【摘要】 本文首先以枯草芽孢杆菌(B.subtilis)穿梭载体pDG150为基础,构建了含有5个克隆位点的表达载体pTD160,并在此基础上构建了带有淀粉酶信号肽的分泌表达载体pTD161和可用于外源蛋白与胞衣蛋白cotG融合表达的表面展示载体pTD162。将脂肪酶基因lipa分别插入pTD161和pTD162,在B.subtilis 168中表达,其LipA酶活性分别为23.4U/mg和15.3U/mg,表明pTD载体具有良好的表达性能。通过无抗生素压力传代培养10代后,发现其稳定性仍为96.5%和95.0%,证明pTD载体具备较高的稳定性。将密码子优化后的猪源表皮生长因子基因pegf分别导入pTD161和pTD162,转化B.subtilis 168获得工程菌Bs168-pTD161-pegf和Bs168-pTD162-pegf,ELISA法证明pEGF在工程菌中表达量分别达到365ng/mL和285ng/mL,证明pEGF获得高效表达和展示。更多还原

【Abstract】 Firstly,based on the shuttle vector pDG150 of Bacillus subtilis,the expression vector pTD160 containing five cloning sites was constructed.Secondly,the expression vector pTD161 harboring a signal peptide of amylase Q and the surface display vector pTD162 which allows fusion expression of foreign proteins with cotG protein were constructed.The LipA gene was inserted into pTD161 and pTD162 respectively and expressed in B.subtilis 168.The activity of LipA was 23.4 U/mg and 15.3 U/mg respectively,indicating that the pTD vectors had good expression performance.The stability of pTD vectors was 96.5%and 95.0%respectively after 10 generations of non-antibiotic pressure subculture.The porcine epidermal growth factor gene(pegf)optimized by codon of B.subtilis was introduced into pTD161 and pTD162 respectively,and then transformed into B.subtilis 168,generating the engineering strains Bs168-pTD161-pegf and Bs168-pTD162-pegf.ELISA assay showed that the expression of pEGF in these engineering strains reached 365 ng/mL and 285 ng/mL respectively,which indicated the pEGF had been well expressed in B.subtilis 168.更多还原