摘要
目的原核表达人溶菌酶(Human lysozyme,hLYZ)和抗菌肽tachyplesins融合蛋白,并检测其抗菌活性。方法人工合成抗菌肽tachyplesins基因和linker,与pMD18-T-hLYZ上切下的hLYZ基因融合,将融合基因克隆至带有GST标签的原核表达载体pGEX-4T-1上,转化大肠杆菌BL21(DE3),IPTG诱导表达,并对表达条件进行优化。表达的融合蛋白经亲和层析纯化后,进行抗菌活性检测。结果重组表达质粒pGEX-4T-1-hLYZ-tachyplesins经PCR、双酶切及测序鉴定,证明构建正确;表达产物经SDS-PAGE分析,在相对分子质量约44000处可见目的蛋白条带,诱导温度在25℃,IPTG终浓度为0.8mmol/L,诱导时间为6h,融合蛋白表达效果较好,主要以可溶性形式存在;纯化的融合蛋白纯度可达90%以上,对金黄葡萄球菌和大肠杆菌具有一定的抑制作用。结论已成功在原核细胞中表达了人溶菌酶-抗菌肽tachyplesins融合蛋白,纯化的融合蛋白具有一定的抗菌活性。
Objective To express the fusion protein of human lysozyme（hLYZ）and tachyplesins, an antimicrobial peptide, in prokaryotic cells, and determine its antimicrobial activity. Methods The gene and linker of tachyplesins were synthesized and fused with the hLYZ gene cut from plasmid pMD18-T-hLYZ, then cloned into prokaryotic expression vector pGEX-4T-1 with GST labeling. The constructed recombinant plasmid pGEX-4T-1-hLYZ-tachyplesins was transformed to E. coli BL21（DE3）for expression under induction of IPTG, and the condition for expression was optimized. The expressed fusion protein was purified by affinity chromatography and determined for antimicrobial activity. Results PCR, restriction analysis and sequencing proved that recombinant plasmid pGEX4T-1-hLYZ-tachyplesins was constructed correctly. The expressed product showed a band with relative molecular mass of about 44 000 on SDS-PAGE profile. The fusion protein was expressed mainly in a soluble form after induction with 0. 8 mmol / L IPTG at 25℃ for 6 h. The purified fusion protein reached a purity of more than 90% and showed a certain inhibitory effect on both Staphylococcus aureus and E. coli. Conclusion The hLYZ-tachyplesins fusion protein was successfully expressed in prokaryotic cells. The purified fusion protein showed a certain antimicrobial activity.
出处
《中国生物制品学杂志》
CAS
CSCD
2010年第8期829-833,共5页
Chinese Journal of Biologicals
基金
吉林省科技发展基金资助项目(20070210)
