摘要
目的原核表达并纯化人胎盘泌乳素(Human placenta lactogen,hPL)。方法从正常产妇新鲜胎盘组织中提取组织总RNA,PCR扩增hPL基因,将其亚克隆至pQE-30载体,构建重组表达质粒,转化感受态大肠杆菌M15(pREP4),经IPTG诱导表达。表达产物经Sephacryl S-200层析柱纯化,纯化产物经SDS-PAGE和western blot鉴定其纯度和特异性。结果重组菌pQE-hPL/M15(pREP4)经双酶切及测序证实构建正确;表达的重组蛋白相对分子质量约23 000,主要以包涵体形式表达,表达量占菌体总蛋白的67%;纯化后目的蛋白纯度可达90%以上;纯化蛋白和hPL包涵体均可与羊抗hPL多克隆抗体特异性结合,具有较强的特异性和抗原性。结论已成功制备了纯度较高的hPL蛋白,为基因工程制备抗体提供了抗原。
Objective To express human placenta lactogen（hPL）in prokaryotic cells and purify the expressed product. Methods The hPL gene was amplified by PCR using the total RNA extracted from fresh placental tissue of healthy lyingin women as a template,and subcloned into expression vector pQE-30.The constructed recombinant plasmid was transformed to competent E.coli M15（pREP4）for expression under induction of IPTG.The expressed product was purified by Sephacryl S-200 chromatography,and identified for purity and specificity by SDS-PAGE and Western blot.Results Both restriction analysis and sequencing proved that recombinant E.coli pQE-hPL/M15（pREP4） was constructed correctly. The expressed recombinant protein,with a relative molecular mass of about 23 000,mainly existed in a form of inclusion body,contained % of total somatic protein and reached a purity of more than 90% after purification.Both the purified protein and hPL inclusion body showed specific bindings to goat anti-hPL polyclonal antibody,indicating high specificity and antigenicity.Conclusion Highly purified hPL was successfully prepared,which provided an antigen for preparation of antibody by gene engineering technique.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第5期661-663,669,共4页
Chinese Journal of Biologicals
基金
吉林省科技计划项目(20110962)
关键词
人胎盘泌乳素
原核细胞
基因表达
纯化
Human placenta lactogen
Prokaryotic cells
Gene expression
Purification
