热休克蛋白70重组腺病毒载体的构建及鉴定

CONSTRUCTION AND IDENTIFICATION OF A RECOMBINANT ADENOVIRUS VECTOR HSP70

目的构建人热休克蛋白70(HSP70)的腺病毒表达载体,为进一步研究HSP70对应激状态下细胞的保护作用提供实验基础。方法采用AdEasy系统构建携带外源性HSP70编码基因的重组腺病毒载体pAd-HSP70,脂质体法转染人胚肾293细胞,包装产生重组腺病毒vAdvHSP。收获病毒进行滴度鉴定,RT-PCR方法检测目的基因在Hela细胞的转录表达。结果PCR、序列测定以及限制性酶切证实HSP70基因正确插入腺病毒表达载体,并在HEK293细胞中包装出重组腺病毒;荧光显微镜下可观察到GFP的表达,收获病毒的滴度为3.2×1012U/L。结论成功构建pAd-HSP70重组腺病毒表达载体,且重组腺病毒在Hela细胞能有效转录。

Objective To construct recombinant adenovirus expression vector containning human HSP70 and provide a base for investigating the protection of HSP70 on the cellunder stringent state. Methods The recombinant adenovirus vector pAd HSP70 carrying HSP70 was constructed with AdEasy system and then transfected 293 ceils to produce recombinant adenovirus vAd-HSP70. The viral titer was tested. The expression of target gene in Hela was tested by RT-PCR. Results It was confirmed by PCR, sequencing identification and restrictive analysis that the target gene was cloned correctly to the shuttle plasmid. The ex pression of GFP could be observed by fluorescence microscope. The viral titer was 3.2 × 10^12 U/L, and RT-PCR indicated that HSP70 could effectively express in Hela. Conclusion Recombinant adenovirus vector was constructed successfully and packed in 293 cells.