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日本血吸虫10.6 kDa膜蛋白基因的克隆及表达
引用本文:沈际佳,蒋作君,余新炳,汪学龙,王维.日本血吸虫10.6 kDa膜蛋白基因的克隆及表达[J].中国寄生虫学与寄生虫病杂志,2001,19(3):157-159.
作者姓名:沈际佳  蒋作君  余新炳  汪学龙  王维
作者单位:1. 安徽医科大学寄生虫学教研室,
2. 中山医科大学寄生虫学教研室,
基金项目:国家自然科学基金(No.39570646)、安徽省科委(0022031)和安徽省教育厅(99j101112)科研基金资助项目
摘    要:目的 寻找新的预防日本血吸虫感染疫苗候选分子。 方法 用具保护性的抗血吸虫膜单抗及免疫血清筛选日本血吸虫 c DNA文库 ,PCR扩增 c DNA插入片段 ,A - T连接法将筛选获得的 3个基因片段克隆到p GEM- T载体上 ,自动测序仪测序 ,序列送 BL AST基因服务站进行同源性分析。重新设计引物扩增编号为 B8克隆的开放阅读框碱基序列 ,先将其克隆入 p GEM- T载体质粒 ,再定向亚克隆入 p BK- CMV表达质粒 ,IPTG诱导表达后用 SDS- PAGE和 Western blotting分析表达产物。 结果 经双酶切及 PCR法均证实基因重组成功。其特异性表达产物约为 10 .6 k Da蛋白抗原。表达产物可被免疫血清及单抗识别。 结论 构建了编码日本血吸虫 10 .6k Da蛋白基因表达克隆。

关 键 词:日本血吸虫  cDNA  基因克隆  表达  疫苗
文章编号:1000-7423(2001)-03-0157-03
修稿时间:2000年5月29日

Cloning and Identification of an Unknown Gene Encoding 10.6 kDa Protein of Schistosoma japonicum
SHEN Ji-jia,JIANG Zuo-jun,YU Xin-bing,WANG Xue-long,WANG Wei.Cloning and Identification of an Unknown Gene Encoding 10.6 kDa Protein of Schistosoma japonicum[J].Chinese Journal of Parasitology and Parasitic Diseases,2001,19(3):157-159.
Authors:SHEN Ji-jia  JIANG Zuo-jun  YU Xin-bing  WANG Xue-long  WANG Wei
Institution:Department of Parasitology, Anhui Medical University, Hefei 230032.
Abstract:OBJECTIVE: To screen a new schistosome vaccine candidate. METHODS: Schistosoma japonicum adult cDNA library was screened using sera from immune rabbits vaccinated with irradiated cercariae and monoclonal antibodies against membrane antigen of S. japonicum schistosomula. Three different fragments of S. japonicum cDNA genes were cloned into pGEM-T vector. The sequences of the inserts were determined using an automatic DNA sequencer and were analysed using Blast program. One of the unknown genes (B8) was selected and its ORF sequence (291 bp) was subcloned into eukaryotic expression vector. The recombinant plasmids were identified by restrictive enzymes and PCR amplification. The positive recombinant plasmids (pBK/SjB8) were transformed into host bacteria XL1-blue, and were then induced by IPTG for expression. SDS-PAGE and Western blotting analysis of total cellular protein from the bacteria were performed to detect the gene products. RESULTS: The results demonstrated that ORF of SjB8 gene was subcloned into the plasmid pBK-CMV and could express as fusion protein in XL1-blue. The results of SDS-PAGE and Western-blot also showed that the molecular weight of the fusion protein with 3 kDa beta-galactosidase was approximately 13.6 kDa and the actual molecular weights of the SjB8 was 10.6 kDa. The expressed fusion product of pBK/Sj-B8 could be recognized by immune serum and McAb. CONCLUSION: A new gene of S. japonicum vaccine candidate (SjB8) was cloned into eukaryotic expression vector pBK-CMV and could express 10.6 kDa schistosome protein. The results provide foundation for further study of the protein for its possibility as candidate vaccine.
Keywords:Schistosoma japonicum  cDNA    gene clone    expression  vaccine
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