L-精氨酸转运体-2的siRNA表达载体的构建及鉴定

目的:构建氨基酸转运体-2(CAT-2)的靶向小分子干扰RNA(siRNA)表达载体质粒,并测序鉴定,为下一步研究靶向siRNA对CAT-2蛋白表达和L-argine/iNOS/NO通路的影响建立基础。方法:根据L-精氨酸转运体CAT-2的mRNA序列及siRNA设计原则,并经BLAST比对,设计合成4对siRNA寡聚单链DNA,退火成双链dsDNA,用载体构建试剂盒BLOCK-iTTMPol II miR RNAi Expression Vector Kit with EmGFP进行重组质粒构建,将4对dsDNA分别插入到siRNA表达载体pcDNATM6.2-GW/EmGFPmiR中,构建4个siRNA表达质粒,并转化至感受态细菌DH5。筛选阳性克隆并提取重组质粒后测序分析。结果:CAT-2的siRNA重组载体质粒测序结果证实表达载体构建成功。结论:L-精氨酸转运体CAT-2 siRNA表达载体质粒成功构建,为下一步研究靶向siRNA对CAT-2蛋白表达和L-arg/iNOS/NO通路的影响建立基础。

Construction and identification of L-arginine transporter CAT-2 siRNA recombinant plasmid

Objective: To construct and identificate the recombinant plasmid expression vector of cationic amino acid transporter-2 siRNA,and establish a foundation to study the influence of target siRNA on the expression of CAT-2 and L-arginine/iNOS/NO pathway.Methods: According to the mRNA sequences of L-arginine transporter CAT-2,siRNA design principles and BLAST Comparison,four pairs single-strand DNA of siRNA were designed and synthesized.Annealing into double-strand DNA,the four pairs of double-strand DNA of the siRNA were inserted into siRNA expression vector pcDNATM 6.2-GW/EmGFPmiR to construct siRNA expression plasmid with vector construction kit BLOCK-iTTM Pol II miR RNAi Expression Vector Kit with EmGFP,and transfected the recombinant plasmid into the competent Escherichia coli DH5.After selecting positive Escherichia coli DH5,and extracting the recombinant plasmids,the sequences of the recombinant plasmids were finally identificated.Results: The sequencing results of the recombinant plasmid expressing CAT-2 siRNA confirmed construction successfully.Conclusion: The recombinant plasmids vectors expressing L-arginine transporter CAT-2 siRNA are successfully constructed,which establish a foundation to study the target unfluence siRNA on the expression of CAT-2 and L-arginine/iNOS/NO pathway.