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
10.3760/cma.j.issn.1003-9279.2018.06.001
- VernacularTitle: 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
- Author:
Wenhui ZHENG
1
;
Zeming QIN
2
;
Xin LI
1
;
Xinyue CAO
1
;
Xiaoyu SHAN
1
;
Hongling WEN
1
;
Zhiyu WANG
1
;
Tao HUANG
3
;
Li ZHAO
1
Author Information
1. Department of Laboratory Science of Sanitary Microbiology, School of Public Health, Shandong University, Key Laboratory of Infectious Diseases Prevention and Control in Colleges and Universities of Shandong Province During the 13th Five-year Plan, Jinan 250012, China
2. Laboratory Center of Preventive Medicine, School of Public Health, Shandong University, Jinan 250012, China
3. Shandong Provincial Center for Disease Control and Prevention, Jinan 250014, China
- Publication Type:Journal Article
- Keywords:
HIV-associated dementia;
HIV-1 tat;
Protein purification;
Human umbilical vein endothelial cells;
Cell activity
- From:
Chinese Journal of Experimental and Clinical Virology
2018;32(6):561-565
- CountryChina
- Language:Chinese
-
Abstract:
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.
