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目的:获得高纯度、高活性的人TLR4胞外段蛋白。方法:采用PCR方法从含人TLR4 cDNA的质粒pCDNA3-TLR4中扩增人TLR4胞外段基因片段,将其插入载体pET32a并导入大肠杆菌BL21(DE3)plysS中表达。用Ni2+-NTA琼脂糖柱纯化融合蛋白。SDS-PAGE和免疫印迹分析目的蛋白的相对分子质量和抗原性,ELISA和FCM鉴定其功能活性。结果:获得1 911 bp的人TLR4胞外段基因片段,构建成重组表达载体pET32a-TLR4并在大肠杆菌中表达。TLR4-Trx融合蛋白的表达量占菌体总蛋白的12%,其纯度较高。ELISA和FCM检测显示,TLR4-TrX融合蛋白不仅与配体LPS结合,还能竞争性抑制LPS与THP1细胞结合。结论:所获TLR4-Trx融合蛋白纯度高,功能活性好,为进一步研究TLR相关的免疫调节机制提供物质基础。
Objective: To obtain highly purified and highly active human TLR4 extracellular domain protein. Methods: The extracellular domain of human TLR4 gene was amplified from plasmid pCDNA3-TLR4 containing human TLR4 cDNA by PCR and inserted into vector pET32a and transformed into E. coli BL21 (DE3) plysS. The fusion protein was purified on a Ni2 + -NTA agarose column. The molecular weight and antigenicity of the target protein were analyzed by SDS-PAGE and Western blotting. The functional activity of the target protein was identified by ELISA and FCM. Results: 1 911 bp human extracellular TLR4 gene fragment was obtained and constructed into recombinant expression vector pET32a-TLR4 and expressed in E. coli. The expression of TLR4-Trx fusion protein accounted for 12% of the total bacterial protein, its purity is higher. ELISA and FCM showed that the TLR4-TrX fusion protein could not only bind LPS but also competitively inhibit the binding of LPS to THP1 cells. CONCLUSION: The obtained TLR4-Trx fusion protein has high purity and good functional activity, and provides the material basis for further study on TLR-related immunoregulatory mechanism.