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

Effects of HIV-1 Tat derived from central nervous system of a HIV-associated dementia patient on the activity of human umbilical vein endothelial cells

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-1Tat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0.47mg/ml。随着Tat浓度的增加,HUVECs活性逐渐降低,与阴性对照组相比,100ng/ml、200ng/ml两组细胞活性差异均无统计学意义(P>0.05),300ng/ml、400ng/ml、500ng/ml、1000ng/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-1Tat蛋白,浓度达到300ng/ml可显著降低HUVECs的活性。

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.MethodsThe 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.ResultsThe 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).ConclusionsThe 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.