人补体受体1型衍生分子融合基因CR1-SCR2D的构建、表达及生物学活性鉴定

Construction,expression,and biologic activity analysis of fusion gene of human complement receptor type 1 derivative CR1-SCR2D

目的构建和表达具有抑制补体生物活性的双功能域人补体受体1型衍生分子CR1-SCR2D。方法基于我室前期工作,将CR1-SCR1-3、人工α螺旋肽段linker、SCR15-17 3个DNA片段依次克隆到改造载体pPIC9k(+)和pPICZαB,构建出表达质粒pPICZαB-CR1-SCR2D;电转化毕赤酵母X-33得到阳性重组子,甲醇诱导目的蛋白CR1-SCR2D表达,SDS-PAGE和Western blot鉴定表达结果;经纯化获得CR1-SCR2D并进行糖基化鉴定后,用CH50和AH50法鉴定目的蛋白在体外抑制补体的生物活性。结果成功构建了目的基因的表达质粒pPICZαB-CR1-SCR2D,基因测序正确;SDS-PAGE和Western blot证实目的基因在毕赤酵母中实现了分泌表达;经镍柱纯化后得到较纯的CR1-SCR2D,糖基化鉴定证明其存在糖基化;CH50法和AH50法结果显示CR1-SCR2D均有较CR1-SCR1-3和CR1-SCR15-17更好的抑制补体生物学活性。结论成功构建了双功能域人补体受体1型衍生分子融合基因CR1-SCR2D,在毕赤酵母中实现了CR1-SCR2D的分泌表达,该融合蛋白对经典/MBL途径及旁路途径补体激活均具有较好的抑制作用。

This study performed to construct and express human complement receptor type 1 derivative CR1- SCR2D, which contains two functional domains that can inhibit complement activity. Based on previous study, PCR fragments of SCR1-3 and SCR15-17 were cloned into transformed plasmid pPIC9k（＋） and an artificial helical peptides linker was inserted between them. The fusion gene was then cloned into pPICZotB to build an expression plasmid pPICZaB-CR1-SCR2D. After identification by PCR, restriction enzymes digestion and DNA sequence, expression plasmid was electrotransported into Pichia pastoris X-33, and then positive recombinants were identified by PCR and induced in shake flask by methanol. Target protein in the supematant was identified by SDS-PAGE and Western-blot, and then purified by Ni-NTA agarose metal-chelate-affinity chromatography. The glycosylation of CR1-SCR2D was also identified. CHS0 and AHS0 inhibition assays showed that CR1-SCR2D had stronger inhibitory activity as compared with CR1-SCR1-3 and CR1-SCR15-17. All the result indicated that CR1-SCR2D is successfully constructed and expressed by Pichia pastoris, which demonstrated stronger inhibitory activity in both complement classical and alternative pathways.