摘要
CDPK是一类钙依赖蛋白激酶,其可参与调控寄生虫入侵,外出宿主细胞,配子的形成及在宿主体内移行等。采用RT-PCR技术,扩增弓形虫RH株目的基因钙依赖蛋白激酶EF模体(CDPK1-EF),然后将其克隆至表达载体pET-32a,以构建重组质粒ET-32a-CDPK1-EF,经IPTG诱导目的蛋白表达,并采用Ni-NTA亲和层析法纯化,最后经SDS-PAGE分析重组表达蛋白。结果,成功地克隆出弓形虫CDPK1-EF基因,序列比对表明,其与GenBank报道的相关序列同源性达100%。采用SDS-PAGE技术对诱导表达蛋白以及纯化产物检测发现37 ku目的蛋白出现,与预期结果相一致。本文在国内首次成功克隆和表达弓形虫RH株CDPK1-EF基因,这为研究该基因功能及分布特征等提供了基础。
CDPK is one of the family of calcium-dependent and non-CAM-dependent protein kinases, which involves in many critical e- vents in parasite, including invasion in host cell and egress, gamete formation and gliding molitity. In this study, the CDPK1-EF gene from Toxoplasma gondii RH strain was amplified by RT-PCR, and the purified product was cloned into the plasmid of pET32a and transformed in- to Escherichia coli to construct the recombinant plasmid pET32a-CDPK1-EF. The expression of recombinant protein was induced by IPTG and the target protein was purified through Ni-NTA affinity chromatography. The results showed that the target gene fragment was successful- ly obtained, and the sequencing analysis showed 100% of identity with the published in GenBank. SDS-PAGE analysis indicated that the size of recombinant protein was about 37 ku as expected. It suggests that the CDPK1-EF gene from T. gondii RH strain has been successfully cloned and expressed, which will be helpful to understand the function of CDPK.
出处
《畜牧与兽医》
北大核心
2014年第2期5-9,共5页
Animal Husbandry & Veterinary Medicine
基金
中国农科院兰州兽医研究所家畜疫病病原生物学国家重点实验室开放基金(sICLVEB20.12KFKTO02)
安徽省生猪产业技术体系项目
