周期型马来丝虫CPI基因克隆和序列分析及编码产物B细胞表位预测  

Cloning, sequencing of CPI gene from periodic Brugia malayi and prediction of B cell epitopes in its amino acid sequence

作　　者：童海燕[1] 方政[1] 张赛楠[1] 徐邦生[1] 方浩[1] 黄为群[1] 谢东方[1] 石佑琴[1] 

机构地区：[1]江苏省南通大学医学院寄生虫学教研室,226001

出　　处：《中国地方病学杂志》2010年第5期515-518,共4页Chinese Jouranl of Endemiology

基　　金：江苏省教育厅自然科学研究项目（04KJD310136）;南通市应用研究计划（K2009027）

摘　　要：目的 克隆周期型马来丝虫半胱氨酸蛋白酶抑制剂（BmCPI）基因,并通过序列测定、分析及编码产物的B细胞表位预测,为进一步研究该基因的功能奠定基础.方法 从周期型马来丝虫虫体中抽提总RNA,以mRNA为模板,采用RT-PCR法体外扩增BmCPI基因,扩增产物经初步鉴定后将其克隆入pGEM-T载体,转化大肠埃希菌（E.coli）DH5α,筛选阳性克隆,进行双酶切及PCR扩增鉴定,获得阳性重组质粒pGEM-TBmCPI,经测序验证,并进行同源性比较.应用5种参数和方法对其编码产物进行B细胞表位预测.结果 RTPCR扩增出一条约621 bp大小的特异性条带,重组质粒双酶切的PCR结果与预期相符,DNA序列分析与GeneBank已知的基因序列同源性为99%.其编码产物经表位预测分析,B细胞表位可能在23～32、50～79、117～126位氨基酸区域.结论 成功构建了周期型马来丝虫半胱氨酸蛋白酶抑制剂重组质粒pGEM-T克隆载体,并进行了序列测定及编码产物的B细胞表位预测,达到预期目标,为进一步研究该基因的功能提供条件.Objective To clone and sequence the cysteine protease inhibitor gene of periodic Brugia malayi（BmCPI） and predict B-cell epitopes in amino acide sequence of BmCPI in order to provide basis for further study the expression of BmCPI and its function. Methods Total RNA was extracted from periodic Brugia malayi.A couple of specific primers were designed on the basis of known sequences of cysteine protease inhibitor gene from BmCPI. The desired gene was amplified by PCR technique from cDNA. The PCR products were purified and cloned into plasmid pGEM-T by T-A cloning method, transformed into Escherichia coli（E, coli） strain DH5α. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification. Five parameters and methods were used to predict B-cell epitopes in amino acide sequence of BmCPI. Results For RT-PCR, a specific band of around 621 bp was amplified. The same band was obtained by double restriction of recombinant plasmids or PCR using recombinant plasmid as template. The result of DNA sequencing showed that BmCPI shares 99% nucleotide sequence identity with that of published sequence. It showed that B-cell epitopes were probably at or adjacent to 23 - 32, 50 - 79 and 117 - 126 in its amino acide sequence. Conclusions pGEM-BmCPI is successfully constructed and sequenced, anticipated objective is reached and conditions is provided for further study of BmCPI expression and its function.

关 键 词：半胱氨酸蛋白酶抑制剂 基因 序列分析 丝虫病 B细胞表位 

分 类 号：R686[医药卫生—骨科学]