EGFRvⅢ蛋白的生物信息学分析及其在HEK293T细胞中的表达
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摘要
目的对表皮生长因子受体Ⅲ(epithelial growth factor receptor variantⅢ,EGFRvⅢ)突变蛋白进行生物信息学分析,并构建表达该突变蛋白的HEK293T细胞系。方法根据NCBI公布的EGFR外显子序列,拼接成EGFRvⅢ编码基因,运用Protparam、ProtScale、SignalP 4. 1 Server、TMHMM Server v. 2. 0、SOPMA、SWISS-MODEL和SMART软件对其编码的氨基酸序列进行生物信息学分析。设计融合PCR引物,克隆EGFRvⅢ基因,并连接到真核表达载体pEGFP-N1上,转染HEK293T细胞,Western blot法检测EGFRvⅢ-EGFP的表达。结果 EGFRvⅢ蛋白含943个氨基酸,预测相对分子质量约为104 335;理论等电点为6. 22;平均亲水性系数为-0. 306,亲水性氨基酸占63. 6%,疏水性氨基酸占36. 4%。EGFRvⅢ蛋白N-端1～24个氨基酸序列为信号肽序列,是一个细胞质膜定位的单次跨膜蛋白。二级结构预测显示,EGFRvⅢ蛋白主要由α螺旋和无规则卷曲组成,三级结构预测与二级结构预测结果一致。结构域预测显示,胞内酪氨酸激酶结构域完整,胞外受体结构域受损。融合PCR克隆出EGFRvⅢ编码基因,并在HEK293T细胞中表达出目的蛋白EGFRvⅢ-EGFP。结论成功获得表达EGFRvⅢ蛋白的HEK293T细胞系,为开展肿瘤浸润淋巴细胞免疫治疗或T细胞受体的研究奠定了基础。
Objective To analyze the bioinformatics of epithelial growth factor receptor variant Ⅲ(EGFRv Ⅲ) and construct a HEK293 T line expressing the variant. Methods According to the exons of EGFR gene published in NCBI,the coding sequence of EGFRvⅢ was spliced,of which the amino acid sequence was analyzed for bioinformatics by using Protparam,ProtScale,SignalP 4. 1 Server,TMHMM Server v. 2. 0,SOPMA,SWISS-MODEL and SMART software.EGFRv Ⅲ gene was cloned by using the designed fusion PCR primers and inserted into eukaryotic expression vector pEGFR-N1. The constructed recombinant plasmid was transfected to HEK293 T cells. The expression of EGFRv Ⅲ-EGFP was determined by Western blot. Results EGFRvⅢ protein,with a predicted relative molecular mass of about 104 335,a theoretical isoelectric point of 6. 22 and a mean hydropathicity coefficient of-0. 306,contained 943 amino acids,in which hydrophilic and hydrophobic amino acids accounted for 63. 6% and 36. 4% respectively. The amino acids 1 ～ 24 at N-terminus of EGFRv Ⅲ protein was signal peptide,which was a single transmembrane protein located in the cell membrane. The secondary structure prediction showed that the EGFRvⅢ protein was mainly composed of alpha helix and random coil. The result of tertiary structure prediction was consistent with that of secondary structure predication. The domain prediction showed that the intrace llular tyrosine kinase domain was complete,while the extracellular receptor domain was impaired. Using fusion PCR,the EGFRvⅢ encoding gene was cloned and the target protein was expressed in HEK293 T cells. Conclusion The HEK293 T cell line for expression of EGFRv Ⅲ protein was successfully obtained,which laid a foundation of developing immunotherapy of tumor infiltrating lymphocytes or investigating the T cell receptor.
Objective To analyze the bioinformatics of epithelial growth factor receptor variant Ⅲ(EGFRv Ⅲ) and construct a HEK293 T line expressing the variant. Methods According to the exons of EGFR gene published in NCBI,the coding sequence of EGFRvⅢ was spliced,of which the amino acid sequence was analyzed for bioinformatics by using Protparam,ProtScale,SignalP 4. 1 Server,TMHMM Server v. 2. 0,SOPMA,SWISS-MODEL and SMART software.EGFRv Ⅲ gene was cloned by using the designed fusion PCR primers and inserted into eukaryotic expression vector pEGFR-N1. The constructed recombinant plasmid was transfected to HEK293 T cells. The expression of EGFRv Ⅲ-EGFP was determined by Western blot. Results EGFRvⅢ protein,with a predicted relative molecular mass of about 104 335,a theoretical isoelectric point of 6. 22 and a mean hydropathicity coefficient of-0. 306,contained 943 amino acids,in which hydrophilic and hydrophobic amino acids accounted for 63. 6% and 36. 4% respectively. The amino acids 1 ～ 24 at N-terminus of EGFRv Ⅲ protein was signal peptide,which was a single transmembrane protein located in the cell membrane. The secondary structure prediction showed that the EGFRvⅢ protein was mainly composed of alpha helix and random coil. The result of tertiary structure prediction was consistent with that of secondary structure predication. The domain prediction showed that the intrace llular tyrosine kinase domain was complete,while the extracellular receptor domain was impaired. Using fusion PCR,the EGFRvⅢ encoding gene was cloned and the target protein was expressed in HEK293 T cells. Conclusion The HEK293 T cell line for expression of EGFRv Ⅲ protein was successfully obtained,which laid a foundation of developing immunotherapy of tumor infiltrating lymphocytes or investigating the T cell receptor.
引文
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[18] HAN J,CHU J,KEUNG CHAN W,et al. CAR-engineered NK cells targeting wild-type EGFR and EGFRvⅢenhance killing of glioblastoma and patient-derived glioblastoma stem cells[J]. Sci Rep,2015,5:11483. doi:10. 1038/srep11483.
[19] LIU K,LIU X,PENG Z,et al. Retargeted human avidin-CAR T cells for adoptive immunotherapy of EGFRvⅢexpressing gliomas and their evaluation via optical imaging[J]. Oncotarget,2015,6(27):23735-23747.
[20] MIAO H,CHOI B D,SURYADEVARA C M,et al. EGFRvⅢ-specific chimeric antigen receptor T cells migrate to and kill tumor deposits infiltrating the brain parenchyma in an invasive xenograft model of glioblastoma[J]. PLoS One,2014,9(4):e94281. doi:10. 1371/journal. pone. 0094281.
[21] O'ROURKE D M,NASRALLAH M P,DESAI A,et al. A single dose of peripherally infused EGFRvⅢ-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma[J]. Sci Transl Med,2017,9(399):pii:eaaa0984. doi:10. 1126/scitranslmed. aaa0984.
[22] DRAPER L M,KWONG M L,GROS A,et al. Targeting of HPV-16+Epithelial Cancer Cells by TCR Gene Engineered T Cells Directed against E6[J]. Clin Cancer Res,2015,21(19):4431-4439.
[2] NORMANNO N,DE LUCA A,BIANCO C,et al. Epidermal growth factor receptor(EGFR)signaling in cancer[J]. Gene,2006,366(1):2-16.
[3] ARTEAGA C L. Epidermal growth factor receptor dependence in human tumors:more than just expression[J]. Oncologist,2002,7(Suppl 4):31-39.
[4] HUANG H S,NAGANE M,KLINGBEIL C K,et al. The enhanced tumorigenic activity of a mutant epidermal growth factor receptor common in human cancers is mediated by threshold levels of constitutive tyrosine phosphorylation and unattenuated signaling[J]. J Biol Chem,1997,272(5):2927-2935.
[5] GAN H K,CVRLJEVIC A N,JOHNS T G. The epidermal growth factor receptor variantⅢ(EGFRvⅢ):where wild things are altered[J]. FEBS J,2013,280(21):5350-5370.
[6] YANG J,YAN J,LIU B. Targeting EGFRvⅢfor glioblastoma multiforme[J]. Cancer Lett,2017,403(9):224-230.
[7] PADFIELD E,ELLIS H P,KURIAN K M. Current therapeutic advances targeting EGFR and EGFRvⅢin glioblastoma[J].Front Oncol,2015,5:5. doi:10. 3389/fonc. 2015. 00005.
[8] VILLA G R,MISCHEL P S. Old player,new partner:EGFRvⅢand cytokine receptor signaling in glioblastoma[J]. Nat Neurosci,2016,19(6):765-767.
[9] GE H,GONG X,TANG C K. Evidence of high incidence of EGFRvⅢexpression and coexpression with EGFR in human invasive breast cancer by laser capture microdissection and immunohistochemical analysis[J]. Int J Cancer,2002,98(3):357-361.
[10] YU H,GONG X,LUO X,et al. Co-expression of EGFRvⅢwith ErbB-2 enhances tumorigenesis:EGFRvⅢmediated constitutively activated and sustained signaling pathways,whereas EGF-induced a transient effect on EGFR-mediated signaling pathways[J]. Cancer Biol Ther,2008,7(11):1818-1828.
[11] OKAMOTO I,KENYON L C,EMLET D R,et al. Expression of constitutively activated EGFRvⅢin non-small cell lung cancer[J]. Cancer Sci,2003,94(1):50-56.
[12] RAE J M,SCHEYS J O,CLARK K M,et al. EGFR and EGFRvⅢexpression in primary breast cancer and cell lines[J]. Breast Cancer Res Treat,2004,87(1):87-95.
[13] PECIAK J,STEC W J,TREDA C,et al. Low incidence along with low m RNA levels of EGFRvⅢin prostate and colorectal cancers compared to glioblastoma[J]. J Cancer,2017,8(1):146-151.
[14] NAGLE S J, GARFALL A L, STADTMAUER E A. The promise of chimeric antigen receptor engineered T cells in the treatment of hematologic malignancies[J]. Cancer J,2016,22(1):27-33.
[15] NEWICK K,MOON E,ALBELDA S M. Chimeric antigen receptor T-cell therapy for solid tumors[J]. Mol Ther Oncolytics,2016,3:16006. doi:10. 1038/mto. 2016. 6.
[16] FRIGAULT M J,MAUS M V. Chimeric antigen receptor-modified T cells strike back[J]. Int Immunol,2016,28(7):355-363.
[17] REN P P,LI M,LI T F,et al. Anti-EGFRvⅢchimeric antigen receptor-modified T cells for adoptive cell therapy of glioblastoma[J]. Curr Pharm Des,2017,23(14):2113-2116.
[18] HAN J,CHU J,KEUNG CHAN W,et al. CAR-engineered NK cells targeting wild-type EGFR and EGFRvⅢenhance killing of glioblastoma and patient-derived glioblastoma stem cells[J]. Sci Rep,2015,5:11483. doi:10. 1038/srep11483.
[19] LIU K,LIU X,PENG Z,et al. Retargeted human avidin-CAR T cells for adoptive immunotherapy of EGFRvⅢexpressing gliomas and their evaluation via optical imaging[J]. Oncotarget,2015,6(27):23735-23747.
[20] MIAO H,CHOI B D,SURYADEVARA C M,et al. EGFRvⅢ-specific chimeric antigen receptor T cells migrate to and kill tumor deposits infiltrating the brain parenchyma in an invasive xenograft model of glioblastoma[J]. PLoS One,2014,9(4):e94281. doi:10. 1371/journal. pone. 0094281.
[21] O'ROURKE D M,NASRALLAH M P,DESAI A,et al. A single dose of peripherally infused EGFRvⅢ-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma[J]. Sci Transl Med,2017,9(399):pii:eaaa0984. doi:10. 1126/scitranslmed. aaa0984.
[22] DRAPER L M,KWONG M L,GROS A,et al. Targeting of HPV-16+Epithelial Cancer Cells by TCR Gene Engineered T Cells Directed against E6[J]. Clin Cancer Res,2015,21(19):4431-4439.