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人工构建的五螺旋蛋白(5-helix)能抑制人类免疫缺陷综合症病毒(HIV)介导的膜融合过程中发卡三聚体的形成,从而抑制病毒感染靶细胞。但5-helix基因在原核细胞中直接表达时易形成包涵体,复性困难,给研究带来不便。本研究探讨了将蛋白结构模拟用于寻找合适的表达载体的方式:通过同源建模,模拟了5-helix在pGEX-6P-1载体及pET44b载体上的融合蛋白形成的最有可能的2种构象。通过对比发现其在pET44b载体中与NusA的融合蛋白的溶剂化能远大于其在pGEX-6P-1中与GST融合蛋白的溶剂化能,且酶切位点位于蛋白表面。应用PCR将5-helix基因从pGEX-6P-1-5H克隆出来,鉴定正确后连接到pET44b载体上,构建成重组载体pET44b-PSP-5Helix。将重组载体转化入大肠杆菌BL21(DE3),于不同温度诱导表达。用镍柱及GST柱纯化蛋白,SDS-PAGE证实成功得到了目的蛋白。用纯化蛋白验证抑制HIV假病毒感染GHOST-CXCR4的活性。结果显示:与NusA融合的5-helix蛋白能够实现高效的可溶表达,且酶切容易;纯化蛋白抑制HIV假病毒感染GHOST-CXCR4的半数抑制浓度为(22.77±5.64)nmol/L,为进一步探讨其在HIV-1病毒感染中的应用奠定了基础。
Artificially constructed 5-helix inhibits HIV-induced membrane-bound trimer formation during membrane fusion and thus inhibits viral infection of target cells. However, the 5-helix gene is easy to form inclusion bodies when expressed directly in prokaryotic cells, resulting in difficulties in refolding and inconvenience to the research. This study explored the way in which protein structural modeling is used to find suitable expression vectors: by homology modeling, the most likely 2-helix formation of fusion proteins on pGEX-6P-1 and pET44b vectors was mimicked Species. By comparison, the solvating energy of the fusion protein with NusA in the pET44b vector was found to be far greater than its solvating energy with the GST fusion protein in pGEX-6P-1, and the restriction site was located on the protein surface. The 5-helix gene was cloned from pGEX-6P-1-5H by PCR and ligated into pET44b vector to construct pET44b-PSP-5Helix. The recombinant vector was transformed into E. coli BL21 (DE3) and induced at different temperatures. The protein was purified by nickel column and GST column, SDS-PAGE confirmed that the target protein was successfully obtained. Purified proteins were used to verify the inhibition of HIV pseudovirus viral infection of GHOST-CXCR4 activity. The results showed that 5-helix protein fused with NusA could express efficiently and easily, and the half-inhibitory concentration of purified protein could inhibit HIV pseudovirus infection of GHOST-CXCR4 (22.77 ± 5.64) nmol / L Explore its application in HIV-1 virus infection laid the foundation.