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背景:酵母双杂交技术是目前应用最成功的研究蛋白质相互作用的技术平台。目的:运用SMART(switching mechanism at5′end of RNA transc-ript)技术构建人肺鳞状细胞癌旁组织的酵母双杂交cDNA文库。设计、时间及地点:单一样本观察,于2006-07/2007-01在四川大学华西基础医学与法医学院病理生理学感染免疫实验室完成。材料:人肺鳞状细胞癌旁组织由四川大学华西医学中心附属第一医院胸外科提供。方法:运用SMART技术,以纯化的Poly(A)+RNA为模板,用Powerscript逆转录酶进行转录,并在mRNA的5’末端添加一段5’-oligo作为延伸后的模板,从而富集全长cDNA。纯化的双链cDNA与线性化pGADT7-Rec质粒载体共同转化酵母菌株感受态AH109,经胞内同源重组形成环状文库质粒。应用营养缺陷的方法在SD/-Leu平板上筛选并收集所有克隆从而获得酵母双杂交的人肺鳞状细胞癌旁组织cDNA文库。主要观察指标:采用琼脂糖凝胶电泳观察人肺鳞状细胞癌旁组织的cDNA文库构建结果,并进行文库容量及多态性鉴定。结果:①总RNA和mRNA纯度较好,比较完整。②双链cDNA文库大小介于0.5～3kb之间。③共获得1.2×106转化子,重组子不同插入片断大小在0.3～2kb之间,文库具有较好的多样性。结论:在酵母菌株AH109成功构建了用于酵母双杂交的人肺鳞状细胞癌旁组织cDNA文库。
Background: Yeast two-hybrid technology is the most successful technology platform for protein interaction. OBJECTIVE: To construct yeast two-hybrid cDNA library of human lung squamous cell carcinoma adjacent tissues by using SMART (switching mechanism at5’end of RNA transc-ript) technology. DESIGN, TIME AND SETTING: A single sample observation was performed at the Pathophysiology Immunization Laboratory, West China School of Fundamental Medicine and Forensic Medicine, Sichuan University from July 2006 to January 2007. MATERIALS: Human lung squamous cell carcinoma adjacent tissues were provided by Department of Thoracic Surgery, First Affiliated Hospital of West China Medical Center, Sichuan University. Methods: Using SMART technology, the purified Poly (A) + RNA was used as a template to transcribe with Powerscript reverse transcriptase and a 5’-oligo was added to the 5 ’end of the mRNA as an extension template to enrich the full length cDNA. The purified double-stranded cDNA was co-transformed into competent yeast strain AH109 with a linearized pGADT7-Rec plasmid vector to form a circular library plasmid by homologous recombination. Methods of Applying Nutritional Defects All clones were screened and collected on SD / -Leu plates to obtain a yeast two-hybrid human lung squamous cell para-cancerous tissue cDNA library. MAIN OUTCOME MEASURES: The cDNA library construction of human lung squamous cell carcinoma adjacent tissues was observed by agarose gel electrophoresis, and the library capacity and polymorphism were identified. Results: ① The total RNA and mRNA purity is good, more complete. ② double-stranded cDNA library size between 0.5 ~ 3kb. ③ A total of 1.2 × 106 transformants were obtained. The sizes of the inserted fragments in the recombinants ranged from 0.3kb to 2kb, and the libraries had good diversity. Conclusion: The human lung squamous cell carcinoma adjacent tissue cDNA library for yeast two-hybrid system was successfully constructed in yeast strain AH109.