大鼠肝细胞系BRL-3A细胞Hsp70过表达条件的优化  

Optimization of Hsp70 over- expression of the BRL- 3A cellsin rat liver cell lines

作　　者：计红[1] 薛琳琳[2] 耿仁德[3] 郭景茹[1] 臧琳[1] 王建发[1] 孔凡志[1] 齐艳萍[1] 杨焕民[1] 郭丽[1] 蒲丽君[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319 [2]黑龙江职业学院,黑龙江双城150111 [3]黑龙江省农垦总局建三江分局勤得力农场第六管理区,黑龙江同江156426

出　　处：《黑龙江畜牧兽医》2014年第3期34-38,209,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：黑龙江省教育厅面上项目(12521371)

摘　　要：为了使热休克蛋白70(heat shock protein 70,Hsp70)在BRL-3A细胞中过表达,试验以带有标签基因的腺病毒感染BRL-3A细胞,根据感染效率初筛病毒感染的最适浓度和作用时间;然后用初筛出的最适浓度(4×107,2×107,1×107,0.5×107,0.25×107pfu/mL)和时间(24 h和48 h)感染BRL-3A细胞,优化Hsp70重组腺病毒感染BRL-3A细胞的条件,并应用荧光定量RT-PCR和免疫蛋白印记方法检测Hsp70的mRNA和蛋白表达量。结果表明:当用1×107pfu/mL病毒AdCMV-Hsp70按照最佳感染条件的感染复数(MOI)=20感染BRL-3A细胞48 h时,能使Hsp70表达量比病毒空载体感染细胞和无病毒的对照组细胞极显著升高(P<0.01)。To make the heat shock protein 70 （ Hsp70 ） over - express in BRL -3A cells, the adenovirus vector with gene tags was used to infect the BRL - 3A cells. The optimal concentrations and action time of the virus infection were preliminary＇carried out according to the infection efficiencies, in which the optimal concentrations were 4×10^7 pfu/mL, 2×10^7 pfu/mL, 1×10^7 pfu/mL, 0.5×10^7 pfu/mL, and 0.25×10^7 pfu/ mL; and the optimal action time was 24 h and 48 h. The optimal concentrations and action time were used to infect the BRL -3A cells, while optimizing the conditions of the Hsp70 adcnovirus infecting the BRL - 3A cells. The mRNA and protein expression levels of Hsp70 were measured using real - time PCR and western blotting. The results showed that when the Ad - CMV - Hsp70 virus at concentration of 1×10^7 pfu/mL with multiplicity of infection （MOI） = 20 was used to infect the BRL -3A cells for 48 h, the expression levels of Hsp70 were extremely signifi- cantly higher （ P 〈 0.01 ） than those of the virus - infected cells with empty vector and the cells without virus in the control group.

关 键 词：热休克蛋白70(Hsp70) BRL-3A细胞 大鼠 过表达 腺病毒 

分 类 号：S858.91[农业科学—临床兽医学]