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

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。

Expression of Recombinant Human Mature TGF-β1 and Its Antigen Epitopes in E. coli and Purification and Immunogenicity of Expressed Product

Objective To prepare human transforming growth factor(TGF)-β_1 autogenous vaccine and induce neutralizing antibody against human TGF-β_1 in mice.Methods Clone the gene fragments encoding human mature TGF-β_1 and its antigen epitopes β_(32) and β_(80) 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-β_1 in sera by ELISA.Test the binding of antisera against hTGF-β_1 to the TGF-β_1 on surface membrane of human hepatoma cell SMMC-7721 strain by flow cytometry(FCM).Results Recombinant plasmids pGEX-TT-β_(32),pGEX-TT-β_(80) and pGEX-TT-hTGF-β_1 were successfully constructed,and recombinant proteins GST-TT-β_(32),GST-TT-β_(80) and GST-TT-hTGF-β_1 were expressed and showed both antigenicities of hTGF-β_1 and GST as proved by Western blot.All the serum neutralizing antibody titers against human TGF-β_1 of immunized mice were not less than 1∶10 000,of which the titer induced by GST-TT-β_(80) was the highest.FCM proved that all the three kinds of recombinant proteins bound to the TGF-β_1 on surface membrane of SMMC-7721 cells.However,the binding rates of GST-TT-β_(80) and GST-TT-hTGF-β_1 were significantly higher than that of GST-TT-β_(32).Conclusion The expressed fusion proteins induced neutralizing antibody against human TGFβ_1 in mice and might be the candidates of human TGF-β_1 autogenous vaccine.