远缘链球菌GTF催化活性蛋白及其多克隆抗体的制备和鉴定

目的:在大肠杆菌中表达重组远缘链球菌葡糖基转移酶(glucosyltransferase,GTF)的催化活性区(catalytic region,CAT)蛋白并制备其多克隆抗体。方法:扩增远缘链球菌OMZ176基因组的CAT区的基因片段,克隆入pQE31载体诱导表达,鉴定表达产物。重组蛋白纯化后免疫小鼠制备多克隆抗体,ELISA测定抗体的效价,West-ern blotting检测抗体的特异性和亲和性。结果:重组质粒成功在大肠杆菌中表达,经抗His tag单克隆抗体免疫印迹法检测,有阳性条带出现,证实重组蛋白有抗原特异性。ELISA测定免疫小鼠所得抗CAT多克隆抗体效价可达到1∶5000。Western blotting检测证明该抗体有较好的针对CAT蛋白的专一性。结论:GTF区CAT基因原核表达质粒构建成功,表达的融合蛋白具有良好的抗原性。抗CAT多克隆抗体特异性和效价良好,能够满足针对CAT免疫印迹和细胞免疫组化检测等实验要求,为深入研究同时包含变形链球菌和远缘链球菌两种致龋菌的主要抗原的新型防龋DNA疫苗奠定了必要的物质基础。

Preparation and Identification of the Fusion Protein and the Polyclonal Antibody of GTF Catalytic Region from S.sobrinus

Objective:To express and purify the catalytic region of glucosyltransferase(GTF)from S.sobrinus in E.coli JM109 and prepare polyclonal antibody against recombinant protein.Methods:The catalytic gene was amplified from the genomic DNA of S.sobrinus OMZ176,and then cloned into the prokaryotic expression vector pQE31.The recombinant plasmid pQE/cat was expressed in E.coli JM109.After induction with IPTG,the expressed recombinant protein rCAT was purified using immobilized metal ion affinity chromatography and identified by SDS-PAGE and Western blotting.The mouse polyclonal antibody against CAT was obtained by immunizing the Balb/c mice with the rCAT protein.The titer and specificity of the mouse antiserum was measured by ELISA and Western blotting.Results:The CAT region was cloned in the plasmid correctly.It was verified by DNA sequencing and restriction enzyme.The recombinant protein rCAT expressed as expectation.The relative molecular mass of the rCAT protein was 42KDa and the specificity of the rCAT protein was confirmed by Western blotting.The specificity of anti-CAT antibody was identified by Western blotting with the use of rCAT protein,revealing,after incubation and coloration,a 43 kDa band on the PVDF membrane.The titer of the antiserum measured by ELISA could achieve 1:5 000.Conclusion:The catalytic region of GTF was successfully cloned into the prokaryotic expression plasmid pQE31.The rCAT protein has the good antigenicity.The CAT polyclonal antibody with high titer and specificity can satisfy Western blotting and immunohistochemistry experiment request.This study could lay a material foundation for the further research of new anti-caries DNA vaccine pGJGAC/VAX encoding antigens of both S.mutans and S.sobrinus.