牛带绦虫乳酸脱氢酶基因原核表达及免疫学特征

Prokaryotic expression of lactate dehydrogenase from Teania saginata and its immunological identification

目的克隆牛带绦虫乳酸脱氢酶(lactate dehydrogenase,LDH)基因在大肠杆菌中表达,研究其免疫学特征。方法将牛带绦虫成虫LDH基因与质粒pET-28a(+)连接构建原核重组质粒,在大肠埃希菌BL21/DE3中用异丙硫代-β-D半乳糖苷诱导表达,表达产物通过十二烷基-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白印迹(Westernblotting)进行分析。结果牛带绦虫乳酸脱氢酶基因编码332个氨基酸,理论分子量为35.4kDa,PCR、双酶切及DNA测序的结果均证明pET-28a(+)-LDH构建成功;SDS-PAGE结果表明,牛带LDH基因在大肠埃希菌高效表达,经过亲和层析高纯度蛋白,且该重组蛋白可以被亚洲带绦虫、牛带绦虫及猪带绦虫感染病人血清识别,表明其具有免疫反应性。结论牛带绦虫LDH基因可在原核系统中有免疫活性的高效表达。

Objective To clone and express the gene encoding lactate dehydrogenase（LDH） protein from Teania saginata and to study its immunological characters.Methods The gene was cloned into prokaryotic expression vector pET-28a（＋）and then transformed to E.coli BL21 with IPTG induction.Thereafter,the gene product was analyzed by SDS-PAGE and Western blotting and purified by Ni-NTA affinity chromatography.Results The gene encodes 332 amino acids with a theoretical molecular weight of 35.4kDa.As demonstrated by PCR,double enzyme digestion and DNA sequencing,the recombinant plasmid pET-28（＋）-LDH was constructed successfully,and high expression of E.coli BL21 was obtained as demonstrated by SDS-PAGE assay.The recombinant protein could be recognized by serum of patients with Taenia asiatica and Taeniarhynchus saginatus infection,indicating its immuno-reactivity.Conclusion Highly purified preparation of the protein with definite immuno-reactivity was obtained through purification with affinity.