摘要
目的获得弓形虫多表位基因在原核系统中可溶性表达产物,为弓形虫病重组抗原试剂盒的制备及疫苗的研究奠定基础。方法以PCR法扩增弓形虫多表位基因,使其两端带有与载体pET32a相匹配的酶切位点,插入载体pET32a,酶切并测序鉴定后,转入大肠杆菌BL21(DE3),以IPTG进行诱导,SDS-PAGE分析和鉴定该基因的表达水平、表达产物的可溶性及其相对分子质量,Western-blot分析表达产物的免疫反应性。结果该基因在原核表达系统中经诱导,得到了Mr31000的可溶性融合表达产物。经Ni-NTA纯化,纯度可达90%以上。免疫印迹实验表明该产物能够被弓形虫B36感染的小鼠血清和弓形虫RH感染的兔血清特异识别。结论弓形虫多表位基因在原核中的可溶性表达产物在弓形虫病诊断及疫苗研究中有潜在的应用价值。
Objective To obtain soluble expression product of immunoreactive recombinant multiepitope antigen of Toxoplasma gondii from E.coli. Methods The gene encoding the multiple epitopes (MEG) of Toxoplasma gondii was amplified by PCR from the original plasmid containing MEG gene and cloned into the prokaryotic soluble expression vector pET32a. After identification by enzyme digestion and sequencing, the positive recombinant plasmid pET32a-MEG was transformed into BL21(DE3), which was induced with IPTG for expression of the target antigen. The relative molecular mass, solubility and antigenicity of the expression products were analyzed by SDS-PAGE and Western blotting. Results The recombinant expression plasmid pET32a-MEG was successfully constructed and the highly efficient expression of the antigen was achieved after IPTG induction of E.coli. Improvement of the induction condition increased the expression product which accounted for about 28% of the total bacterial protein. The target protein, with good solubility and a relative molecular mass of about 31 000, was purified by immobilized metal affinity chromatography (Ni-NTA resin) and could be well recognized by mouse and rabbit antisera derived by infection of the animals with Toxoplasma gondii B36 and RH, respectively. Conclusion The recombinant multiepitope antigen has good antigenicity and potential value in diagnosis and vaccine development of toxoplasmosis.
出处
《第一军医大学学报》
CSCD
北大核心
2005年第5期528-530,544,共4页
Journal of First Military Medical University
基金
广东省自然科学基金(20020041)
广州市科技计划项目基金(2002Z-T24011)~~
关键词
弓形虫
多表位
大肠杆菌
蛋白表达
免疫反应性
expression, multiepitope, antigen, Toxoplasma gondii, immunoreactivity
