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目的:克隆我国地方品种姜曲海猪TLR5基因,构建该基因的原核表达质粒,获得融合表达蛋白并鉴定其免疫特性,为进一步研制TLR5单克隆抗体(mAb)提供免疫原。方法:从全血中提取姜曲海猪的基因组,设计特异性引物,利用PCR方法扩增得到TLR5基因片段,PCR产物克隆到原核表达载体pET30a(+)中,构建重组表达质粒pET-TLR5,将重组表达质粒转化E.coli BL21,0.5 mmol/L IPTG诱导表达目的蛋白,应用SDS-PAGE和Western blot分析鉴定蛋白活性。结果:成功扩增出猪TLR5基因片段,大小为2 571 bp。酶切鉴定结果表明,猪的TLR5基因成功克隆入原核表达载体pET30a(+)中,重组质粒pET-TLR5在大肠杆菌中获得表达,SDS-PAGE结果显示出相对分子质量(Mr)大小约95 200;Western blot结果表明,表达产物与兔抗小鼠TLR5 mAb具有良好的反应性。结论:成功克隆并表达具有较好免疫原性的猪TLR5分子,为其mAb的制备提供生物材料。
OBJECTIVE: To clone the TLR5 gene of Jiangquhai pig in China and construct the prokaryotic expression plasmid of the gene. The fusion protein was identified and its immunogenicity was identified. The TLR5 monoclonal antibody (mAb) was provided as an immunogen. Methods: The genomic DNA of Jiangquhai pig was extracted from whole blood and specific primers were designed. The TLR5 gene fragment was amplified by PCR. The PCR product was cloned into prokaryotic expression vector pET30a (+), and the recombinant plasmid pET-TLR5 was constructed. The expression plasmid was transformed into E. coli BL21 and induced with 0.5 mmol / L IPTG. The protein was identified by SDS-PAGE and Western blot. Results: The TLR5 gene fragment was amplified successfully and its size was 2 571 bp. The results of restriction enzyme digestion showed that the porcine TLR5 gene was successfully cloned into the prokaryotic expression vector pET30a (+), and the recombinant plasmid pET-TLR5 was expressed in E. coli. SDS-PAGE showed that the relative molecular mass was about 95 200 ; Western blot results showed that the expression product and rabbit anti-mouse TLR5 mAb has good reactivity. Conclusion: The porcine TLR5 molecule with good immunogenicity was successfully cloned and expressed, which provided biological material for the preparation of mAb.