HIV gp41七肽重复区五螺旋蛋白的高效表达及对病毒融合的抑制

High level expression of 5-helix protein in HIV gp41 heptad repeat regions and its virus fusion-inhibiting activity

摘要:

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。

Abstract:

The artificial 5-helix can inhibit the formation of trimer-of-hairpins structure during the course of HIV-directed membrane fusion and then inhibit human immunodeficiency virus (HIV) infecting target cells. But 5-helix was apt to form inclusion body when expressed directly in prokaryotic cell and was difficult to renature, which causes inconvenience to future study. We found a proper expression vector by simulating protein structure. We simulated its proper conformation in two vectors pGEX-6P-1 and pET44b by homology modeling. The contrast of conformations showed that the energy of salvation of its fusion protein with NusA in vector pET44b was higher than its fusion protein with glutathione-S-transferase (GST) in pGEX-6P-1 and its restriction site lay on the surface of its fusion protein in vector pET44b. 5-helix gene was amplified from pGEX-6P-1-5H by PCR, and was ligated to pET44b to construct recombinant vector pET44b-PSP-5Helix after tested correctly by enzymes digestion. The recombinant vector was transformed into Escherichia coil BL21 (DE3) to express 5-helix protein at different temperatures. Aim protein was purified with Ni column and GST column, and was determined by SDS-PAGE. Then the purified 5 -Helix was used to test the inhibitive activity of pseudo HIV virus infecting GHOST-CXCR4. Results show that its fusion protein with NusA can be effectively soluble expressed and easier to be cleaved, and that the purified 5-helix can efficiently inhibit pseudo HIV virus infecting GHOST-CXCR4 and its IC50 value is (22.77±5.64) nmol/L, which lay the foundation to further discuss the application in HIV-1 infection.