酸敏感离子通道基因重组质粒的构建、表达及其活性测定

Construction,expression and activity detection of the recombinant plasmids of acid-sensing ion channels genes

为了构建含有小鼠酸敏感离子通道(ASIC1a、ASIC2a)基因全长cDNA的重组质粒,并表达有生物学活性的ASIC1a和ASIC2a融合蛋白,本研究通过逆转录聚合酶链反应(RT-PCR)分别获得小鼠ASIC1a和ASIC2a全长cDNA,并将其克隆入真核表达载体pEGFP-N3中,构建含小鼠全长ASIC1a和ASIC2a基因的重组质粒pEGFP-ASIC1a和pEGFP-ASIC2a,经DNA测序鉴定序列正确。脂质体法分别将其转染至中国仓鼠卵巢细胞(CHO),荧光显微镜下观察小鼠ASIC1a和ASIC2a融合蛋白在细胞内的表达分布,Westernblot检测其蛋白表达。酸性处理转染重组质粒细胞,并比较其细胞存活率及乳酸脱氢酶(LDH)的释放来评价融合蛋白的生物学活性。结果显示:本研究成功地分别将小鼠ASIC1a和ASIC2a全长cDNA克隆入pEGFP-N3载体中,并将其转染至CHO细胞,荧光显微镜下观察到CHO细胞膜周围呈强绿色荧光条带,Westernblot显示小鼠ASIC1a和ASIC2a融合蛋白分别在90kD和88kD处有表达。经酸性处理的转染ASIC1a和ASIC2a重组质粒的CHO细胞,分别较其在pH7.4条件下的细胞存活率明显降低,LDH释放量明显增高,且通道阻断剂可抑制该效应。上述结果提示,我们已成功构建出含小鼠ASIC1a和ASIC2a全长cDNA的重组质粒pEGFP-ASIC1a和pEGFP-ASIC2a,并使有生物学活性的ASIC1a和ASIC2a融合蛋白在CHO细胞中表达。

To construct the recombinant plasmids containing the full length cDNA of mouse ASICla and ASIC2a genes and to express ASICI a and ASIC2a fusion proteins with biological activity,the full length cDNA of mouse ASIC1 a and ASIC2a genes were obtained by RTPCR and cloned into the eukaryotie expression vector pEGFP-N3 respectively in the present study. Then,recombinant plasmids pEGFPAS/C1 a and pEGFP-ASIC2a were constructed. After identified by DNA sequencing,the recombinant plasmids were transfected into Chinese hamster ovary（CHO） cells respectively with lipofectamine reagent. The distribution of the fusion protein expression in the cells was detected by fluorescence microscope and Western-blot after transfection. After the cells with recombinant plasmids were acidified, cell viability and LDH release were determined to evaluate the biological activity of fusion protein. Our results showed that the full length cDNA of mouse ASICla and AS1C2a genes had been cloned into the vector pEGFP-N3 and transfected into CHO cells. Obvious green fluorescence was de- tected around CHO cell membrane with recombinant plasmids. Western blot showed that ASICla and ASIC2a fusion proteins were respec- tively expressed at about 90 kD and 88 kD. Compared with pH7.4-treated cells, cell viability was reduced and LDH release increased significantly in the acidified cells with recombinant plasmids, which could be inhibited by ASICs blockers. The present results suggest that the recombinant plasmids containing the full length cDNA of mouse pEGFP-ASIC1a and pEGFP-ASIC2a genes were successfully constructed and expressed with biological activity in CHO cells.