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

目的P53作为转录因子在细胞应激时呈活化型，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P53呈非活化状态，其中包括P53C-末端负调控序列的作用。该研究目的是制备携带垒长和缺失这些负调控序列P53两种重组腺病毒和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。材料和方法利用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。三种不同浓度病毒分别感染人肺癌801D细胞，FCM分析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）没有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p53两种重组腺病毒，Ad—p53（del），Ad-p53（wtp）及空载体Ad-（empty）。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 P53 is an activated form as a transcription factor in cell stress regulating cell cycle and programmed cell death to inhibit tumor growth. P53 usually keeps a non-activated form through various mechanisms, including the action of P53 C-terminal negative regulatory sequences. The objective was to prepare the two types P53 recombinant adenoviruses carrying 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 diagram. Material and Methods Using pAdEasy-Track vector system, the P53 recombinant plasmids was constructed and the homologous recombinants in E. coli was produced. Three kinds of recombinant adenovirus were produced by transfection to L293 cell. Three different concentration virus infected human lung cancer 801D cells respectively, and exogenous GFP expression was detected by FCM scatter plot. Result P53 recombinant adenovimses named Ad-P53（wtp）, Ad-P53（del） and Ad-（empty carrier） were produced. Sequencing indicated that the Ad-P53（del） was loss of 111 bases before stop codon TGA and of 3＇untranslated region at P53. The Ad-P53（wtp） had no loss of any p53 base, and the Ad（empty carrier） no p53 sequence. FCM scatter plot showed the percentage of expressed GFP produced by 801D cells with three kinds of viral infection was almost same and increased with the virus concentration. 801D contained cells with different ratio of fluorescence intensity. Conclusion Recombinant adenovirus Ad-p53（del）, pA-p53（wtp） and Ad-（empty） were constructed. The results of FCM scatter plot proved that the virus system is reliable,and can detect quantitatively exogenous GFP expression which can provide an accurate method of selecting concentration for virus infecting cells.