日本血吸虫亮氨酸氨基肽酶的原核表达及纯化

目的原核表达并纯化日本血吸虫亮氨酸氨基肽酶(Leucine aminopeptidase of Schistosoma japonicum,SjLAP)。方法从日本血吸虫成虫中RT-PCR扩增LAP基因,克隆至原核表达载体pET-28a中,转化感受态E.coli BL21(DE3),IPTG诱导表达,表达的重组SjLAP蛋白(rSjLAP)经SDS-PAGE和Western blot分析后,经His Binding Purification Kit层析纯化,纯化的rSjLAP蛋白经SDS-PAGE分析纯度,Bradford法测定浓度。结果克隆的SjLAP基因与GenBank中登录的基因序列比较有2个碱基发生置换,导致对应的2个氨基酸发生置换,但均不在关键区域;重组表达质粒pET-28a/SjLAP经双酶切鉴定证实构建正确;表达的rSjLAP蛋白相对分子质量约45 000,诱导4 h表达量最高;纯化的rSjLAP蛋白纯度较高,浓度达5.0 mg/ml,并可被抗小鼠His-tag单抗及血吸虫感染的患者血清和兔血清特异性识别,具有良好的反应原性。结论成功在大肠杆菌中表达了rSjLAP蛋白,纯化的rSjLAP纯度较高,为血吸虫病的诊断和预防等研究奠定了基础。

Prokaryotic expression and purification of leucine aminopeptidase of Schistosoma japonicum

Objective To express leucine aminopeptidase(LAP) of Schistosoma japonicum(Sj) in prokaryotic cells and purify the expressed product.Methods LAP gene was amplified from the adult worm of Sj by RT-PCR and inserted into prokaryotic expression vector pET-28a.The constructed recombinant plamid pET-28a / SjLAP was transformed to competent E.coli for expression under induction of IPTG.The expressed recombinant SjLAP(rSjLAP) was identified by SDS-PAGE and Western blot,then purified by His Binding Purification Kit,and analyzed for purity by SDS-PAGE,and for concentration by Bradford method.Results Compared with the SjLAP gene reported in GenBank,two base substitutions were observed in the cloned gene,resulting the substitutions of two corresponding amino acids.However,neither the substitutions was located in dominant domain.Restriction analysis proved the recombinant plasmid pET-28a / SjLAP was constructed correctly.The expression level of rSjLAP,with a relative molecular mass of about 45 000,reached the maximum 4 h after induction.The purified rSjLAP reached a high purity and a concentration of 5.0 mg / ml,and was recognized by monoclonal antibody against mouse His-tag as well as human and rabbit sera infected with Sj,indicating a high reactogenicity.Conclusion Recombinant SjLAP was successfully expressed in E.coli and reached a high purity after purification,which laid a foundation of diagnosis and prevention of schistosomiasis.