·8%和76·7%,氨基酸同源性分别为99·2%、90·2%和67·0%。经Ni-NTA-His系统纯化,得到纯净的单一蛋白。结论已成功克隆了PG1的LppB蛋白基因,并进行了表达及纯化。

Gene Cloning, Expression and Purification of Lipoprotein LppB of Mycoplasma mycoide subsp. Mycoides SC

Objective To clone and express the gene encoding lipoprotein LppB of Mycoplasma mycoide subsp.Mycoides SC(MmmSC) and purify the expressed product.Methods Amplify the whole LppB gene of standard strain PG1 of MmmSC by PCR and insert into vector pMDT18.Identify the recombinant plasmid by sequencing.Select a 903 bp fragment between the two TGAs of the amplified LppB gene and insert into vector pET30a for expression under induction of IPTG.Purify the expressed product by Ni-ATA-His system.Results The LppB gene at a length of about 1 869 bp was amplified,which was consistent with that expected.The homologies of the amplified gene sequence to those of Afade,Y-goat and Bovine 7 strains reported in NCBI were 99.8%,92.8% and 76.7%,and those of deduced amino acid sequence were 99.2%,90.2% and 67.0% respectively.After purification,the expressed product showed a single protein band on gel electrophoretic profile.Conclusion The LppB gene of MmmSC was successfully cloned and expressed,and the expressed product was purified.