人补体受体1型SCR1-3功能域基因的克隆表达及生物活性鉴定

Cloning,expression of the SCR1-3 domains of human complement receptor type 1 and analysis of its activities

目的实现人补体受体1型功能域SCR1-3基因的克隆、表达及生物学活性鉴定。方法从人外周血中提取总RNA,通过逆转录获得cDNA,PCR扩增获得编码CR1-SCR1-3基因序列;克隆到毕赤酵母分泌表达质粒pPIC9K,构建重组质粒pPIC9K-CR1-SCR1-3,菌落PCR、双酶切鉴定后测序;线性化重组质粒电转化毕赤酵母KM71基因组中,经菌落PCR技术筛选在G418平板上生长的多拷贝阳性转化子;摇瓶发酵和甲醇诱导,SDS-PAGE和Western-blot鉴定目的蛋白表达;镍柱亲和层析纯化目的蛋白;用体外抑制补体溶血反应实验测定目的蛋白的生物活性。结果获得了人CR1-SCR1-3编码区序列,测序结果与与GenBank中的相应序列一致;SDS-PAGE和Western-blot表明目的基因在毕赤酵母中实现了分泌表达;体外实验证实经纯化后的CR1-SCR1-3能够明显抑制补体溶血。结论成功构建重组表达质粒pPIC9K-CR1-SCR1-3,在毕赤酵母中实现了CR1-SCR1-3的分泌表达,该蛋白具有较高的抑制补体生物活性。

We aim to clone and express the first three short consensus repeat modules of human complement receptor type 1 in Pichia pastoris,and characterize its bioactivities.Firstly,the total RNA was prepared from human peripheral blood,and the gene fragment of CR1-SCR1-3 was amplified by RT-PCR,then the fragment was cloned into Pichia pastoris secretory expression vector pPIC9K to construct pPIC9K-CR1-SCR1-3.After identification by colony PCR,restriction enzyme digestion and DNA sequence,the linearized plasmid was electrotransported into the Pichia pastoris.Positive clones were selected at different concentration of G418 in plates of yeast extract peptone Dextrose medium（YPD） and identified by colony direct PCR.Then positive recombinants were fermented in shake flask induced by methanol.SDS-PAGE and Western-Blot indicated that the target protein was secreted in the supernatant of culture medium.NI-NTA agarose metal-chelate-affinity chromatography was used for protein purification.Then SRBC haemolytic assay in vitro demonstrated that the purified protein could inhibit complement hemolysis in vitro.All the results show that the recombinant plasmid pPIC9K-CR1-SCR1-3 is successfully constructed,and the CR1-SCR1-3 protein could be highly expressed in the Pichia pastoris,which is proved to possess well complement control activity.