小鼠S1PR3慢病毒表达载体的构建及表达

Construction of the recombinant expression lentivirus vector carrying S1PR3 gene

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m RNA并逆转录得到c DNA,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p Lent-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c DNA中扩增出大小约1 100 bp的目的片段;双酶切后成功将S1PR3克隆到慢病毒表达载体p Lent-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p Lenti6.3-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。

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.