重组人sCR1真核表达载体的构建、表达及其生物学活性  

Construction expression and biologic activity of recombinant human sCR1 eucaryotic cell

作　　者：王广兰[1] 宫璀璀[1] 王文丽[1] 张长远[1] 刘亚利[1] 何培霞[1] 郑淑娜[1] 陈湘林[1] 

机构地区：[1]解放军第153中心医院济南军区检验中心,河南郑州450042

出　　处：《细胞与分子免疫学杂志》2008年第5期453-456,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：济南军区重点课题资助(02Z27)

摘　　要：目的:采用酵母细胞分泌型载体pPIC9k表达人可溶性补体受体(sCR1),研究重组人sCR1融合蛋白的体外生物学活性。方法:从人外周血中提取总RNA,应用RT-PCR获得人sCR1全长cDNA,然后将其克隆入毕赤酵母细胞分泌型表达载体pPIC9k中,构建含人sCR1的重组质粒(pPIC9k-sCR1),经测序鉴定正确,电转化入毕赤酵母细胞SMD1168中,将经G418抗性筛选出的重组sCR1酵母细胞株进行PCR鉴定,经甲醇诱导,表达产物经SDS-PAGE分析和Western blot鉴定,通过Ni2+-NTA agarose亲和层析纯化后进行生物学活性鉴定。结果:获得毕赤酵母细胞分泌型表达载体pPIC9k-sCR1,经G418筛选及PCR鉴定得到高拷贝整合的重组酵母细胞株,经甲醇诱导含pPIC9k-sCR1的酵母SMD1168细胞表达出重组sCR1融合蛋白。此蛋白在SDS-PAGE上表现为Mr约31000的蛋白区带,在Western blot分析中可被sCR1的CD35单克隆抗体(mAb)识别。经Ni2+-NTA agarose亲和层析纯化后得到较纯的sCR1融合蛋白及较高的生物学活性。结论:人sCR1融合蛋白在酵母细胞表达系统中的高水平表达,并且有与人体天然蛋白相同的抗原性及其生物学活性。AIM： To express human soluble complement receptor type 1 （ sCR1 ） protein using ferment cell secreting type carrier and study the extraorgan biologic activity of recombinant human sCR1 fusion protein. METHODS： Total human RNA was extracted from peripheral blood. The full length cDNA of human sCR1 gene was obtained by RT-PCR and them, cloned into Pichia pastoris eukaryotic expression vector pPIC9k to construct the recombinant plasmid pPIC9k- sCR1 containing human sCR1. After identified by DNA sequencing, the recombinant plasmid pPIC9k-sCR1 was transformed into Pichia pastoris SMDl168. The ferment cell line of the recombinant sCR1 which was chosen by G418 resistanca was identified by PCR, After methanol induction, the expressed protein products were verified by SDS-PAGE and Western blot, purified by Ni^2＋ -NTA agarose affinity chromatography, and its biologic activity was identified. RESULTS： The obtained Pichia pastoris secretion type yeast carrier pPIC9k-sCR1 was chosen by G418 and identified by PCR to get a highly copied and integral recombinant ferment cell line. The recombinant human sCR1 fusion protein was ex- pressed by yeast cells containing pPIC9k-sCR1 induced by methanol. It was a protein band about Mr 31 000 in gel, which could be identified by CD35 of anti-sCR1 protein monoclonal antibody with Western blotting technique. The highly purified sCR1 fusion protein and its biologic activity were detected obtained by Ni^2＋ -NTA agarose affinity chromatography. CONCLUSION： The recombinant human sCR1 fusion protein can be highly expressed in the Pichia pastods expression system, which resembles the human natural proteinS antigenicity and biologic activity.

关 键 词：可溶性补体受体1 毕赤酵母细胞 生物学活性 真核表达载体 

分 类 号：R392.11[医药卫生—免疫学]