牛乳溶菌酶的原核表达及初步纯化

目的原核表达并初步纯化牛乳溶菌酶(Bos taurus lysozyme1,LYZ1)。方法人工设计并合成LYZ1基因CDS序列,将其克隆至表达载体pET-32a,构建重组表达质粒pET-32a-LYZ1,转化大肠杆菌BL21(DE3),筛选阳性菌株,经IPTG诱导,初步纯化表达产物,并对其进行SDS-PAGE和Western blot分析。结果所构建的重组表达质粒经PCR、双酶切及测序鉴定正确;SDS-PAGE分析显示,目的蛋白相对分子质量约为32000,主要以包涵体形式存在,表达量约占菌体总蛋白的70%以上,切胶纯化后纯度达95%;Western blot分析显示,重组融合蛋白可与小鼠Anti-HisTag单抗特异结合。结论已成功原核表达并初步纯化了LYZ1,为后续研究与应用奠定了基础。

Prokaryotic Expression and Preliminary Purification of Bos taurus Lysozyme

Objective To express Bos taurus lysozyme(LYZ1)in prokaryotic cells and preliminarily purify the expressed product.Methods The CDS sequence of LYZ1 gene was designed and synthesized,then cloned into expression vector pET-32a.The constructed recombinant plasmid pET-32a-LYZ1 was transformed to E.coli BL21(DE3),and the positive clones were screened for expression under induction of IPTG.The expressed product was preliminarily purified and identified by SDS-PAGE and Western blot.Results PCR,restriction analysis and sequencing proved that recombinant plasmid pET-32a-LYZ1 was constructed correctly.SDSPAGE proved that the expressed protein,with a relative molecular mass of about 32 000,mainly existed in a form of inclusion body,contained more than 70% of total somatic protein and reached a purity of 95% after purification.Western blot showed specific binding of the expressed fusion protein to mouse anti-His Tag McAb.Conclusion LYZ1 was successfully expressed in prokaryotic cells and preliminarily purified,which laid a foundation of further study.