大鼠UT-B重组腺病毒载体的构建及体外转染Caco-2细胞后基因表达

Construction of mouse UT-B recombinant adenovirus vector and gene expression after of transfected into Caco-2 cells in vitro

目的采用GatewayTM技术构建含大鼠尿素转运蛋白B(Urea transporter B,UT-B)和增强型绿色荧光蛋白(Enhanced green fuorescent protein,EGFP)的重组腺病毒载体,并以该载体体外转染Caco-2细胞,观察Caco-2细胞内UT-B的表达。方法分别以pMD18-UT-B和IRES2-EGFP质粒为模板,PCR扩增UT-B和IRES-EGFP序列,通过引物重叠区进行PCR拼接,并在UT-B-IRES-EGFP两端引入attB1、attB2重组臂序列。PCR产物经BP重组系统将UT-B-IRES-EGFP融合基因重组到入门载体pDONR221中,构建入门克隆。入门克隆与表达载体pAD/CMV/V5-DEST使用LR重组系统进行体外重组,构建表达克隆pAD-UT-B-IRES-EGFP,pAD-UT-B-IRES-EGFP转化DH-5α,筛选阳性克隆,PCR及测序验证。pAD-UT-BIRES-EGFP用Pac I酶切线性化后由Lipofectamine 2000介导转染293A细胞,包装病毒,收集病毒液并测定滴度。重组的腺病毒体外感染培养的Caco-2细胞,RT-PCR观察Caco-2细胞中UT-B mRNA的表达。结果成功构建了pAD-UT-B-IRES-EGFP载体,经PCR及测序验证,表达载体包含UT-B基因,且序列与GeneBank中收录的大鼠UT-B序列一致。重组的腺病毒感染Caco-2细胞后可检测到UT-B mRNA的表达。结论利用GatewayTM系统成功构建了包含UT-B和IRES-EGFP的腺病毒表达载体,为进一步研究其功能奠定了基础。

Objective To construct a recombinant adenovirus vector containing rat urea transporter B （UT-B） and enhanced green fluorescent protein （EGFP） by Gateway rM system and detect UT-B expression in Caco-2 cells after infection with recombinant adenovirus. Methods UT-B and IRES-EGFP genes were obtained respectively by PCR amplification using pMD18-UT-B and IRES2-EGFP vectors as templates. UT-B and IRES-EGFP genes were spliced by overlap-extension PCR, the attBl and attB2 sequences were added at two ends of UT-B-IRES-EGFP recombinant gene. The entry clone was constructed after recombination of entry vector pDONR221 and UT-B-IRES-EGFP using BP recombination system. Then the entry clone and the expression vector pAd/CMV/V5-DEST were recombined together in vitro to create the expression clone pAd-UT-B-IRES-EGFP using LR recombination system. The positive clone was selected after pAd- UT-B-IRES- EGFP transformed DH-5 a. The vector was extracted and confirmed by PCR and sequencing. After linearized by the restriction enzyme Pac I, pAd-UT-B-IRES-EGFP was transfected into 293A cells with lipofectamine 2000 to be packaged into adenovirus stock.The expression of UT-B mRNA in Caco-2 cells was detected by RT-PCR after infection with the recombined adenovirus. Results The target gene of UT-B was transferred into pAd-UT-B-IRES-EGFP vector correctly and was confirmed by RT-PCR and sequencing, pAd-UT-B-IRES-EGFP was packaged into matured adenovirus successfully. The expression of UT-B mRNA in caco-2 cells was detected successfully by RT-PCR after infection with recombined adenovirus. Conclusion Recombinant adenovirus vector pAd-UT-B-IRES-EGFP which coding for the full length of rat UT-B gene was successfully constructed, which lays the experimental foundation for further studying on the function of UT-B.