重组人成熟型转化生长因子β_1及其抗原表位在大肠杆菌中的表达、纯化及其免疫原性鉴定

Expression of Recombinant Human Mature TGF-β_1 and Its Antigen Epitopes in E. coli and Purification and Immunogenicity of Expressed Product

目的研制人转化生长因子(TGF)-β1自体蛋白疫苗,并诱导小鼠产生抗人TGF-β1的中和抗体。方法将hTGF-β1及其抗原表位基因β32和β80分别克隆入pGEX-4T-1原核表达载体,并引入T细胞辅助表位(TT),转化大肠杆菌,经IPTG诱导表达。应用GST亲和层析纯化重组融合蛋白,并经Westernb lot鉴定,用流式细胞术(FCM)检测抗hTGF-β1抗血清与人肝癌细胞株SMMC-7721表面膜型TGF-β1的结合状况。用纯化蛋白免疫BALB/c小鼠,通过ELISA检测血清中抗hTGF-β1抗体的效价。结果成功构建了人成熟型TGF-β1及其不同表位基因片段的重组表达质粒pGEX-TT-β32、pGEX-TT-β80和pGEX-TT-hTGF-β1,表达的3种重组蛋白(GST-TT-β32、GST-TT-β80和GST-TT-hTGF-β1)经Western blot鉴定显示,同时具有hT-GF-β1和GST的抗原性。经纯化后免疫BALB/c小鼠,血清抗人TGF-β1抗体的效价均达到1∶104,其中GST-TT-β80的抗体效价最高。FCM显示3种重组蛋白免疫的小鼠抗血清均能与高表达膜型TGF-β1的人SMMC-7721结合,但重组蛋白GST-TT-β80和GST-TT-hTGF-β1免疫组抗血清的结合率明显高于GST-TT-β32组。结论构建的人成熟型TGF-β1及其不同表位片段的不同融合蛋白疫苗均能够诱导BALB/c小鼠产生抗人TGF-β1的中和抗体。

Objective To prepare human transforming growth factor（TGF） -β1 autogenous vaccine and induce neutralizing antibedy against human TGF-β1 in mice. Methods Clone the gene fragments encoding human mature TGF-β1 and its antigen epitopes β32 and β80 into prokaryotic expression vector pGEX-4T-1 ,with a tetanus toxoid epitope introduced, respectively. Transform the constructed recombinant plasmids to E. coli for expression under induction of IPTG. Purify the expressed protein by GST affinity chromatography and identify by Western blot. Immunize BALB/c mice with the purified protein and determine the titers of neutrazlizing antibody against hTGF-β1 in sere by ELISA. Test the binding of antisem against hTGF-β1 to the TGF-β1 on surface membrane of human hepatoma cell SMMC-7721 strain by flow cytometry （ FCM ）. Results Recombinant plasmids pGEX-TF-β32, pGEX-TF-β80 and pGEX-TF-hTGF-β1 were successfully constructed, and recombinant proteins CST-TT-β32, GST-TT-β80 and GST-TT-hTGF-β1 were expressed and showed beth antigenicities of hTGF-β1 and GST as proved by Western blot. All the serum neutralizing antibody titers against human TGF-β1of immunized mice were not less than 1：10 000,of which the titer induced by GST-TT-β80 was the highest. FCM proved that all the three kinds of recombinant proteins bound to the TGF-β1 on surface membrane of SMMC-7721 cells. However,the binding rates of GST-TT- β80 and C, ST-TT-hTGF-β1 were significantly higher than that of GST-TT-β32. Conclusion The expressed fusion proteins induced neu- tralizing antibody against human TGF-β1 in mice and might be the candidates of human TGF-β1 autogenous vaccine.