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目的 应用重组和克隆技术在大肠杆菌中表达葡萄球菌肠毒素B(SEB)突变体。方法 利用PCR和PCR致变技术从产SEB的标准株扩增SEB基因 ,与原核表达载体 pTrc99A重组后引入大肠杆菌JM1 0 9。通过序列测定鉴定突变基因 ;用聚丙烯酰胺凝胶电泳 (SDS PAGE)和单向免疫扩散试验分别检测表达产物和表达量。结果 获得了 3株重组菌NJM1 0 9、M2 3JM1 0 9、M1 5 0JM 1 0 9。测序表明 ,SEB N序列与已报道的天然seb完全一致 ;SEB M 2 3第 2 3位天冬酰胺密码子AAT发生定向突变 ,即由丝氨酸密码子AGT替代 (N2 3S) ;SEB M1 5 0第1 5 0位氨基酸发生非定向突变 ,由苏氨酸密码子ACT突变为丙氨酸密码子GCT(T1 5 0A)。表达SEB M1 5 0和SEB M2 3的重组细菌裂解液经SDS PAGE ,在 2 80 0 0处可见有表达条带 ,非重组菌则无。单向免疫扩散试验和蛋白浓度测定表明 ,重组菌表达目的蛋白量为 5 μg/ml培养液 ,表达量占菌体总蛋白量的 3.5 %。 结论 获得了2株能表达SEB突变体的工程菌 ,为进一步作功能研究奠定了基础
Objective To express staphylococcal enterotoxin B (SEB) mutants in E. coli using recombinant and cloning techniques. Methods The SEB gene was amplified from the standard strain of SEB by PCR and PCR mutagenesis, and then introduced into E. coli JM1 0 9 after recombination with prokaryotic expression vector pTrc99A. Mutant genes were identified by sequence analysis. Expression products and expression levels were detected by polyacrylamide gel electrophoresis (SDS PAGE) and unidirectional immunodiffusion assays. Results Three recombinant strains NJM1 0 9, M2 3JM1 0 9 and M1 5 0JM 1 0 9 were obtained. Sequencing showed that the sequence of SEB N was completely identical with the reported native seb. The asparagine codon AAT at position 2 3 of SEB M 2 3 was mutated by the serine codon AGT (N2 3S); SEB M1 5 0 A non-directional mutation of the 150th amino acid was made from the threonine codon ACT to the alanine codon GCT (T150A). The recombinant bacterial lysate expressing SEB M1 50 and SEB M2 3 showed an expressed band at 8080 by SDS PAGE and no non-recombinant bacterium. One-way immunodiffusion test and protein concentration determination showed that the recombinant protein expressed the target protein in 5 μg / ml culture medium, the expression amount accounted for 3.5% of the total bacterial protein. Conclusion Two strains of engineered bacteria expressing SEB mutant were obtained, which laid the foundation for further study on the function of SEB