CAJ | 학술논문

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.
목적 극륭주기형마래사충반광안산단백매억제제(BmCPI)기인,병통과서렬측정、분석급편마산물적B세포표위예측,위진일보연구해기인적공능전정기출.방법 종주기형마래사충충체중추제총RNA,이mRNA위모판,채용RT-PCR법체외확증BmCPI기인,확증산물경초보감정후장기극륭입pGEM-T재체,전화대장애희균(E.coli)DH5α,사선양성극륭,진행쌍매절급PCR확증감정,획득양성중조질립pGEM-TBmCPI,경측서험증,병진행동원성비교.응용5충삼수화방법대기편마산물진행B세포표위예측.결과 RTPCR확증출일조약621 bp대소적특이성조대,중조질립쌍매절적PCR결과여예기상부,DNA서렬분석여GeneBank이지적기인서렬동원성위99%.기편마산물경표위예측분석,B세포표위가능재23～32、50～79、117～126위안기산구역.결론 성공구건료주기형마래사충반광안산단백매억제제중조질립pGEM-T극륭재체,병진행료서렬측정급편마산물적B세포표위예측,체도예기목표,위진일보연구해기인적공능제공조건.
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.