南极假丝酵母脂肪酶B(CALB)基因在乳酸乳球菌MG1363中的整合及表达  

Expression and Integration of Candida antarctica Lipase B Gene in Lactococcus lactis MG1363

作　　者：尹建洪[1] 罗立新[1] 王成[1] 

机构地区：[1]华南理工大学生物科学与工程学院,广州510006

出　　处：《生物技术通报》2012年第5期105-109,共5页Biotechnology Bulletin

摘　　要：根据南极假丝酵母脂肪酶B(CALB)的基因序列将CALB基因进行TA克隆、酶切鉴定及测序后,亚克隆至大肠杆菌-乳酸乳球菌穿梭表达载体pMG36e-NisI中,构建重组表达载体pMG36e-NisI-CALB。设计特异性引物P3和P4,对重组质粒pMG36e-NisI-CALB进行红霉素抗性基因的敲除,以构建食品级表达载体pMG36N-CALB,后再将两种重组质粒分别电转化入乳酸乳球菌MG1363,以Nisin为选择压力,考察CALB在MG1363中的表达情况。结果显示,成功构建了表达载体pMG36e-NisI-CALB及pMG36N-CALB,两株重组菌在含有20 IU Nisin/mL的培养基中均生长情况良好,遗传性能稳定,且经水解圈鉴定,CALB能够进行活性表达。进一步研究发现,CALB基因整合到乳酸乳球菌MG1363染色体中。It has become a research focus in molecular biology of lactic acid bacteria that exploring some genes with practical applications significance by cloning and expression in lactic acid bacteria and constructing a food-grade selection marker in Lactococcus lactis expression system. According to the sequence of CALB, the CALB fragment was amplified by polymerase chain reaction with pMD19-CALB as template. After sequenced, the amplicon was confirmed by Blast from NCBI. Then, the nisI was subcloned into the E. coli-L, lactis shuttle vector pMG36e-NisI, resulting in the plasmid pMG36e-NisI-CALB. In order to construct a food-grade vector pMG36N-CALB, erythromycin resistance gene from the recombinant plasmid pMG36e-NisI-CALB was knocked out by redesigning the pair of specific primers. The recombinant strain MG1363/pMG36N-CALB and MG1363/pMG36e-NisI-CALB were obtained when the plasmid pMG36N-CALB and pMG36e-NisI-CALB were transformed into L. lactis MG1363 competent cell by electroporation respectively. In a result, the expression vector pMG36e-NisI-CALB and pMG36N-CALB were constructed. When the medium 20 IU Nisin/mL, the recombinant strain carrying pMG36N-CALB showed the same growth curve and genetic stability as L. lactis MG1363. CALB can be expressed actively and the CALB gene was integrated into the chromosome of Lactococcus lactis MG1363.

关 键 词：乳酸乳球菌 食品级表达载体 CALB 基因敲除 整合 活性测定 

分 类 号：Q78[生物学—分子生物学]