猪TLR7基因胞外区部分片段的克隆、表达及其重组蛋白的纯化分析

目的]获得高表达量的蛋白，为进一步研制单克隆抗体提供较好的免疫原，同时也可为TLR7胞外区该片断蛋白未知区域的结构和功能研究奠定基础。方法]应用PCR技术从重组质粒pcDNA3．1／CT-GFP-pTLR7上扩增出编码猪TLR7基因胞外区N端第27—202位氨基酸序列，利用BamHI和HindⅢ酶切位点将其插入到原核表达载体PE130a中，重组质粒转化BL21（DE3）后，在不同条件下诱导表达。结果]经SDS．PAGE分析表明，重组菌表达出约26kD的融合蛋白，并证实主要以包涵体形式表达，其最佳诱导表达条件是37℃、0．5mm01／LIPTG诱导6h，表达量可接近50％；纯化的重组蛋白免疫小鼠后，间接ELISA检测抗体效价，结果该蛋白免疫BALB／c小鼠效价达10^5．结论]TLR7胞外区该片断重组蛋白具有很好的抗原性，可以作为单克隆抗体研制的免疫原。

Research on the Clone and Expression of the Extracellular Domain Fragment of Porcine TLR7 Gene and Analysis of the Purification of its Recombinant Protein

Objective] To obtain a high expression of the protein,in order to further the development of monoclonal antibodies into Lunogen to provide better immune-derived.but also for the extracellular domain of TLR7 protein fragments of unknown structure and function of the region lay the foundation for research.Methods] PCR technology from the recombinant plasmid was amplified pcDNA3.1/CT-GFP-pTLR7 porcine TLR7 gene encoding the extracellular domain of N-section 27-202 of amino acid sequence,using BamH Ⅰ and Hind Ⅲ restriction sites will be its inserted into the prokaryotic expression vector PET30a,the recombinant plasmid into BL21(DE3) after induction of expression under different conditions.Results] by SDS-PAGE analysis showed that the expression of recombinant bacteria of about 26 kD fusion protein and confirmed that the main form of expression in inclusion bodies,the best conditions induced the expression of 37 ℃,0.5 mmol/L IPTG induced 6 h,expression of volume close to 50%;purified recombinant protein immol/Lunized mice,the indirect ELISA detection of antibody titers,the results of the protein in the immol/Lune BALB/c mice up to 105 small price.Conclusion] TLR7 extracellular domain of the recombinant protein fragment of the antigen has a very good,and the development of monoclonal antibodies can be used as the i mmol/Lunogen.