【摘要】 [目的]克隆周期型马来丝虫半胱氨酸蛋白酶(cysteine protease of periodic Brugiamalayi,BmCP)基因到原核载体中,测定其序列,为进一步的研究奠定基础。[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法体外扩增出BmCP基因序列,扩增产物经初步鉴定后将其克隆入pMD18-T载体,转化大肠杆菌(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pMD18-T-BmCP,经测序验证,并进行同源性比较。[结果]RT-PCR扩增出一条约1201bp大小的特异性条带,重组质粒双酶切和以质粒为模板的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%。[结论]成功构建了周期型马来丝虫半胱氨酸蛋白酶重组质粒pMD18-T克隆载体,为进一步研究该基因的功能提供了条件。更多还原

【Abstract】 [Objective]To clone and sequence the cysteine protease gene of periodic Brugia malayi(BmCP)for immunological prevention.[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 gene from B. malayi. The desired gene was amplified by PCR technique from cDNA. The PCR products were purified and cloned into plasmid pMD18-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.[Results]For RT-PCR,a specific band of around 1 201 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 BmCP shared 99% nucleotide sequence identity with that of published sequence.[Conclusion]pMD18-BmCP is successfully constructed and it provide the basis for further study on expression of BmCP protein and its function.更多还原