制备两种p53重组腺病毒和流式细胞仪定量外源绿荧光蛋白表达

Preparation of Two Types p53 Recombinant Adenovirus and Quantitative Exogenous Expression of Green Fluorescence Protein by Flow Cytometry

背景与目的p53作为转录因子,在细胞应激时呈活化型,可调控细胞周期和程序性死亡抑制肿瘤生长,通常通过各种机制可使p53呈现非活化状态,其中包括p53C-末端负调控序列的作用。本研究旨在制备携带全长和缺失这些负调控序列p53的两种重组腺病毒,并采用流式细胞仪散点图(flow cytometry scatter plot,FCM)检测人肺癌细胞外源绿荧光蛋白(green fluorescence protein,GFP)表达。方法利用pAdEasy-Track载体系统,构建两种p53重组质粒并在细菌中产生重组体,转染L293细胞产生三种重组腺病毒,测序证明。三种不同浓度病毒分别感染人肺癌801D细胞,FCM scatter plot检测其GFP表达。结果测序证明重组腺病毒:Ad-p53(del)缺失p53C-末端终止密码子前111个碱基和非编码区,Ad-p53(wtp)无p53碱基缺失。Ad-(empty carrier)无p53。FCM scatter plot显示三种病毒感染801D细胞表达GFP百分率接近并随病毒浓度递增。801D包含了不同荧光强度比率的细胞。结论构建和制备了去C-末端p53和全长p53的两种重组腺病毒:Ad-p53(del)、Ad-p53(wtp)及空载体Ad-(empty carrier)。流式细胞仪散点图证明该病毒试验系统可靠,可定量外源GFP表达为病毒感染细胞选择浓度提供准确方法。

Background and objective The p53 as a transcription factor in cell stress was activated to regulate cell cycle and programmed cell death to inhibit tumor growth. Usually, p53 is kept in non-activated state through various mechanisms, including the action of p53 C-terminal negative regulatory sequences. The purpose of the study is to prepare the two types p53 recombinant adenoviruses that carry full-length p53 as well as deletion of negative regulatory sequences at p53 C-terminus and to detect exogenous GFP expression in human lung cancer cell infected-virus by FCM scatter plot. Methods Using pAdEasy-Track vector system the p53 recombinant plasmids was constructed and the homologous recombinants in E.coli was produced. The three kinds of recombinant adenovirus in L293 cells was generated, sequencing proved. Exogenous GFP expression in human lung cancer 801D cells infectedvirus was detected by FCM scatter plot. Results p53 recombinant adenoviruses named Ad-p53（wtp）, Ad-p53（del） and Ad-（empty carrier） were produced. Results of sequences indicate that the Ad-p53（del） was deletion of 111 bases before stop codon TGA and of 3 untranslated region at p53, the Ad-p53（wtp） no loss of any p53 base, the Ad-（empty carrier） no p53 sequence. FCM scatter plot indicate the percentage of 801D cells expressed GFP with three kinds of viral infection was almost same and was increased with the virus density. 801D contains ratio of cells with different fluorescence intensity. Conclusion The preparation of recombinant adenovirus, Ad-p53（del）, pA-p53（wtp） and Ad-（empty carrier）. The cells expressed-GFP can be quantitatively detected by FCM scatter plot. It was provide that the reliability of the virus system and accurate method for selecting viruses density to infecting cells.