大鼠组织激肽释放酶8重组腺病毒载体的构建及鉴定

目的构建大鼠组织激肽释放酶8（KLK8）的腺病毒表达载体,为进一步研究KLK8对心肌细胞的作用提供实验基础。方法采用AdEasy系统构建携带外源性KLK8编码基因的重组腺病毒载体pAd-KLK8,转染AD-293细胞包装产生重组腺病毒Ad-KLK8,TCID50法对收获病毒进行滴度鉴定,RT-PCR及Western-blot方法检测目的基因在Hela细胞的表达。结果 PCR、序列测定以及限制性酶切证实KLK8基因正确插入腺病毒表达载体,并在AD-293细胞中包装出重组腺病毒;收获病毒的滴度为3.16×1012 IU/mL。结论成功构建pAd-KLK8重组腺病毒表达载体,且重组腺病毒在Hela细胞能有效表达。

Construction and identification of a recombinant adenovirus vector KLK8

Objective To construct recombinant adenovirus expression vector containing rat KLK8and provide a base for investigating the function of KLK8on myocardial cells.Methods The recombinant adenovirus vector pAd-KLK8carrying KLK8was constructed with AdEasy system and then transfected AD-293cells to produce recombinant adenovirus Ad-KLK8.TCID50was used to measure the virus titers.The expression of target gene in Hela was tested by RT-PCR and Western-blot.Results It was confirmed by PCR,sequencing identification and restrictive analysis that the target gene was cloned correctly to the shuttle plasmid.The titer of the recombinant adenovirus was 3.16×1012IU/mL.Conclusion Recombinant adenovirus vector is constructed successfully and recombinant adenovirus Ad-KLK8can efficiently expressed in Hela.