人早幼粒细胞cDNA文库的构建及其鉴定  

Construction of human promyelocytic cDNA library and its identification

作　　者：张曦 潘玉卿[2] 李静芳[1] 杨伟[1] 高艳章[1] 魏颖 石琼[1] 夏全松 杨丽[1] ZHANG Xi;PAN Yuqing;LI Jingfang;YANG Wei;GAO Yanzhang;WEI Ying;SHI Qiong;XIA Quansong;YANG Li(Department of Clinical Laboratory,Caner Hospital of Yunnan Province,Third Affiliated Hospital,Kunming Medical University,Kunming 650118,China;Department of Clinical Laboratory,First Affiliated Hospital,Kunming Medical University,Yunnan Institute of Experimental Diagnosis,YunnanKey Laboratory of Laboratory Medicine, Kunming 650032,China)

机构地区：[1]云南省肿瘤医院·昆明医科大学第三附属医院检验科,云南昆明650118 [2]昆明医科大学第一附属医院检验科云南省实验诊断研究所云南省检验医学重点实验室,云南昆明650032

出　　处：《吉林大学学报（医学版）》2019年第5期1015-1019,共5页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金地区项目资助课题(81760426);云南省科技厅-昆明医科大学联合专项基金资助课题(2015FB070);云南省教育厅科学研究基金项目资助课题(2018JS228)

摘　　要：目的:构建人早幼粒细胞HL60的cDNA文库,阐明文库内相关基因在急性早幼粒细胞白血病(APL)发生发展中的相关机制。方法:采用Trizol法提取HL60细胞总RNA,离心柱法分离纯化样本中的mRNA,逆转录PCR法合成cDNA第一链,酶促法直接合成cDNA第二链,胶回收目的长度的cDNA片段后将其与pPR3-N载体连接,通过同源重组方法在酵母Y187菌株中构建人早幼粒细胞cDNA文库。将培养的菌液涂布于LB平板进行库容量鉴定,PCR法鉴定插入片段大小及分布。结果:提取到的HL60细胞RNA条带清晰无降解且弥散度低,以纯化后的细胞mRNA为模板成功合成cDNA,经胶回收cDNA片段后顺利将其与pPR3-N载体连接。将重组载体转化至酵母菌株Y187,通过同源重组方法在菌株中成功构建人早幼粒细胞cDNA文库。在原始电转化菌稀释100倍后取10μL涂板,共长约250个克隆子,库容量为2.5×10^6CFU·mL^-1,转化后原始菌液共计5mL,总库容量为1.25×10^7CFU。文库的插入片段电泳检测,平均插入片段大于1200bp,阳性率为100%。结论:成功构建人早幼粒细胞的cDNA文库,为白血病的发病机制及治疗机制的研究奠定了基础。Objective : To construct a cDNA library of human promyelocytic leukemia (HL60), and to elucidate the mechanism of related genes in the pathogenesis and development of acute promyelocytic leukemia (APL). Methods : The total RNA of HL60 cells was extracted by Trizol method. The mRNA of samples were isolated and purified by centrifugal column method. The first strand of the cDNA was synthesized by reverse transcriptase-polymerase chain reaction (RT-PCR) while the second strand was synthesized by enzyme-catalyzed method. The cDNA fragments were recovered and then linked to the pPR3-N vector. The human promyelocytic cDNA library was constructed by homologous recombination in yeast strain Y187. The culture fluid was coated with LB plate to identify the library capacity, and PCR method was used to identify the size and distribution of the inserted fragment. Results : The RNA strand extracted from HL60 cells was clear, non-degradable and had low dispersion. The purified cell mRNA was used as template to synthesize the cDNA. After recovering the cDNA fragments, the cDNA fragments were successfully linked to the pPR3-N vector. The recombinant vector was transformed into yeast strain Y187, and the human promyelocytic cDNA library was successfully constructed by homologous recombination. The original electro transformation bacteria were diluted 100 times and 10 μL coating plate was taken, about 250 clones were generated. The total library capacity was 2.5 ×10^6 CFU mL^-1 and the total library capacity was 1.25×10^7CFU for 5 mL of transformed original bacterial solution. The average insertion fragment was more than 1 200 bp , and the positive rate was 100%. Conclusion : The human promyelocyic cDNA library is successfully constructed, and it lays a foundation for studying the pathogenesis and therapeutic mechanism of leukemia.

关 键 词：白血病 人早幼粒细胞 酵母双杂交 CDNA文库 

分 类 号：R733.7[医药卫生—肿瘤]