新型分泌蛋白VSTM1-v2高效稳定表达株的构建及纯化
刘绘绘1，李婷1，惠觅宙2，陈英玉1，韩文玲1
基金项目：高等学校博士学科点专项科研基金(20110001110016)；重大新药创制基金（2011ZX09506-005） 作者简介：刘绘绘（1989-），女，在读博士生，主要研究方向细胞因子和免疫细胞膜分子 通信联系人：李婷（1980-），女，中级，主要研究方向细胞因子和免疫细胞膜分子.

（1. 北京大学基础医学院免疫学系，卫生部医学免疫学重点实验室，北京大学人类疾病基5 因研究中心，北京100191； 2. 杭州安瑞普生物制品研究有限公司，杭州 310020）
摘要：VSTM1-v2是通过免疫基因组学策略发现的潜在新细胞因子，为一经典分泌蛋白，其主要表达于免疫系统，重组VSTM1-v2蛋白能促进Th17细胞分化和活化。为了获得大量重组人VSTM1-v2蛋白进行功能实验，本文利用重叠PCR方法将小鼠IgGκ信号肽序列拼接到VSTM1-v2成熟蛋白编码序列的N端，并克隆到pcDNA3.1/myc-His(-)B表达载体中。瞬时转染发现置换信号肽后VSTM1-v2表达量显著提高。然后将IgGκ信号肽-VSTM1-v2-myc-His的序列亚克隆到高效真核表达载体pMH3中，命名为pMH3-IgGκSP-VSTM1-v2。利用电击转染该质粒到CHO细胞，G418筛选阳性单克隆，通过Western blot1筛选鉴定其表达。经过两轮加压筛选，获得5株高效稳定表达重组VSTM1-v2蛋白的细胞株。挑选三株悬浮驯化后进行30ml小量表达及纯化，得到表达量高且生长状态最好的细胞株，将此细胞株扩大到1L，连续流加培养，亲和纯化后得到7mg纯度为90%的蛋白。结论：置换VSTM1-v2的信号肽后，其表达量显著提高；成功建立了稳定、高效表达重组人VSTM1-v2蛋白的CHO细胞株，并对其1L培养上清进行了纯化，得到7mg高纯度重组蛋白。
关键词：免疫学；细胞因子；VSTM1-v2；高效稳定表达；CHO细胞。
中图分类号：R392
Stable high-level expression and purification of VSTM1-v2, 25 a novel secretory protein
LIU Huihui1, LI Ting1, HUI Mizhou2, CHEN Yingyu1, HAN Wenling1
(1. Department of Immunology，School of Basic Medical Sciences，Key Laboratory of Medical Immunology， Ministry of Health，Center for Human Disease Genomics，Peking University，Beijing 100191，China; 2. Hangzhou amprotein biological product Co. Ltd. Hangzhou 310020，China)
Abstract: VSTM1-v2 is a classical secretory protein and a potential novel cytokine, which was identified by the strategy of Immunogenomics. VSTM1-v2 is mainly expressed in immune system, and recombinant human VSTM1-v2 can promote the differentiation and activation of Th17 cells. To obtain a large amount of rhVSTM1-v2 for functional studies, the cDNA fragment encoding  mouse IgGκ signal peptide was ligated to the N terminus of the cDNA fragment encoding mature VSTM1-v2 through overlapping PCR, and this fused fragment was subcloned into pcDNA3.1/myc-His(-)B vector. IgGκ signal peptide replacement significantly increased VSTM1-v2 expression and secretion after transient transfection. Then IgGκSP-VSTM1-v2-myc-His fragment was subcloned into a high efficiency eukaryotic  expression vector pMH3 (named pMH3-IgGκ-VSTM1-v2). The constructed plasmid was transfected into CHO cells with electroporation and the secretion of VSTM1-v2 in G418-resistant clones was analyzed by Western blot. Five cell clones with stable high-expression level were obtained after two rounds of selection. Three higher-expression clones were acclimated for suspension culture. The highest-expression cell strain was selected, cultured in 30ml serum-free