目的 构建截短型人转化生长因子βⅡ型受体胞外域( thTβRⅡ)原核表达载体,诱导表达纯化thTβRⅡ蛋白,并分析其生物学活性.方法 以本实验室前期构建的TβRⅡ全长基因片段( pGEX-4T1-TβRⅡ)为模版,常规PCR扩增出带有 NdeI和BamHI酶切位点的thTβRⅡ目的基因,并插入原核表达载体pET28a,命名为pET28a-thTβRⅡ.将经过PCR、双酶切和DNA测序正确的pET28a-thTβRⅡ转化至大肠杆菌Rossta诱导表达,用Ni2+亲和层析纯化获得高纯度thTβRⅡ目的蛋白,经SDS-PAGE进行纯度分析,MTT法观察其对转化生长因子-β1(TGF-β1)活化的人肝星状细胞LX-2增殖活性的影响,real-time RT-PCR及Western blot检测其对TGF-β1、纤连蛋白(FN)mRNA及FN、α-平滑肌肌动蛋白(α-SMA)蛋白表达的影响.结果 pET28a -thTβRⅡ原核表达载体构建成功,宿主菌 Rossta/pET28a -thTβRⅡ在37℃经0.8 mmol/L异丙基-β-D-硫代半乳糖苷诱导5 h获得目的蛋白thTβRⅡ的大量表达,目的蛋白主要以包涵体的形式存在,通过Ni2+亲和层析成功获得高纯度thTβRⅡ,SDS-PAGE鉴定分子量约为18.0 kD, MTT显示150 μg/mL的重组蛋白thTβRⅡ能明显抑制活化的人肝星状细胞LX-2增殖,real-time RT-PCR及Western blot显示200 μg/mL的重组蛋白thTβRⅡ其能显著减少TGFβ1、FNmRNA;FN、α-SMA蛋白的表达.结论 成功构建thTβRⅡ原核表达载体,诱导表达纯化并获得高纯度重组蛋白thTβRⅡ,其能抑制活化的LX-2 细胞增殖并具有抗纤维化作用,为进一步体内外功能研究打下基础.%Objective To construct the prokaryotic expression vector containing the extracellular domain of trans -forming growth factor-beta receptor type Ⅱ(thTβRⅡ, so as to evaluate its expression and characteristics .Methods The gene fragment of thTβRⅡfrom recombinant vector pGEX -4T1-TβRⅡwas amplified by regular PCR with specific primers, and cloned into the prokaryotic expression vector pET 28a.The recombinant protein pET28a-thTβRⅡwere in-duced with IPTG, purified with Ni2+-IDA affinity chromatography and characterized with SDS -PAGE and MTT assay. Expression of TGFβ1, FN mRNA, FN and α-SMA were assessed by real -time RT-PCR and Western blotting.Re-sults The PCR product of thTβRⅡdigested with two restriction enzymes (NdeI and BamHI) was cloned into the expres-sion vector pET28a, and confirmed by PCR, restriction endonuclease digestion and DNA sequence .The optimal expres-sion of recombinant protein thT βRⅡwas induced with 0.8 mM IPTG at 37℃for 5 h.The high-level expressed protein in the form of inclusion bodies was refolded by dialysis refolding procedures and purified by Ni 2+-IDA affinity chromatog-raphy, identified by SDS-PAGE.The recombinant protein thTGF -βRⅡ, at concentrations of 150 μg/mL, could signif- icantly inhibit the proliferation of TGF -β1-induced LX-2 cells; and at concentrations of 200 μg/mL, could signifi-cantly reduce the expression level of TGF β1, FN mRNA and FN, α-SMA protein.Conclusion The pET28a-thTβRⅡexpression vector is constructed , and the production of active recombinant protein thT βRⅡcould inhibit the prolifera-tion of TGF-β1-induced LX -2 cells, reduce the mRNA and protein expression levels of fibrosis -related factors, which lays the foundation for exploring the effect of the recombinant protein thT βRⅡin vitro and in vivo.
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