rAd-preS1基因治疗载体的构建及对肝细胞嗜向性检测

Construction of gene therapy vector rAd-preS1 and identification of liver cell targeting function

摘要:

目的：制备携带PreS1基因的重组腺病毒基因治疗载体，研究其对肝细胞的嗜向性。方法：设计合成含PreS1的腺病毒纤毛Fiber基因片段，置换野生型骨架质粒Fiber片段，同源重组法构建重组体rAd-PreS1，酶切鉴定后HEK293包装生成腺病毒，抽提蛋白Western blot检测目的蛋白PreS1表达。重组腺病毒rAd-preS1分别转染人正常肝细胞L02、人肺癌细胞 A549、人喉癌细胞Hep2、人卵巢癌细胞SKOV3，同时野生腺病毒rAd作为阴性对照，荧光显微镜下观察各细胞内绿色荧光蛋白（green fluorescent protein，GFP）表达程度，用细胞的病毒滴度值表示，检测其对肝细胞的嗜向性。结果：经酶切鉴定成功构建rAd-preS1载体，Western blot示蛋白分子量大小均约为63 kD目的条带。rAd-preS1感染L02细胞组GFP表达程度明显强于A549、Hep2及SKOV3细胞组（P均是0.000），而A549、Hep2及SKOV3各细胞组间GFP表达差异无统计学意义（P >0.05）；对照组各细胞组间GFP表达差异无统计学意义（P >0.05）。结论：含PreS1的重组腺病毒rAd-preS1显示出对人肝细胞具有较强嗜向性，而对非肝细胞无明显效果，这为进一步研究肝脏疾病靶向基因治疗奠定了基础。

Abstract:

Objective：To prepare the recombinant adenoviruses carrying gene fragment PreS1 and to detect tropism of liver cell infect－ed-virus. Methods：Fiber DNA gene fragment containing PreS1 gene was designed and amplified and inserted into the pAdEasy-1. Fiber DNA gene fragment replaced wild fiber DNA fragment then produced rAd-preS1 by homogenous recombination. After being confirmed by restriction enzyme digesion，human embryonic kidney cell lines were transfected with pAd-preS1 to get rAd-preS1. Protein expression of preS1 was detected by Western blot. Human normal live cell L02，human lung cancer cell A549，human larngy－neal cancer cell Hep2，human overian cancer cell SKOV3 were infected with rAd-preS1 respectively and rAd was taken as negative control group. Green fluorescent protein（GFP） expression of each cell were observed and manifested by virus titers. Results：Restric－tion digestion confirmed the consistent of recombinant vertor. Western blot results showed that protein molecular weight was about 63 kD fragment stripe. Transfection results showed that GFP expression were higher in L02 group than in A549，Hep2，SKOV3 groups（P value were 0.000）. There was no staitical differnece in GFP expression among A549，Hep2 and SKOV3 groups（P >0.05）. There was no staitical differnece in GFP expression among control groups（P >0.05）. Conclusions：Recombinant adenovirus vector rAd-preS1 demonstrate its function of tropism to liver cell in vitro，which will be the foundation for the research of gene therapy of PreS1 for hepatic diseases.