T7噬菌体展示人肝癌cDNA文库筛选人CD8~+T淋巴细胞特异结合分子
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摘要
目的:利用噬菌体表面展示技术从人肝癌T7 cDNA文库中筛选出能与人CD8~+T淋巴细胞特异结合的蛋白分子。方法:以人CD8~+T淋巴细胞为筛选靶标,对人肝癌T7 cDNA噬菌体肽库进行4轮"吸附-洗脱-扩增"生物淘选与富集,ELISA鉴定特异结合的噬菌体单克隆;对获得的阳性克隆裂解液进行PCR扩增及DNA测序,然后进行生物信息学分析,确定所编码的结合蛋白。结果:经过4轮筛选,结合人CD8~+T淋巴细胞的噬菌体克隆得到富集,通过测序与同源性比对分析获得17个特异性结合的多肽序列;生物信息学分析发现阳性噬菌体克隆的同源蛋白中,可能与肝癌免疫逃逸相关的分子有Unc-51样自噬活化激酶1(ULK1)、一般受体磷酸肌醇相关支架蛋白1(GRASP)、中间α球蛋白抑制因子H1(ITIH1)、富含亮氨酸重复8家族成员D(LRRC8D)和CD164等。结论:从人肝癌T7 cDNA噬菌体肽库中筛选获得了能与人CD8~+T淋巴细胞特异结合的分子,为阐明人肝癌发生发展过程中肿瘤免疫逃逸相关机制奠定了基础。
Objective: To screen human hepatoma T7 phage cDNA library for proteins binding with human CD8~+T lymphocytes by using phage display technique.Methods: By using human CD8~+T lymphocytes as the selective target, the T7 select human hepatoma cDNA library was biopanned for four times and positive phage clones were selected. The positive plaques were identified by ELISA and DNA sequencing. Homology analysis and bioinformatics analysis were used to investigate the function of the candidate molecules. Results: T7 phage cDNA inserts were acquired after 4 rounds of biopanning. We have obtained a series of tumor-associated molecules that can specifically bind human CD8~+T lymphocytes, such as ULK1, GRASP, ITIH1, LRRC8 D and CD164. Conclusion: We obtained some candidate molecules that may interact with human CD8~+T lymphocytes through cDNA library phage display method. The present study laid a foundation for further exploring the mechanism of the effect of cancer cells on CD8~+T lymphocytes and targeted therapy for hepatoma.
Objective: To screen human hepatoma T7 phage cDNA library for proteins binding with human CD8~+T lymphocytes by using phage display technique.Methods: By using human CD8~+T lymphocytes as the selective target, the T7 select human hepatoma cDNA library was biopanned for four times and positive phage clones were selected. The positive plaques were identified by ELISA and DNA sequencing. Homology analysis and bioinformatics analysis were used to investigate the function of the candidate molecules. Results: T7 phage cDNA inserts were acquired after 4 rounds of biopanning. We have obtained a series of tumor-associated molecules that can specifically bind human CD8~+T lymphocytes, such as ULK1, GRASP, ITIH1, LRRC8 D and CD164. Conclusion: We obtained some candidate molecules that may interact with human CD8~+T lymphocytes through cDNA library phage display method. The present study laid a foundation for further exploring the mechanism of the effect of cancer cells on CD8~+T lymphocytes and targeted therapy for hepatoma.
引文
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[3]Baitsch L,Fuertes-Marraco S A,Legat A,et al.The three main stumbling blocks for anticancer T cells[J].Trends Immunol,2012,33(7):364-372.
[4]Yi J S,Cox M A,Zajac A J.T-cell exhaustion:characteristics,causes and conversion[J].Immunology,2010,129(4):474-481.
[5]Rosenberg A,Mierendorf R.T7 Select phage display system:a powerful new protein display system based on bacteriophage T7[J].Novation,1996,6:1-6.
[6]Altschul S F,Madden T L,Schaffer A A,et al.Gapped BLAST and PSI-BLAST:a new generation of protein database search programs[J].Nucleic Acids Res,1997,25(17):3389-3402.
[7]Samoylova T I,Morrison N E,Globa L P,et al.Peptide phage display:opportunities for development of personalized anticancer strategies[J].Anticancer Agents Med Chem,2006,6(1):9-17.
[8]Kehoe J W,Kay B K.Filamentous phage display in the new millennium[J].2005,105(11):4056-4072.
[9]Krag D N,Shukla G S,Shen G P,et al.Selection of tumor-binding ligands in cancer patients with phage display libraries[J].Cancer Res,2006,66(15):7724-7733.