CAJ | 학술논문

目的：构建小鼠1-磷酸鞘氨醇受体3(S1PR3)慢病毒表达载体，并检测其表达情况。方法小鼠心肌组织mRNA并逆转录得到cDNA，设计并合成引物，采用亚克隆方式将目的基因克隆至慢病毒表达载体pLent-IRES-EGFP中，将表达载体与包装质粒共转293T细胞，进行目的基因的过表达慢病毒包装，随后收集培养成功病毒，使用病毒感染小鼠L929细胞，检测感染后L929细胞表达S1PR3情况。结果从小鼠心机组织cDNA中扩增出大小约1100 bp的目的片段；双酶切后成功将S1PR3克隆到慢病毒表达载体pLent-IRES-EGFP，表达质粒与包装质粒供转染293细胞后48 h就可以看到细胞中成功表达绿色荧光。收集培养成功病毒感染小鼠L929细胞，成功表达出S1PR3。结论本研究成功构建了可以高表达S1PR3的pLenti6.3-S1PR3-IRES-EGFP慢病毒表达载体，为进一步调控S1PR3相关的细胞功能奠定了基础。
목적：구건소서1-린산초안순수체3(S1PR3)만병독표체재체，병검측기표체정황。방법소서심기조직mRNA병역전록득도cDNA，설계병합성인물，채용아극륭방식장목적기인극륭지만병독표체재체pLent-IRES-EGFP중，장표체재체여포장질립공전293T세포，진행목적기인적과표체만병독포장，수후수집배양성공병독，사용병독감염소서L929세포，검측감염후L929세포표체S1PR3정황。결과종소서심궤조직cDNA중확증출대소약1100 bp적목적편단；쌍매절후성공장S1PR3극륭도만병독표체재체pLent-IRES-EGFP，표체질립여포장질립공전염293세포후48 h취가이간도세포중성공표체록색형광。수집배양성공병독감염소서L929세포，성공표체출S1PR3。결론본연구성공구건료가이고표체S1PR3적pLenti6.3-S1PR3-IRES-EGFP만병독표체재체，위진일보조공S1PR3상관적세포공능전정료기출。
Objective To construct the recombinant lentivirus vector carrying sphingosine-1-phosphate re-ceptor 3 (S1PR3) gene. Methods The fragment of S1PR3 gene from the cDNA of the cardiac muscle of mouse was cloned into the lentivirus vector pLent-IRES-EGFP. Then the recombinant vector and the Packaging Mix were cotrans-fected into the 293T cells. Filtered culture media were harvested and the viral titer was checked by observing the ex-pression of green fluorescent protein (GFP). Mouse L929 cells were infected with the lentivirus and the expression of the S1PR3 of the L929 cells was detected by the real time PCR. Results A 1 100 bp fragment was amplified from cDNA of mouse myocardial tissue. Then after double enzyme digestion, S1PR3 was successfully cloned into the lenti-viral expression vector pLent-IRES-EGFP. Green fluorescence was observed 48 h after transfection into 293 cells. L929 cells were collected and cultured successfully, and S1PR3 was successfully expressed. Conclusion The recom-binant lentivirus vector carrying S1PR3 gene (pLenti6.3- S1PR3-IRES-EGFP) with high expression of S1PR3 was constructed successfully in the mouse L929 cells successfully, laying basis for regulating S1PR3-related cell function.