结缔组织生长因子CT结构域的原核表达及分离纯化  

Prokaryotic expression and purification of connective tissue growth factor(CT domain) in E.coli

作　　者：薛秀蕾[1] 范小波[1] 吴国球[2] XUE Xiu-lei;FAN Xiao-bo;WU Guo-qiu(Medical School of Southeat University,Nanjing 210009, China;ZhongdaHospital,Southeast University,Nanjing 210009,China)

机构地区：[1]东南大学医学院,江苏南京210009 [2]东南大学附属中大医院,江苏南京210009

出　　处：《东南大学学报（医学版）》2017年第2期230-234,共5页Journal of Southeast University(Medical Science Edition)

摘　　要：目的:构建pET32a-TrxA-His-CTGF-CT原核表达载体,实现重组CTGF-CT的可溶性表达及分离纯化,以用于制备CTGF-CT抗体。方法:基因合成CTGF-CT序列,利用NcoI和XhoI限制性内切酶双酶切后插入pET32a载体,构建pET32a-TrxA-His-CTGF-CT重组质粒,阳性质粒转化E.coli strain BL21(DE3)细胞,通过异丙基β-D-硫代半乳糖苷(IPTG)诱导获得重组融合蛋白,利用镍柱亲和纯化及肠激酶酶切纯化获得目的蛋白CTGF-CT,SDS-PAGE鉴定蛋白纯度并进行Western blotting分析。结果:所构建pET32a-TrxA-His-CTGF-CT重组质粒验证正确。经诱导表达获得了与预期分子量(28 kD)一致的融合蛋白,融合蛋白经肠激酶切割及两次镍柱亲和纯化获得目的蛋白CTGF-CT,纯化后纯度约95%,Western blotting分析表明纯化目的蛋白为CTGF-CT。结论:通过构建原核pET32a-TrxA-His-CTGF-CT表达载体实现了重组CTGF-CT蛋白的表达及分离纯化,为CTGF-CT抗体的制备和CTGF-CT功能的深入研究打下了基础。O bjective:To construct the recombinant pET32a_TrxA-His-CTGF-CT plasmid and to express and purifyCT domain of connective tissue growtli factor(CTGF)in order to prepare CTGF-CT polyclonal antibody.M ethods:The nucleotide sequence encoding CTGF-CT was chemically synthesized and inserted(+)vector.The constructed pET32a_TrxA_His-CTGF-CT plasmid was transformed into E.coli strain BL21(DE3).And the TrxA-CTGF-CT fusion protein was induced expression by IPTG;Then CTGF-CT was purified by enterokinasecleavage and Ni_NTA purification and was identified by SDS-PAGE and Western blotting.R esults%The authenticityof tlie recombinant pET32a T rx A_CTGF_CT-His plasmid was verified by DNA sequencing and it was identifiedcontainning a400bp fragment by Ncol and Xhol double digestion.The molecular weightwas about28kD after IPTG induced expression.After enterokinase cleavage and Ni-NTA purification,the was obtained as expected molecular weight of14kD.Western blotting proved that tlie protein was CTGF-CT.The purity of tlie recombinant CTGF-CT was about95%.C on clu sion:The construction of pE T32aT rxA-H is-CTGF-CT and the expression and purification of CTGF-CT are successfully achieved.That will preparation of CTGF-CT antibody and the further study on the function of CTGF-CT.

关 键 词：结缔组织生长因子 原核表达 蛋白纯化 

分 类 号：R-33[医药卫生] R393