[日本血吸虫膜蛋白四跨膜蛋白2-A的克隆、表达及鉴定]

[Cloning, expression and identification of membrane protein Tetraspanin 2-A of Schistosoma japonicum].

作者信息

Wang Yang-Yang, Liu Miao, Zhu Shao-Chun, Ren Cui-Ping, Shen Ji-Jia

机构信息

Department of Microbiology and Parasitology, Anhui Medical University, Hefei 230032, China.

出版信息

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2008 Feb 28;26(1):21-4.

PMID:18637579

Abstract

OBJECTIVE

To clone and express a membrane protein (Tetraspanin 2) gene of Schistosoma japonicum (SjTsp2).

METHODS

A pair of primers was designed to amplify the SjTsp2 gene which was subcloned into prokaryotic plasmid pET28a(+). The recombinant plasmid was transformed into E. coli BL21(D3) and followed by expression of the protein induced by IPTG. The protein was purified by affinity chromatography and used to immunize BALB/c mice. Dilution of antibody against SjTsp2 was determined by ELISA. The protein was also identified by Western blotting.

RESULTS

Big loop of SjTsp2-A, 228 bp, was amplified in vitro by PCR. Its deduced amino sequence shared 52% similarity with SmTsp2. The soluble recombinant SjTsp2-A was expressed in the experiment and high dilution antibody against the recombinant (1:32,000 in maximum) was produced in immunized mice. SjTsp2-A reacted positively with sera of acute and chronic schistosomiasis patients but not with sera from healthy persons by Western blotting.

CONCLUSION

SjTsp2 has been expressed and shows certain antigenicity.

摘要

目的

克隆并表达日本血吸虫的一种膜蛋白（四跨膜蛋白2，SjTsp2）基因。

方法

设计一对引物扩增SjTsp2基因，将其亚克隆至原核质粒pET28a(+)。重组质粒转化至大肠杆菌BL21(D3)，经异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达蛋白。通过亲和层析纯化蛋白并用于免疫BALB/c小鼠。采用酶联免疫吸附测定（ELISA）法测定抗SjTsp2抗体的稀释度。同时通过蛋白质印迹法对该蛋白进行鉴定。

结果

通过聚合酶链反应（PCR）体外扩增出大小为228 bp的SjTsp2-A大环状片段。其推导的氨基酸序列与曼氏血吸虫Tsp2（SmTsp2）有52%的相似性。实验中表达出可溶性重组SjTsp2-A，免疫小鼠后产生了高稀释度的抗重组蛋白抗体（最高可达1:32,000）。蛋白质印迹法显示，SjTsp2-A与急性和慢性血吸虫病患者血清反应呈阳性，而与健康人血清无反应。