摘要
目的构建天蚕素A(1～8)-蛙皮素(1～12)杂合基因抗菌肽(CA-MA杂合肽)突变体,在大肠杆菌中融合表达,并进行抗菌活性检测。方法采用PCR体外定点突变技术,设计1对方向相反的引物,其中1个引物引入突变点,应用高保真的PolybestDNA多聚酶进行重组表达质粒pGEX-4T-1-CA-MA的PCR扩增,使CA-MA杂合肽第16位密码子由AGT突变为TGG。将扩增片段自身连接,构建CA-MA杂合肽突变重组表达质粒pGEX-4T-1-W16-CA-MA,转化E.coliBL21,IPTG诱导表达突变体蛋白W16-CA-MA,并对表达产物进行纯化。分离GST融合蛋白后,进行抗菌活性检测。结果重组突变表达质粒DNA测序结果表明,在预期位点发生了突变;突变蛋白在大肠杆菌中的表达量约占菌体总蛋白的18%;纯化后蛋白纯度可达75%以上,并具有一定的抗菌活性。结论已成功获得了具有抗菌活性的杂合肽突变体W16-CA-MA。
Objective To construct cecropin A （ 1 - 8 ）-magainin （ 1 - 12） （CA-MA） hybrid peptide mutant for fusion expression in E. coli and determine the antibacterial activity of expressed product. Methods Design a pair of primers, to one of which mutation site was introduced, and amplify the gene fragment encoding CA-MA hybrid peptide mutant in which the codon at site 16 was changed from AGT to TGG by PCR with high fidelity Polybest DNA polymerase, using recombinant expression vector pGEX-4T-1-CAMA as a template. Recombinant plasmid pGEX-4T-1-W^16-CA-MA was constructed by linking the PCR products using blunting kination kit and transformed to E. coli BL21 for expression under induction of IPTG. The expressed mutant protein W^16-CA-MA was purified, cleaved from GST fusion protein and determined for antibacterial activity. Results The DNA sequencing of recombinant plasmid pGEX-4T-1-W^16-CA-MA proved mutation at the expected site. The expressed W^16-CA-MA protein contained 18% of total somatic protein, reached a purity of more than 75% after purification and showed a certain inhibitory effect on Staphylococcus aureus. Con- clusion The W^16-CA-MA protein with antibacterial activity was successfully expressed.
出处
《中国生物制品学杂志》
CAS
CSCD
2008年第9期752-755,共4页
Chinese Journal of Biologicals
基金
国家"863"计划资助项目(2006AA10A208-1-4)
关键词
天蚕素A-蛙皮素杂合肽
突变体
融合表达
抗菌活性
Cecropin A （ 1 - 8 ）-magainin （ 1 - 12 ） （ CA-MA ） hybrid peptide
Mutant
Fusion expression
Antibacterial activity
