结缔组织生长因子CT结构域的原核表达及分离纯化

摘要

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.%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.