树鼩干细胞生长因子的原核表达、纯化及免疫原性

Prokaryotic expression,purification and immunogenicity of tree shrew stem cell factor

目的原核表达、纯化树鼩干细胞生长因子(stem cell factor,SCF),并检测其免疫原性。方法根据GenBank预测的树鼩SCF基因序列设计引物,PCR扩增SCF基因,克隆至原核表达载体p ET-30a(+)中,构建重组表达质粒pET-30a(+)-SCF,转化大肠埃希菌BL21(DE3),IPTG诱导表达。表达的融合蛋白经Ni柱纯化后,免疫家兔,Western blot和ELISA法检测其免疫原性,并检测SCF在树鼩不同组织中的分布情况。结果重组表达质粒pET-30a(+)-SCF经酶切及测序证实构建正确;表达的重组蛋白相对分子质量约为34 500,表达量约占菌体总蛋白的35. 6%,以包涵体形式存在;纯化后的重组蛋白纯度达90%以上,可与兔抗人SCF多克隆抗体特异性结合,且免疫原性良好,抗血清效价为1∶256 000。在鼻、气管、肺、脾、肾、食管中能检测到SCF的分布。结论在大肠埃希菌中高效表达了重组SCF蛋白,纯化复性后具有良好的免疫原性。

Objective To express the stem cell factor(SCF) of tree shrew in prokaryotic cells,purify the expressed product and determine its immunogenicity.Methods Primers were designed according to the predicted SCF gene sequence in GenBank,with which SCF gene was amplified and cloned into prokaryotic expression vector p ET-30a(+).The constructed recombinant plasmid pET-30a(+)-SCF was transformed to E.coli BL21(DE3) and induced with IPTG.The expressed fusion protein was purified by nickel ion column chromatography and immunized to rabbits,of which the immunogenicity was analyzed by Western blot and ELISA.Meanwhile,the distribution of SCF in various tissues of tree shrew was determined.Results Restriction analysis and sequencing proved that recombinant plasmid pET-30a(+)-SCF was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 34 500,contained about 35.6% of total somatic protein,existed in a form of inclusion body,reached a purity of more than 90% after purification,and showed specific binding to anti-human SCF polyclonal antibody.The protein showed good immunogenicity,of which the antiserum titer was 1:256 000.SCF was detected in nose,trachea,lung,spleen,kidney and esophagus of tree shrew.Conclusion Recombinant SCF was highly expressed in E.coli,which showed good immunogenicity after purification and re-naturalization.