具有免疫反应性的弓形虫多表位抗原在大肠杆菌中的可溶性表达和鉴定

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (05): 528-530,544.

具有免疫反应性的弓形虫多表位抗原在大肠杆菌中的可溶性表达和鉴定

杨培梁¹, 陈晓光², 王元占¹, 李华², 周晓红², 吴焜², 言慧²

1. 南方医科大学南方医院实验动物研究中心, 广东, 广州, 510515;
2. 南方医科大学寄生虫学教研室, 广东, 广州, 510515

出版日期:2005-05-20 发布日期:2005-05-20
基金资助:
收稿日期:2004-8-15。
基金项目:广东省自然科学基金(20020041);广州市科技计划项目基金(2002Z-T24011)
作者简介:杨培梁(1972- ),男,硕士,助理研究员,电话:020-61641037,E-mail:plyang927@hotmail.com

Soluble expression and characterization of the immunoreactive multiepitope antigen of Toxoplasma gondii in E.coli

YANG Pei-liang¹, CHEN Xiao-guang², WANG Yuan-zhan¹, LI Hua², ZHOU Xiao-hong², WU Kun², YAN Hui²

1. 南方医科大学南方医院实验动物研究中心, 广东, 广州, 510515;
2. 南方医科大学寄生虫学教研室, 广东, 广州, 510515

Online:2005-05-20 Published:2005-05-20

摘要/Abstract

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pET2a相匹配的酶切位点,插入载体pET2a,酶切并测序鉴定后,转入大肠杆菌BL21(DE),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M_r1000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B6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.

Abstract: 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.

中图分类号:

Q785
R382.5

杨培梁¹, 陈晓光², 王元占¹, 李华², 周晓红², 吴焜², 言慧². 具有免疫反应性的弓形虫多表位抗原在大肠杆菌中的可溶性表达和鉴定[J]. 南方医科大学学报, 2005, 25(05): 528-530,544.

YANG Pei-liang¹, CHEN Xiao-guang², WANG Yuan-zhan¹, LI Hua², ZHOU Xiao-hong², WU Kun², YAN Hui². Soluble expression and characterization of the immunoreactive multiepitope antigen of Toxoplasma gondii in E.coli[J]. Journal of Southern Medical University, 2005, 25(05): 528-530,544.

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