标题：小鼠S1PR3慢病毒表达载体的构建及表达

      作者：王 航 1，蔡克银 1，黄 浩 2
    (1.广州军区武汉总医院干部病房二科，湖北 武汉 430070；
2.深圳市合一康生物科技有限公司临床医学中心，广东 深圳 513000)

      卷次： 2015年26卷18期

      【摘要】 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。

      【关键词】 慢病毒；1-磷酸鞘氨醇受体3；血管损伤；再内皮化

      【中图分类号】 R-332 【文献标识码】 A 【文章编号】 1003—6350（2015）18—2653—04


Construction of the recombinant expression lentivirus vector carrying S1PR3 gene.
WANG Hang 1, CAI Ke-yin 1,
HUANG Hao 2. 1. Second Department of Geratology, Wuhan General Hospital of Guangzhou Command, Wuhan 430070,
Hubei, CHINA; 2. Clinical Medical Center, Shenzhen Hornetcorn Biotechnology Co. Ltd. Shenzhen 513000, Guangdong,
CHINA

【Abstract】 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.

      【Key words】 Lentivirus; Sphingosine-1-phosphate receptor 3 (S1PR3); Vascular injury; Reendothelialization
基金项目：国家自然科学基金(编号：81300153)