【摘要】 目的构建携带颗粒裂解肽G13结构域的原核表达质粒,在大肠杆菌BL21(DE3)中表达目的蛋白,并检测其对大肠杆菌活力的影响。方法人工合成G13结构域编码DNA序列,PCR扩增后,用T-A克隆法与pBAD/TOPOThioFusion表达载体连接。通过PCR鉴定及测序筛选阳性重组质粒,转化大肠杆菌BL21(DE3),经阿拉伯糖诱导表达,SDS-PAGE检测表达情况,同时监测诱导表达前后工程菌A600值的变化及菌液中的突变体。结果工程菌经诱导,表达出相对分子质量约15000的特异蛋白,表达量占菌体总蛋白的3%左右。随着外源蛋白的表达,菌液A600值下降,随后又缓慢上升。提取的质粒测序结果表明,其启动子的-35区和-10区均发生了突变,随着诱导时间的增加,突变细胞的比例不断增加。结论已成功构建了G13结构域原核表达质粒,并在大肠杆菌中表达出G13融合蛋白。该异源蛋白的表达导致工程菌的活力降低。更多还原

【Abstract】 Objective To construct a prokaryotic expression vector for G13 domain of granulysin, express in E. coli BL21 (DE3) and study the effect of expressed product on the activity of host E. coli. Methods Synthesize the DNA sequence encoding G13 domain of granulysin, amplify by PCR and clone into expression vector pBAD/TOPO ThioFusion by T-A cloning. Screen positive recombinant plasmid by PCR and sequencing, and transform to E. coli BL21(DE3) for expression under induction of L-arabinose. Identify the expressed product by SDS-PAGE and determine the A600 values of host E. coli before and after induction. Test the recombinant E. coli after induction for mutant. Results The specific protein with a relative molecular mass of about 15 000 was expressed, which contained about 3% of total somatic protein. The A600 value of host E. coli decreased with the increasing time for induction, and increased gradually after expression of foreign protein. The sequencing result of extracted plasmid showed mutation at both -35 and -10 domains. The proportion of mutant cells increased with the increasing time for induction. Conclusion The prokaryotic expression vector for G13 domain of granulysin was successfully constructed, and G13 fusion protein was expressed in E. coli. The expression resulted the decrease of activity of host E. coli.更多还原