结缔组织生长因子CT结构域的原核表达及分离纯化-东南大学学报医学版

结缔组织生长因子CT结构域的原核表达及分离纯化

单位：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

分类号：R-33;R393

出版年·卷·期（页码）：2017·36·第二期（230-234）

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。

Objective: To construct the recombinant pET32a-TrxA-His-CTGF-CT plasmid and to express and purify CT domain of connective tissue growth factor(CTGF) in order to prepare CTGF-CT polyclonal antibody. Methods: The nucleotide sequence encoding CTGF-CT was chemically synthesized and inserted into the pET-32a(+) 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 enterokinase cleavage and Ni-NTA purification and was identified by SDS-PAGE and Western blotting. Results: The authenticity of the recombinant pET32a-TrxA-CTGF-CT-His plasmid was verified by DNA sequencing and it was identified containning a 400 bp fragment by NcoI and XhoI double digestion. The molecular weight of TrxA-CTGF-CT was about 28 kD after IPTG induced expression. After enterokinase cleavage and Ni-NTA purification, the CTGF-CT was obtained as expected molecular weight of 14 kD. Western blotting proved that the protein was CTGF-CT. The purity of the recombinant CTGF-CT was about 95%. Conclusion: The construction of pET32a-TrxA-His-CTGF-CT and the expression and purification of CTGF-CT are successfully achieved. That will lay the foundations for preparation of CTGF-CT antibody and the further study on the function of CTGF-CT.

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