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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。
Objective To prokaryotic express and purify Leucine aminopeptidase of Schistosoma japonicum (SjLAP). Methods The LAP gene was amplified by RT-PCR from adult Schistosoma japonicum and cloned into prokaryotic expression vector pET-28a. The recombinant plasmid was transformed into competent E. coli BL21 (DE3) and induced by IPTG. The expressed recombinant SjLAP protein (rSjLAP) After PAGE and Western blot analysis, purified by His Binding Purification Kit, the purity of the purified rSjLAP protein was analyzed by SDS-PAGE and the concentration was determined by Bradford method. Results The nucleotide substitutions of 2 bases occurred in the cloned SjLAP gene compared with those registered in GenBank, which resulted in the replacement of the corresponding 2 amino acids, but none of them was in the critical region. The recombinant plasmid pET-28a / SjLAP was verified by double enzyme digestion The expressed rSjLAP protein had a relative molecular mass of about 45 000 and showed the highest expression level at 4 h after induction. The purified rSjLAP protein was highly purified at a concentration of 5.0 mg / ml and could be infected with anti-mouse His-tag mAb and schistosome Of patient serum and rabbit serum specific recognition, has a good response to the original. Conclusion The rSjLAP protein was successfully expressed in Escherichia coli and the purified rSjLAP was highly purified, which laid the foundation for the diagnosis and prevention of schistosomiasis.