HIV相关脑病患者中枢神经系统来源的HIV-1 Tat蛋白对HUVECs活性的影响

摘要

Objective BG-derived HIV-1 Tat protein from an HIV-associated dementia (HAD) patient was expressed in E. coli BL21(DE3) and purified in order to research the effects on human umbilical vein endothelial cells (HUVECs) activity. Methods The recombinant plasmid pGEX-KG-tat with HIV-1 tat stored in our laboratory was amplified by PCR. The PCR product was cloned into pET-32a-tat. The recombinant plasmid pET-32a-tat was transfected into E. coli, and Tat protein was expressed in BL21 (DE3), which was induced by IPTG. Then it was purified by Ni-chelating chromatography column and gel filtration preloaded column, and identified by SDS-PAGE and Western blot(WB). The concentration was determined by BCA Kit. Different concentrations of Tat were added into HUVECs to detect their effects on cell activity by cck-8. Results The Tat with high purity was efficiently expressed in BL21 ( DE3) and obtained by using the Ni-chelating chromatography column and gel filtration preloaded column. The concentration was 0. 47 mg/ ml by using BCA Kit. As the concentration of Tat increased, HUVECs activity decreased. There was no significant difference in cells viability between negative control with 100 ng/ ml and 200 ng/ ml group (P > 0. 05). There was significant difference in cells viability between negative control with 300 ng/ ml, 400 ng/ ml, 500 ng/ ml and 1000 ng/ ml group (P < 0. 05). But the difference between 300 ng/ ml, 400 ng/ ml, 500 ng/ ml and 1000 ng/ ml group was not statistically significant ( P > 0. 05 ). Conclusions The HIV-1 Tat with biological activity was efficiently expressed in BL21 ( DE3), and the activity of HUVECs was significantly decreased when the concentration reached 300 ng/ ml.%目的 在原核细胞中表达并纯化HIV相关脑病(HIV-associated dementia,HAD)患者基底核(basal ganglia,BG)来源HIV-1 Tat蛋白,研究其对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)活性的影响.方法 以本室保存的重组质粒pGEX-KG-tat为模板扩增tat,构建重组表达载体pET-32a-tat.转化BL21(DE3)并用IPTG诱导表达Tat蛋白.Tat蛋白经镍亲和层析柱和凝胶过滤预装柱纯化、SDS-PAGE和Western blot(WB)鉴定后浓缩,测定BCA蛋白浓度,cck-8检测不同浓度Tat对HUVECs活性的影响.结果 在BL21大肠杆菌中表达、纯化得到了纯度较高的Tat蛋白,BCA测定浓度为0.47 mg/ml.随着Tat浓度的增加,HUVECs活性逐渐降低,与阴性对照组相比,100 ng/ml、200 ng/ml两组细胞活性差异均无统计学意义(P>0.05),300 ng/ml、400 ng/ml、500 ng/ml、1000 ng/ml四组细胞活性差异均有统计学意义(P<0.05),但四组组间差异无统计学意义(P>0.05).结论 在原核细胞中高效表达了具有生物学活性的HIV-1 Tat蛋白,浓度达到300 ng/ml可显著降低HUVECs的活性.