人源受体相关蛋白在大肠杆菌中表达的优化及纯化  

Research on Optimal Expression and Purification of Human Receptor Associated Protein in E. coli

作　　者：张红[1] 刘志国[2] 屈伸[1] 

机构地区：[1]华中科技大学同济医学院基础医学院生物化学与分子生物学系,武汉430030 [2]武汉工业学院生物与化学工程系,武汉430023

出　　处：《华中科技大学学报（医学版）》2004年第3期265-268,272,共5页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目 (No 39970 30 7)

摘　　要：目的　获得人源受体相关蛋白 (RAP) ,对表达条件进行优化。方法　RAPcDNA被插入到原核表达质粒pT7 PL和pET 2 8a (+)中 ,分别构建成RAP重组表达载体 ,转化两种BL2 1菌株后 ,转化菌用异丙基硫代半乳糖苷 (IPTG)诱导 ,选择 pT7 PL RAP质粒进行表达条件的优化 ,采用镍离子亲和层析纯化所获得的RAP ,通过检测与巨噬细胞的结合能力评估其生物活性。结果　两个重组质粒均获得了RAP的高表达 ,对于pT7 PL RAP质粒 ,RAP在BL2 1(DE3,plysS)菌株中的表达显著高于在BL2 1(DE3)菌株的表达 ;有氯霉素存在时表达高于无氯霉素时 ;诱导表达的RAP大部分为可溶性的 ;镍离子亲和层析树脂纯化所获得的RAP ,可与富含低密度脂蛋白受体(LDLR)家族受体的小鼠巨噬细胞系RAW 2 6 4 7结合。结论　本实验获得了有生物学功能的RAP ,为进一步开发生产此蛋白打下良好的基础。Objective To obtain human receptor associated protein and to optimize its expression conditions. Methods RAP cDNA was inserted into prokaryotic expression vector-pT7-PL and pET-28a (+) to construct two RAP recombinant human RAP expression plasmids. After they were transformed to the BL21 strains, the transformed strains were induced with isopropylthiogalactoside (IPTG). The pT7-PL-RAP was selected for optimal expression, and Ni +-nitrilotriacetic acid (Ni-NTA) affinity chromatogram was applied for purification of RAP. The biological activity of RAP was assessed by detecting its binding ability to phagocytes. Results The high expression of RAP was detected with the two recombinant plasmids: pT7-PL and pET-28a (+). The expression level of pT7-PL-RAP in BL21 (DE3, plysS) strain was significantly higher than in BL21 (DE3) strain. The expression level of RAP in the presence of chloramphenicol was higher than in the absence of chloramphenicol when pT7-PL-RAP in BL21 (DE3, plysS) strain was used for RAP expression. Most of the inducible expressed RAP was soluble. The RAP purified by Ni-NTA resin could strongly bind with the RAW264.7 cell richly containing low density lipoprotein receptor (LDLR) family receptors.Conclusion Functional RAP was obtained through the above strategy. All of these are the basis for the farther production of RAP that is applied as a researchful tool.

关 键 词：RAP 表达条件 受体相关蛋白 PL 巨噬细胞 纯化 亲和层析 原核表达质粒 IPTG 诱导表达 

分 类 号：R392[医药卫生—免疫学] Q786[医药卫生—基础医学]