Construction and quality identification of T7 recombination expression cDNA library form human lung cancer

Objective:Currently,only a limited numbers of tumor markers for non small lung cancer(NSCLC) diagnosis,new biomarker,such as serum autoantibodies may improve the early detection of lung cancer.Our objective is construction human lung squamous carcinoma and adenocarcinoma T7 phage display cDNA library from the tissues of NSCLC patients.Methods:mRNA was isolated from a pool of total RNA extract from NSCLC tissues obtained from 5 adenocarcinomas and 5 squamous carcinomas,and then mRNA was reverse transcribed into double stranded cDNA.After digestion,the cDNA was inserted into T7Select 10-3 vector.The phage display cDNA library was constructed by package reaction in vitro and plate proliferation.Plaque assay and PCR were used to evaluate the library.Results:Two T7 phage display cDNA library were established.Plaque assay show the titer of lung squamas carcinoma library was 1.8 × 106 pfu,and the adenocarcinoma library was 5 × 106 pfu.The phage titer of the amplified library were 3.2 × 1010 pfu/mL and 2.5 × 1010 pfu/mL.PCR amplifica-tion of random plaque show insert ratio were 100%(24/24) in adenocarcinoma library and 95.8% in human lung squamas carcinoma library(23/24).Insert range from 300 bp to 1 500 bp.Conclusion:Two phage display cDNA library from NSCLC were constructed.

应用SEREX技术筛选和鉴定非小细胞肺癌肿瘤相关抗原

背景与目的目前用于非小细胞肺癌诊断的标志物为数不多,为寻找更多的早期诊断和靶向治疗相关的标志物,本研究应用SEREX技术筛选与鉴定非小细胞肺癌肿瘤相关抗原。方法使用非小细胞肺癌患者血清对人肺鳞癌和腺癌噬菌体展示文库进行生物淘选和SEREX筛选。PCR扩增阳性克隆插入片段后测序,结果与GeneBank数据库中已知基因进行同源性比较,分析其生物学特性。结果用非小细胞肺癌血清对噬菌体展示文库进行筛选,共获得31个阳性克隆,测序后发现其代表15个基因,其中12个与已知cDNA序列同源,与肺癌或其它肿瘤的发生、发展关系密切;另外3个未发现有同源基因,可能是新基因。结论应用SEREX技术发现15个肺癌相关抗原和3个肺癌相关抗原的候选基因,为进一步的深入研究打下良好的基础。

应用SEREX技术筛选和鉴定非小细胞肺癌肿瘤相关抗原

背景与目的目前用于非小细胞肺癌诊断的标志物为数不多，为寻找更多的早期诊断和靶向治疗相关的标志物，本研究应用SEREX技术筛选与鉴定非小细胞肺癌肿瘤相关抗原。方法使用非小细胞肺癌患者血清对人肺鳞癌和腺癌噬菌体展示文库进行生物淘选和SEREX筛选。PCR扩增阳性克隆插入片段后测序，结果与GeneBank数据库中已知基因进行同源性比较，分析其生物学特性。结果用非小细胞肺癌血清对噬菌体展示文库进行筛选，共获得31个阳性克隆，测序后发现其代表15个基因，其中12个与已知cDNA序列同源，与肺癌或其它肿瘤的发生、发展关系密切；另外3个未发现有同源基因，可能是新基因。结论应用SEREX技术发现15个肺癌相关抗原和3个肺癌相关抗原的候选基因，为进一步的深入研究打下良好的基础。

Screening study on new tumor marker periplakin for lung cancer

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.

非小细胞肺癌T7噬菌体展示文库的构建

背景与目的SEREX(Serological analysis of recombinant expression libraries)是近年来发展起来的一种寻找肿瘤相关抗原的方法,通过与噬菌体展示技术结合,可以有效和快速的筛选肿瘤相关抗原。本研究拟构建人肺鳞癌和肺腺癌T7噬菌体展示cDNA文库,为进一步筛选和鉴定非小细胞肺癌相关抗原打下基础。方法诊断明确的肺鳞癌和腺癌患者手术标本各5例,Trizol试剂提取总RNA,Straight A's系统分离鳞癌和腺癌mRNA,逆转录合成双链cDNA后,与T7Select 10-3载体连接,体外包装并扩增得到人肺鳞癌和腺癌T7噬菌体展示cDNA文库。结果构建肺鳞癌和肺腺癌T7噬菌体展示cDNA文库各一个,原始文库重组克隆数分别为鳞癌1.8×106pfu,腺癌5×106pfu。扩增后滴度为3.2×1010pfu/mL和2.5×1010pfu/mL。随机从2个文库中各抽取24个克隆PCR扩增插入片断,电泳结果显示肺腺癌文库所有克隆均有插入片段,肺鳞癌文库插入率超过95%(23/24)。两个文库插入片断大小均在(300-1500)bp之间。结论成功构建了人肺鳞癌和腺癌T7噬菌体展示cDNA文库,为下一步筛选和鉴定非小细胞肺癌相关基因奠定基础。

非小细胞肺癌T7噬菌体展示文库的构建

背景与目的SEREX（Serological analysis of recombinant expression libraries）是近年来发展起来的一种寻找肿瘤相关抗原的方法，通过与噬菌体展示技术结合，可以有效和快速地筛选肿瘤相关抗原。本研究拟构建人肺鳞癌和肺腺癌T7噬菌体展示cDNA文库，为进一步筛选和鉴定非小细胞肺癌相关抗原打下基础。方法诊断明确的肺鳞癌和腺癌患者手术标本各5例，Trizol试剂提取总RNA，Straight A’s系统分离鳞癌和腺癌mRNA，逆转录合成双链cDNA后，与T7Select10-3载体连接，体外包装并扩增得到人肺鳞癌和腺癌T7噬菌体展示cDNA文库。结果构建肺鳞癌和肺腺癌T7噬菌体展示cDNA文库各一个，原始文库重组克隆数分别为鳞癌1．8×10^5pfu，腺癌5×10^6pfu。扩增后滴度为3．2×10^10pfu／ml和2．5×10^10pfu／ml。随机从2个文库中各抽取24个克隆PCR扩增插入片断，电泳结果显示肺腺癌文库所有克隆均有插入片段，肺鳞癌文库插入率超过95％（23／24）。两个文库插入片断大小均在300～1500bp之间。结论成功构建了人肺鳞癌和腺癌T7噬菌体展示cDNA文库，为下一步筛选和鉴定非小细胞肺癌相关基因奠定基础。