人LY6D基因真核表达载体的构建及蛋白的表达和定位
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摘要
目的:构建人LY6D真核表达载体并证实融合蛋白在细胞内的表达和定位。方法:提取HEK293T细胞的mRNA,反转录为cDNA。PCR扩增LY6D基因的CDS序列,并将其亚克隆至pEGFP-C1表达载体中。进一步将构建的重组质粒进行酶切和测序鉴定,并转染到工具细胞HEK293T中,提取细胞总蛋白进行Western blot检测。然后利用激光扫描共聚焦显微镜观察LY6D在HEK293T细胞内的定位,最后Q-PCR检测过表达LY6D真核表达载体影响EMT关键基因的表达。结果:LY6D基因cDNA的编码区序列克隆到了真核表达载体pEGFP-C1中,酶切鉴定片断为387 bp,并进一步测序成功。Western blot检测到了pEGFP-LY6D融合蛋白的表达,分子量约为41 kDa。pEGFP-LY6D融合蛋白在HEK293T细胞中主要定位于细胞膜和细胞质。过表达LY6D真核表达载体减少E-cadherin的表达。结论:成功构建了LY6D基因cDNA的CDS序列的真核表达载体,pEGFP-LY6D蛋白在HEK293T细胞中主要定位于细胞膜和细胞质,LY6D过表达可以降低E-cadherin的表达。
Objective:To construct human LY6 D eukaryotic expression vector and confirm the expression and localization of fusion protein in cells.Methods:The mRNA of HEK293 T cells were extracted and reverse transcribed into cDNA.The CDS sequence of the LY6 D gene was PCR amplified and subcloned into the pEGFP-C1 expression vector.The recombinant plasmid was further digested and sequenced,and transfected into HEK293 T cells.The total protein was extracted and detected by Western blot.Then,laser scanning confocal microscopy was used to observe the localization of LY6 D protein in HEK293 T cells.Finally,the expression of key EMT genes were affected when overexpressed LY6 D eukaryotic expression vector into HEK293 T cells by Q-PCR assay.Results:The coding region of the LY6 D gene cDNA was cloned into the eukaryotic expression vector pEGFP-C1.The restriction fragment was 387 bp and further sequenced successfully.Western blot showed the expression of GFP-LY6 D fusion protein with a molecular weight of approximately 41 kDa.pEGFP-LY6 D protein is mainly localized in the cell membrane and cytoplasm in the HEK293 T cells.The E-cadherin mRNA level was decreased when overexpression of the LY6 D eukaryotic expression vector into HEK293 T cells.Conclusion:The eukaryotic expression vector of LY6 D gene cDNA was successfully constructed.pEGFP-LY6 D protein was mainly located in cell membrane and cytoplasm in HEK293 T cells,and overexpression of LY6 D can reduce the expression of E-cadherin.
Objective:To construct human LY6 D eukaryotic expression vector and confirm the expression and localization of fusion protein in cells.Methods:The mRNA of HEK293 T cells were extracted and reverse transcribed into cDNA.The CDS sequence of the LY6 D gene was PCR amplified and subcloned into the pEGFP-C1 expression vector.The recombinant plasmid was further digested and sequenced,and transfected into HEK293 T cells.The total protein was extracted and detected by Western blot.Then,laser scanning confocal microscopy was used to observe the localization of LY6 D protein in HEK293 T cells.Finally,the expression of key EMT genes were affected when overexpressed LY6 D eukaryotic expression vector into HEK293 T cells by Q-PCR assay.Results:The coding region of the LY6 D gene cDNA was cloned into the eukaryotic expression vector pEGFP-C1.The restriction fragment was 387 bp and further sequenced successfully.Western blot showed the expression of GFP-LY6 D fusion protein with a molecular weight of approximately 41 kDa.pEGFP-LY6 D protein is mainly localized in the cell membrane and cytoplasm in the HEK293 T cells.The E-cadherin mRNA level was decreased when overexpression of the LY6 D eukaryotic expression vector into HEK293 T cells.Conclusion:The eukaryotic expression vector of LY6 D gene cDNA was successfully constructed.pEGFP-LY6 D protein was mainly located in cell membrane and cytoplasm in HEK293 T cells,and overexpression of LY6 D can reduce the expression of E-cadherin.
引文
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[13] Mansson R,Zandi S,Welinder E,et al.Single-cell analysis of the common lymphoid progenitor compartment reveals functional and molecular heterogeneity[J].Blood,2010,115(13):2601-2609.
[14] Adolfsson J,Mansson R,Buza-Vidas N,et al.Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment[J].Cell,2005,121(2):295-306.
[15] Borghesi L,Hsu LY,Miller JP,et al.B lineage-specific regulation of V(D)J recombinase activity is established in common lymphoid progenitors[J].Exp Med,2004,199(4):491-502.
[16] Welner RS,Esplin BL,Garrett KP,et al.Asynchronous RAG-1 expression during B lymphopoiesis[J].Immunol,2009,183(12):7768-7777.
[17] Mansson R,Zandi S,Anderson K,et al.B-lineage commitment prior to surface expression of B220 and CD19 on hematopoietic progenitor cells[J].Blood,2008,112(4):1048-1055.
[18] Alberti-Servera L,von Muenchow L,Tsapogas P,et al.Single-cell RNA sequencing reveals developmental heterogeneity among early lymphoid progenitors[J].EMBO J,2017,36(24):3619-3633.
[19] Kong HK,Park JH.Characterization and function of human Ly-6/uPAR molecules[J].BMB Rep,2012,45(11):595-603.
[20] Ishikawa H,Imano M,Shiraishi O,et al.Phase I clinical trial of vaccination with LY6K-derived peptide in patients with advanced gastric cancer[J].Gastric Cancer,2014,17(1):173-180.
[21] Asundi J,Crocker L,Tremayne J,et al.An antibody-drug conjugate directed against lymphocyte antigen 6 complex,locus E (LY6E) provides robust tumor killing in a wide range of solid tumor malignancies[J].Clin Cancer Res,2015,21(14):3252-3262.
[22] Yoshitake Y,Fukuma D,Yuno A,et al.Phase II clinical trial of multiple peptide vaccination for advanced head and neck cancer patients revealed induction of immune responses and improved OS[J].Clin Cancer Res,2015,21(2):312-321.
[2] Choi SH,Kong HK,Park SY,et al.Metastatic effect of LY-6K gene in breast cancer cells[J].Int J Oncol,2009,35(3):601-607.
[3] Liao XH,Xie Z,Guan CN.MiRNA-500a-3p inhibits cell proliferation and invasion by targeting lymphocyte antigen 6 complex locus K (LY6K) in human non-small cell lung cancer[J].Neoplasma,2018,65(5):673-682.
[4] Upadhyay G,Yin Y,Yuan H,et al.Stem cell antigen-1 enhances tumorigenicity by disruption of growth differentiation factor-10 (GDF10)-dependent TGF-beta signaling[J].Proc Natl Acad Sci USA,2011,108(19):7820-7825.
[5] Ruud H Brakenhoff,Martijn Gerretsen,Ellen MC Knippels,et al.The human E48 antigen,highly homologous to the murine Ly-6 antigen ThB,is a GPI-anchored molecule apparently involved in keratinocyte cell-cell adhesion [J].J Cell Biol,1995,129(6):1677-1689.
[6] Coia Romeu,Xavier Farre,Antonio Cardesa,et al.Expression of Ep-CAM,but not of E48,associates with nodal involvement in advanced squamous cell carcinomas of the larynx[J].Histopathology,2013,62(6):954-961.
[7] Barros-Silva JD,Linn DE,Steiner I,et al.Single-cell analysis identifies LY6D as a marker linking castration-resistant prostate luminal cells to prostate progenitors and cancer[J].Cell Rep,2018,25(12):3504-3518.
[8] Mayama A,Takagi K,Suzuki H,et al.OLFM4,LY6D and S100A7 as potent markers for distant metastasis in estrogen receptor-positive breast carcinoma[J].Cancer Sci,2018,109(10):3350-3359.
[9] Quak JJ,Balm AJM,van Dongen GAMS.A 22-Kd surfaceantigen detected by monoclonal antibody-E-48 is exclusively expressed in stratified squamous and transitional epithelia[J].Am J Pathol,1990,136(1):191-197.
[10] Schrijvers AHGJ,Gerretsen M,Fritz JM.Evidence for a role of the monoclonal antibody-E48 defined antigen in cell celladhesion in squamous epithelia and head and neck squamouscell carcinoma[J].Exp Cell Res,1991,196(2):264-269.
[11] Torenbeek R,Blomjous CEM,Quak JJ,et al.Use of monoclonal-antibody E48 in diagnosing transitional cell-carcinoma of urinary-bladder[J].Clin Pathol,1992,45(4):303-307.
[12] Colnot DR,Nieuwenhuis EJC,Kuik DJ.Clinical significance of micrometastatic cells detected by E48 (Ly-6D) reverse transcription-polymerase chain reaction in bone marrow of head and neck cancer patients[J].Clin Cancer Res,2004,10(23):7827-7833.
[13] Mansson R,Zandi S,Welinder E,et al.Single-cell analysis of the common lymphoid progenitor compartment reveals functional and molecular heterogeneity[J].Blood,2010,115(13):2601-2609.
[14] Adolfsson J,Mansson R,Buza-Vidas N,et al.Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment[J].Cell,2005,121(2):295-306.
[15] Borghesi L,Hsu LY,Miller JP,et al.B lineage-specific regulation of V(D)J recombinase activity is established in common lymphoid progenitors[J].Exp Med,2004,199(4):491-502.
[16] Welner RS,Esplin BL,Garrett KP,et al.Asynchronous RAG-1 expression during B lymphopoiesis[J].Immunol,2009,183(12):7768-7777.
[17] Mansson R,Zandi S,Anderson K,et al.B-lineage commitment prior to surface expression of B220 and CD19 on hematopoietic progenitor cells[J].Blood,2008,112(4):1048-1055.
[18] Alberti-Servera L,von Muenchow L,Tsapogas P,et al.Single-cell RNA sequencing reveals developmental heterogeneity among early lymphoid progenitors[J].EMBO J,2017,36(24):3619-3633.
[19] Kong HK,Park JH.Characterization and function of human Ly-6/uPAR molecules[J].BMB Rep,2012,45(11):595-603.
[20] Ishikawa H,Imano M,Shiraishi O,et al.Phase I clinical trial of vaccination with LY6K-derived peptide in patients with advanced gastric cancer[J].Gastric Cancer,2014,17(1):173-180.
[21] Asundi J,Crocker L,Tremayne J,et al.An antibody-drug conjugate directed against lymphocyte antigen 6 complex,locus E (LY6E) provides robust tumor killing in a wide range of solid tumor malignancies[J].Clin Cancer Res,2015,21(14):3252-3262.
[22] Yoshitake Y,Fukuma D,Yuno A,et al.Phase II clinical trial of multiple peptide vaccination for advanced head and neck cancer patients revealed induction of immune responses and improved OS[J].Clin Cancer Res,2015,21(2):312-321.