目的构建人卵巢癌cDNA噬菌体表达文库。方法培养人卵巢肉瘤样癌细胞系细胞,提取总RNA,在RNA转录本5′末端开关机制(switching mechanismat5′end of RNAtranscript,SMART)寡核苷酸参与下,反转录合成cDNA第一链,用长距离聚合酶链式反应(l...目的构建人卵巢癌cDNA噬菌体表达文库。方法培养人卵巢肉瘤样癌细胞系细胞,提取总RNA,在RNA转录本5′末端开关机制(switching mechanismat5′end of RNAtranscript,SMART)寡核苷酸参与下,反转录合成cDNA第一链,用长距离聚合酶链式反应(long-distance PCR,LD-PCR)合成cDNA第二链并扩增双链DNA,鉴定扩增产物。采用层析离心柱除去小于500bp的片段后,与去磷酸化的SfiⅠ酶消化的λTriplEx2噬菌体载体连接,用GigapackⅢGold包装提取物体外包装噬菌体,感染宿主菌XL1-Blue,滴定原始文库以确定库容量大小,用蓝白斑试验测定重组率。随机挑取12个白色噬斑,采用平板裂解法扩增,用λ噬菌体DNA纯化试剂盒提取、纯化DNA,用SfiⅠ酶切鉴定插入片段大小。扩增原始库后保存备用。结果 PCR产物琼脂糖凝胶电泳在400bp^7kb的弥散背景下,可见到数条对应于优势转录本的特征性条带,符合哺乳动物细胞的特征。构建成含5×106pfu/ml的人卵巢癌cDNA噬菌体表达文库,重组率为99.0%,插入外源cDNA片段大小范围500bp^4kb,平均长度约1.79kb。扩增库滴度2.7×109pfu/ml。结论成功构建人卵巢癌cDNA噬菌体表达文库,可进一步用于筛选卵巢癌相关抗原基因。展开更多
目的为筛选食管癌相关的差异表达基因和基因组，建立哈萨克族食管癌表达文库。方法应用DAzLE系统构建哈萨克族食管癌cDNA表达文库，包括Trizol一步法提取总RNA，Oligo（dT）纤维素层析柱纯化mRNA，Super Script TMChoice System For cDN...目的为筛选食管癌相关的差异表达基因和基因组，建立哈萨克族食管癌表达文库。方法应用DAzLE系统构建哈萨克族食管癌cDNA表达文库，包括Trizol一步法提取总RNA，Oligo（dT）纤维素层析柱纯化mRNA，Super Script TMChoice System For cDNA Synthesis Kit合成cDNA，分级分离，纯化cDNA与质粒载体pSPORT1连接，电转化导入E.coliDH10B。结果14-18回收管合并，得到1.3×10^6个克隆，同时合成了肿瘤组和对照组的cDNA探针。结论成功构建的哈萨克族食管癌cDNA表达文库，可用于进一步筛选食管癌相关的差异表达基因，而且为搭建差异表达技术平台做好了准备工作。展开更多
Objective: The aim of this study was to use lung cancer targeting binding polypeptide ZS-9 to screen cDNA library of human lung cancer and obtain ZS-9 specific ligand to confirm tumor marker of non small-cell lung can...Objective: The aim of this study was to use lung cancer targeting binding polypeptide ZS-9 to screen cDNA library of human lung cancer and obtain ZS-9 specific ligand to confirm tumor marker of non small-cell lung cancer. Methods: Artificially synthesize biotin labeled peptide ZS-9, anchored ZS-9 in the enzyme label plate coupled by avidin, used ZS-9 as probe to screen cDNA library of human lung cancer, after screening, obtained bacteriophage clone specifically binding with anchored polypeptide ZS-9. Extracted plasmid of bacteriophage and performed sequencing after amplified by PCR. Results: It was demonstrated by bioinformatic analysis on the sequence of ligand binded by lung cancer specific peptide ZS-9 that the ligand was the cytoskeletal protein periplakin on the surface of lung cancer cells, suggesting that periplakin might be a new marker for non-small-cell lung cancer in lung cancer. Conclusion: Use specific lung cancer binding peptide to screen new tumor marker periplakin in lung cancer and further studies on its biologic functions in genesis and development of lung cancer are still needed.展开更多
目的构建人肺癌细胞A549λ噬菌体cDNA文库,为抗肺癌药物的筛选和药物作用分子机理的研究奠定基础。方法提取人肺癌细胞A549λ总RNA,用DnaseⅠ处理后,采用RNA 5’末端移动反转录(switching mechanism at 5′end of RNA transcript,SMART...目的构建人肺癌细胞A549λ噬菌体cDNA文库,为抗肺癌药物的筛选和药物作用分子机理的研究奠定基础。方法提取人肺癌细胞A549λ总RNA,用DnaseⅠ处理后,采用RNA 5’末端移动反转录(switching mechanism at 5′end of RNA transcript,SMART)技术合成双链cDNA,切胶回收去除短片段,依次用蛋白酶K和SfiⅠ进行酶切,并与经过酶切的λTriplEx2载体连接后进行体外包装,得到初级文库。初级文库扩增后,测定扩增文库滴度,并通过PCR鉴定插入片段。结果构建的人肺癌细胞A549λ噬菌体初级文库库容为1.88×10^(7)pfu/mL,扩增文库滴度为2.50×10^(9)pfu/mL,插入cDNA片段为500~3000 bp。结论成功构建了人肺癌细胞A549λ噬菌体cDNA文库,为进一步筛选治疗肺癌的药物作用分子机理奠定了基础。展开更多
文摘目的为筛选食管癌相关的差异表达基因和基因组，建立哈萨克族食管癌表达文库。方法应用DAzLE系统构建哈萨克族食管癌cDNA表达文库，包括Trizol一步法提取总RNA，Oligo（dT）纤维素层析柱纯化mRNA，Super Script TMChoice System For cDNA Synthesis Kit合成cDNA，分级分离，纯化cDNA与质粒载体pSPORT1连接，电转化导入E.coliDH10B。结果14-18回收管合并，得到1.3×10^6个克隆，同时合成了肿瘤组和对照组的cDNA探针。结论成功构建的哈萨克族食管癌cDNA表达文库，可用于进一步筛选食管癌相关的差异表达基因，而且为搭建差异表达技术平台做好了准备工作。
基金Supported by grants from the Science and Technology Planning Project of Guangdong Province (No. 2010B031600066 No. 2010B031500034+2 种基金 No. 2008B030303008)the Key Scientific Subject Foundation of Ministry of Education of China (No. 208105)the National Science and Technology Major Projects for New Drugs (No. 2011zx09102-001-31)
文摘Objective: The aim of this study was to use lung cancer targeting binding polypeptide ZS-9 to screen cDNA library of human lung cancer and obtain ZS-9 specific ligand to confirm tumor marker of non small-cell lung cancer. Methods: Artificially synthesize biotin labeled peptide ZS-9, anchored ZS-9 in the enzyme label plate coupled by avidin, used ZS-9 as probe to screen cDNA library of human lung cancer, after screening, obtained bacteriophage clone specifically binding with anchored polypeptide ZS-9. Extracted plasmid of bacteriophage and performed sequencing after amplified by PCR. Results: It was demonstrated by bioinformatic analysis on the sequence of ligand binded by lung cancer specific peptide ZS-9 that the ligand was the cytoskeletal protein periplakin on the surface of lung cancer cells, suggesting that periplakin might be a new marker for non-small-cell lung cancer in lung cancer. Conclusion: Use specific lung cancer binding peptide to screen new tumor marker periplakin in lung cancer and further studies on its biologic functions in genesis and development of lung cancer are still needed.
文摘目的构建人肺癌细胞A549λ噬菌体cDNA文库,为抗肺癌药物的筛选和药物作用分子机理的研究奠定基础。方法提取人肺癌细胞A549λ总RNA,用DnaseⅠ处理后,采用RNA 5’末端移动反转录(switching mechanism at 5′end of RNA transcript,SMART)技术合成双链cDNA,切胶回收去除短片段,依次用蛋白酶K和SfiⅠ进行酶切,并与经过酶切的λTriplEx2载体连接后进行体外包装,得到初级文库。初级文库扩增后,测定扩增文库滴度,并通过PCR鉴定插入片段。结果构建的人肺癌细胞A549λ噬菌体初级文库库容为1.88×10^(7)pfu/mL,扩增文库滴度为2.50×10^(9)pfu/mL,插入cDNA片段为500~3000 bp。结论成功构建了人肺癌细胞A549λ噬菌体cDNA文库,为进一步筛选治疗肺癌的药物作用分子机理奠定了基础。
