SMART 技术构建肾乳头状腺癌及癌旁组织cDNA文库.docVIP

SMART 技术构建肾乳头状腺癌及癌旁组织cDNA文库 　 作者：成瑜 李旭 陈葳 杨玉琮 【关键词】 肾肿瘤 　　Construction of cDNA libraries of renal papillary adenocarcinoma tissue and its side tissue with SMART technology 　　【Abstract】 AIM: To construct the cDNA libraries of renal papillary adenocarcinoma tissue and its side tissue by SMART technology. METHODS: Switch mechanism at 5′end of mRNA template technology, SMART technology, was used to construct the libraries. The purified Poly(A)+RNA was used as template, Powerscript reverse transcriptase was used to transcribe and 5′oligo as a short extended template was added to the 5′ end of mRNA to enrich the fulllength cDNAs. After amplification, the ds cDNA was digested by sfiI, sizefractionated by columns (CHROMA SPIN400) and then recombined to the λTripIEx2 vector. After package, the libraries were tittered, the rate of recombination ( blue/white) was determined and then the cDNA libraries were amplified. RESULTS: The titer of the cDNA libraries of renal papillary adenocarcinoma tissue and its side tissue were 1.4×106 pfu#12539;mL-1 and 2.6×106 pfu#12539;mL-1, respectively. After amplification, the rates increased to 6×1011 pfu#12539;mL-1 and 9×1011 pfu#12539;mL-1, respectively. CONCLUSION: Our successfully constructed cDNA libraries are fulllength libraries with high efficiency and can be screened by probes and antibodies to find the associated genes of the renal papillary adenocarcinoma. 　　【Keywords】 kidney neoplasms; cDNA library; SMART 　　【摘要】 目的： 运用SMART 技术构建肾乳头状腺癌及癌旁组织的cDNA文库. 方法： 运用mRNA 5′末端的模板转换方法，即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. cDNA扩增后， 经sfiI酶切、CHROMA SPIN400洗脱，与λTripIEx2载体连接并用载体蛋白包装后，测定滴度和重组率，并扩增，建成cDNA文库. 结果： 构建的肾乳头状腺癌及癌旁组织cDNA文库滴度分别为1.4×106 pfu#12539;mL-1和2.6×106 pfu#12539;mL-1，重组率均gt;98%，扩增后滴度分别达6×1011 pfu#12539;mL-1和9×1011 pfu#12539;mL-1. 结论： 我们构建的人肾乳头状腺癌及癌旁组织cDNA文库为高效、全长cDNA文库，可以用做探针、抗体等免疫学筛选, 进一步探寻与肾乳头状腺癌相关的基因. 　　【关键词】 肾肿瘤；cDNA文库;SMART技术 　　0引言 　　肾癌是泌尿系统最常见的恶性肿瘤之一，生物学行为极为多变，其发病机制及发生发展的分子生物