pEGFP-C2-ASIC2a真核表达载体的构建及其在大鼠关节软骨细胞中的表达  

Construction of pEGFP-C2-ASIC2a eukaryotic expression vector and its expression in articular cartilage cells of rats

作　　者：倪文琳[1] 唐杰[1] 潘春晓[1] 葛金芳[1] 陈飞虎[1] 

机构地区：[1]安徽医科大学药学院,合肥230032

出　　处：《安徽医科大学学报》2014年第3期304-308,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81271949)

摘　　要：目的通过构建真核表达pEGFP-C2-ASIC2a质粒,转染大鼠关节软骨细胞,建立ASIC2a在关节软骨细胞中过表达的模型,观察ASIC2a mRNA及其蛋白在细胞中的表达。方法从大鼠脑组织中提取目的基因ASIC2a,并用EcoRⅠ和KpnⅠ进行双酶切,同时用这两种酶双酶切质粒pEGFPC2,将其酶切产物按常规方法连接并转化入大肠杆菌DH5a,挑单克隆菌进行培养,提取质粒,再通过双酶切鉴定及测序后,用Lipofectamine 2000将所构建质粒转染入关节软骨细胞,在荧光显微镜下观察绿色荧光蛋白的表达,使用RT-PCR和Western blot法检测ASIC2a mRNA和蛋白表达,以鉴定模型建立成功与否。结果进行双酶切鉴定目的条带清晰准确,转染后可在荧光显微镜下观察到绿色荧光表达,通过RT-PCR可发现mRNA转录,Western blot法可发现目的蛋白表达。结论成功构建重组pEGFP-C2-ASIC2a表达载体,将用于进一步观察ASICs对软骨细胞的影响。Objective To construct eukaryotic expression vector of acid sensing ion channel-2 a （ ASIC2 a ） , and transfect it into the articular cartilage cells of rats to make the model of overexpression of ASIC2a. Methods Got the cDNA of ASIC2a from rat brain, then ASIC2a cDNA was amplified by PCR and cut with double enzyme EcoR I and Kpn I, then inserted into the eukaryotic expression vetor pEGFP-C2. The recombinant vector was verified by PCR, restriction enzymes cut and sequencing identified. We used Lipofectamine 2000 transfection reagent to trans-fect the plasmid into the cartilage cells of rats, then observed GFP expression under the fluorescence microscope. We also determined the relative expression of the mRNA and protein of ASIC2 a by RT-PCR and Western blot to i-dentify whether the model of overexpression was constructed successfully. Results The plasmid was identified by enzyme cutting where the purpose gene was included, and the electrophoretic stripes were accurate and distinct, al-so GFP could be detected in the transfected the articular cartilage cells of rats. ASIC2a gene expression could be detected by PCR, also its protein expression was detected by Western blot. Conclusion The model of overexpres-sion is constructed successfully which can be used to observe the effect on cartilage cells of the acid sensing ion channels expression.

关 键 词：ASIC2a PEGFP-C2 软骨细胞 基因表达 蛋白表达 

分 类 号：R593.22[医药卫生—内科学]