沉默过氧化物酶体增殖子活化受体γ表达降钙素基因相关肽基因重组载体的构建与鉴定

Construction and identification of the recombinant vector silencing peroxisome proliferator-activated receptor-γand expressing calcitonin gene-related peptide gene

目的 构建与鉴定沉默过氧化物酶体增殖子活化受体γ（PPARγ）同时表达降钙素基因相关肽（CGRP）基因重组载体.方法 选用pGFP-V-RS载体,将PPARγ小干扰RNA（siRNA）寡核苷酸靶序列连接入pGFP-V-RS载体,酶切鉴定及测序正确后得到重组载体pGFP-V-RS-siPPARγ;将CGRP片段与Mlu Ⅰ酶切,并与末端羟基化的线性质粒pGFP-V-RS重组连接,得到重组载体pGFP-V-RS-exCGRP.将获取的CGRP基因片段连接入线性化重组载体pGFP-V-RS-siPPARγ,酶切鉴定及测序正确后得到重组载体pGFP-V-RS-siPPARγ-exCGRP.收集第3代HEK-293细胞,将细胞分为4组：CON组：转染空载体pGFP-V-RS质粒,作为对照；SI组：转染重组载体pGFP-V-RS-siPPARγ质粒；EX组：转染重组pGFP-V-RS-exCGRP质粒；DOU组：转染重组双基因载体pGFP-V-RS-siPPARγ-exCGRP质粒.每组电极杯中加入5x106个细胞,并分别加入上述质粒2.5μl,给予直流电低压脉冲1次,时间15 ms.培养48 h后收集各组细胞,用逆转录-聚合酶链反应（RT-PCR）与Western blot检测细胞内PPAR、CGRP mRNA及蛋白的表达.结果 重组双基因载体pGFP-V-RS-siPPARγ-exCGRP经酶切鉴定及测序结果与设计完全一致.电转染HEK-293细胞后,DOU组细胞PPARγ基因呈低表达,PPARγmRNA与其蛋白的相对表达量为0.036±0.003、0.108 ±0.031,与EX组（0.156±0.014、0.778±0.103）及CON组（0.148±0.014、0.742 ±0.143）比较,差异有统计学意义（P＜0.05）,与SI组（0.054±0.005、0.135 ±0.039）比较,差异无统计学意义（P＞0.05）.DOU组细胞CGRP基因呈高表达,CGRP mRNA与其蛋白的相对表达量为1.144±0.106、1.244±0.184,与SI组（0.320±0.030、0.370±0.089）及CON组（0.444 ±0.041、0.274±0.062）比较,差异有统计学意义（P＜0.05）,与EX组（1.021 ±0.095、1.221±0.181）比较,差异无统计学意义（P＞0.05）.结论 成功构建出沉默PPARγ基因并表达CGRP基因的重组载体pGFP-V-RS-siPPARγ-exCGRP,能够有效阻断PPARγ基因表达,同时促进CGRP基因表达.

Objective To construct the recombinant vector silencing peroxisome proliferator-activated receptor-γ （PPARγ） and expressing calcitonin gene-related peptide （CGRP） gene.Methods The pGFP-V-RS vector we selected contains a small interfering RNA （siRNA） expressing unit,and also can express exogenous gene inserted.The PPARγ siRNA oligonucleotides sequence was ligated into pGFP-VRS vector after annealling,and then PPAR siRNA recombinant vector （pGFP-V-RS-siPPARγ） was obtained by restriction analysis and DNA sequencing.The CGRP gene fragment was ligated into linear plasmids pGFP-V-RS-siPPARγrecombinant vector,and then the recombinant vector pGFP-V-RS-siPPARγ-exCGRP was obtained by restriction analysis and DNA sequencing.The HEK-293 cells were collected and divided into 4 groups at random.In control group,the cells were only transfected with vector pGFP-V-RS plasmid.In SI group,the cells were transfected with recombinant vector pGFP-V-RS-siPPARγplasmid.In EX group,the cells were transfected with recombinant vector pGFP-V-RS-exCGRP plasmid.In DOU group,the cells were transfected with pGFP-V-RS-siPPARγ-exCGRP plasmid.There were 5 ＆#215; 106 HEK-293 cells in electrode cup of each group,and 2.5 μl above-mentioned plasmids were added respectively.The cells were given DC voltage pulse one time （15 ms）.The cells were harvested in each group after culture for 48 h,and CGRP and PPARγmRNA and protein expression levels were detected by using reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting.Results The results of restriction analysis and DNA sequencing were completely matched with those of the designed sequences after the PPARγsiRNA oligonucleotides sequence and CGRP gene fragment were cloned into pGFP-V-RS vector.After recombinant vector pGFP-V-RS-siPPARγ-exCGRP was transfected into HEK-293 cells,the expression levels of PPARγ mRNA and protein were reduced in DOU group DOU （0.036 ±0.003 and 0.108 ±0.031）as compared with EX group （0.156 ±0.014 and 0.778 ±0.103） and control group （0.148 ±0.014 and 0.742 ± 0.143） （P 〈 0.05）,but showed no significant difference in comparison to SI group （0.054 ± 0.005and 0.135±0.039） （P〉0.05）.The expression level of CGRP mRNA and protein was significantly increased in DOU group （1.144 ±0.106 and 1.244 ±0.184） as compared with IS group （0.320 ±0.030 and 0.370 ±0.089） and control group （0.444 ±0.041 and 0.274 ±0.062） （P 〈0.05）,but there was no significant difference in comparison to EX group （1.021 ±0.095 and 1.221 ±0.181） （P 〉0.05）.Conclusion We successfully constructed the recombinant vector pGFP-V-RS-siPPARγ-exCGRP that silencing PPARγand expressing CGRP gene,and the recombinant vector can efficaciously suppress PPARγgene expression and promote CGRP gene expression simultaneously.