热带病与寄生虫学

热带病与寄生虫学 ›› 2013, Vol. 11 ›› Issue (2): 77-79.doi: 10.3969/j.issn.1672-2302.2011.01.005

• 论著 • 上一篇    下一篇

日本血吸虫四跨膜蛋白6编码基因的克隆、表达与鉴定

叶芳   汪玉玲   王宏志   汪学龙*   

  1. 安徽医科大学病原生物学教研室（叶芳、汪玉玲、王宏志、汪学龙），中科院合肥研究院医学物理中心检验科（叶芳），*通讯作者
  • 收稿日期:2013-02-21 出版日期:2013-02-25 发布日期:2013-06-08

Molecular cloning, expression and identification of encoding Sj-Tsp6 gene from Schistosoma japonicum

Ye Fang, Wang Yuling, Wang Hongzhi, Wang Xuelong*.   

  1. Department of Parasitology, Anhui Medical University, Hefei 230032, China. * Corresponding author.
  • Received:2013-02-21 Online:2013-02-25 Published:2013-06-08

摘要： 目的 为了寻找血吸虫病新的免疫学诊断候选分子，在大肠杆菌中高效表达日本血吸虫Sj-Tsp6样融合蛋白。 方法 设计特异性引物，PCR法扩增出日本血吸虫Sj-Tsp6样蛋白基因编码基因序列，T载体连接，再将其亚克隆入pET28a(+)表达载体，经酶切、测序证实正确后，转化入大肠杆菌BL21中，并用IPTG对该重组菌诱导表达，用SDS-PAGE和Western blot方法观察表达结果。 结果 PCR法扩增出一条大小约为702bp大小的Sj-Tsp6样蛋白的DNA片断，将目的基因转入表达质粒pET28a(+)，经SDS-PAGE可见一条约28kDa大小的带有HIS标签的融合蛋白条带，此蛋白可被血吸虫病兔血清所识别。 结论 本实验成功克隆日本血吸虫Sj-Tsp6 样抗原的编码基因，并在原核细胞中进行表达，为进一步验证其免疫诊断价值奠定了基础。

关键词: 日本血吸虫, Sj-Tsp6样蛋白, 克隆, 表达

Abstract:

Objective To discover new candidate diagnosis molecules for the diagnosis of schistosomiasis, the Sj-Tsp6 protein gene of Schistosoma japonicum was cloned and expressed in the prokaryotic cell. Methods The specific primers were designed and synthesized. The target gene was amplified by PCR. The product from PCR was cloned into PMD-T vector, and then subcloned into the expression vector pET28a (+). After induced by IPTG, the cells were collected to analyze by SDS-PAGE and Western blot. Results A specific band of around 702 bp was amplified by PCR. A band of 28kDa fusion protein with the label of HIS was revealed by SDS-PAGE after the target gene subcloned into the expression vector pET28a (+). The fusion protein could be recognized by the sera of rabbit infected with schistosomiasis. Conclusion The cDNA encoding sj-Tsp6 protein of S. japanicum cloned and expressed in the prokaryotic cell provided basis for further study on value of SjTsp6 immunological diagnosis.

Key words: Schistosoma japonicum, Sj-Tsp6 protein, Gene cloning, Gene expression