Construction and Identification of Fluorescent Protein Expression Vector with Chimeric Molecule in T Lymphocytes_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

XU Hongyong ¹ , XU Li ² , LI Kaizong ³ , DOU Kefeng ³

1.Department of Hepatobiliary Surgery, The 451st Hospital of Chinese PLA, Xi’an 710054, China;;
2.Institute of Digestive Disease, Xijing Hospital, The Fourth Medical University, Xi’an 710032, China;;
3.Department of Hepatobiliary Surgery, Xijing Hospital, The Fourth Medical University, Xi’an 710032, ChinaCorresponding Author: XU Hong-yong, E-mail: hongyongxu@medmail.com.cn;

Corresponding author：

Keywords：

Single chain variable fragment; CD28; pEGFP-C3 vector; Tumor associated glycoprotein 72; Transfection

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Abstract Full text Figures/Tables Video References Cited by

Objective 　To construct a green fluorescent protein expression plasmid pEGFP-C3-anti-TAG72 scFv-CD28, containing anti-TAG72 single chain variable fragment (scFv) fused into the transmembrane and intracellular domain of the signal-transducing chain of CD28 gene, and to transfect it into peripheral blood mononuclear cells．
Methods 　Recombinant transmembrane and intracellular domain of CD28 cDNA and anti-TAG72 scFv cDNA fragment was subcloned into pEGFP-C3 vector. Recombinant clones were selected by Kanamyein， and then identified by PCR， enzyme digestion analysis and DNA sequencing. The recombinant plasmid was transfected into peripheral blood mononuclear cells by means of lipofection. The recombinant protein expression was confirmed by immunocytochemistry, laser scanning confocal microscope, PCR and Western blot analysis.
Results 　The fused gene fragment of anti-TAG72 scFv-CD28 was successfully inserted into pEGFP-C3 plasmid, and it was confirmed by enzyme digestion and DNA sequencing. The fused anti-TAG72 scFv-CD28 gene and its protein was identified in peripheral blood mononuclear cells．
Conclusion 　The eukaryotic expression plasmid pEGFP-C3-anti-TAG72 scFv-CD28 was successfully constructed and transiently expressed in peripheral blood mononuclear cells， which would lay a foundation for further studies on the role of it to activate tumor-associated antigen-specific T lymphocyte, for generating of modified T lymphocytes targeting gastrointestinal tumors.

Citation： XU Hongyong,XU Li,LI Kaizong,DOU Kefeng. Construction and Identification of Fluorescent Protein Expression Vector with Chimeric Molecule in T Lymphocytes. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2010, 17(10): 1046-1049. doi: Copy

1.	徐宏勇, 徐立, 李开宗, 等. 靶向性肿瘤相关抗原TAG-72的scFv-CD28融合基因的真核表达载体的构建 [J]. 中国普外基础与临床杂志, 2002; 9(6): 398-401.
2.	徐立, 徐宏勇, 樊代明. 胃肠道肿瘤靶向抗癌胚抗原单链抗体与T淋巴细胞表面受体基因的融合分子构建 [J]. 中华消化杂志, 2004; 24(2): 75-77.
3.	徐宏勇, 徐立, 高建宏, 等. 嵌合锚定T细胞致癌胚抗原阳性胃癌细胞的凋亡效应 [J]. 中华医学杂志, 2007; 87(15): 1053-1057.
4.	Chmielewski M, Hombach A, Heuser C, et al. T cell activation by antibody-like immunoreceptors: increase in affinity of the single-chain fragment domain above threshold does not increase T cell activation against antigen-positive target cells but decreases selectivity [J]. J Immunol, 2004; 173(12): 7647-7653.
5.	Alvarez-Vallina L, Hawkins RE. Antigen-specific targeting of CD28-mediated T cell co-stimulation using chimeric single-chain antibody variable fragment-CD28 receptors [J]. Eur J Immunol, 1996; 26(10): 2304-2309.
6.	Hombach A, Heuser C, Marquardt T, et al. CD4+ T cells engrafted with a recombinant immunoreceptor efficiently lyse target cells in a MHC antigen- and Fas-independent fashion [J]. J Immunol, 2001; 167(2): 1090-1096.
7.	Hombach A, Sent D, Schneider C, et al. T-cell activation by recombinant receptors: CD28 costimulation is required for interleukin 2 secretion and receptor-mediated T-cell proliferation but does not affect receptor-mediated target cell lysis [J]. Cancer Res, 2001; 61(5): 1976-1982.
8.	Heuser C, Hombach A, Lsch C, et al. T-cell activation by recombinant immunoreceptors: impact of the intracellular signalling domain on the stability of receptor expression and antigen-specific activation of grafted T cells [J]. Gene Ther, 2003; 10(17): 1408-1419.
9.	Hombach AA, Schildgen V, Heuser C, et al. T cell activation by antibody-like immunoreceptors: the position of the binding epitope within the target molecule determines the efficiency of activation of redirected T cells [J]. J Immunol, 2007; 178(7): 4650-4657.
10.	Alonso-Camino V, Sánchez-Martín D, Compte M, et al. Lymphocyte display: a novel antibody selection platform based on T cell activation [J]. PLoS One, 2009; 4(9): e7174.
11.	Compte M, Cuesta AM, Sánchez-Martín D, et al. Tumor immunotherapy using gene-modified human mesenchymal stem cells loaded into synthetic extracellular matrix scaffolds [J]. Stem Cells, 2009; 27(3): 753-760.
12.	徐宏勇, 徐立, 李开宗, 等. 癌胚抗原嵌合锚定T细胞对胃癌的体内外抑癌效应观察 [J]. 中华外科杂志， 2008； 46(2): 148-149.
13.	冯百岁, 姚雪华, 赵国强. 幽门螺杆菌细胞毒素相关基因A绿色荧光蛋白真核表达载体的构建 [J]. 中华医学杂志， 2007; 87(8): 570-572.

1. 　徐宏勇, 徐立, 李开宗, 等. 靶向性肿瘤相关抗原TAG-72的scFv-CD28融合基因的真核表达载体的构建 [J]. 中国普外基础与临床杂志, 2002; 9(6): 398-401.
2. 　徐立, 徐宏勇, 樊代明. 胃肠道肿瘤靶向抗癌胚抗原单链抗体与T淋巴细胞表面受体基因的融合分子构建 [J]. 中华消化杂志, 2004; 24(2): 75-77.
3. 　徐宏勇, 徐立, 高建宏, 等. 嵌合锚定T细胞致癌胚抗原阳性胃癌细胞的凋亡效应 [J]. 中华医学杂志, 2007; 87(15): 1053-1057.
4. 　Chmielewski M, Hombach A, Heuser C, et al. T cell activation by antibody-like immunoreceptors: increase in affinity of the single-chain fragment domain above threshold does not increase T cell activation against antigen-positive target cells but decreases selectivity [J]. J Immunol, 2004; 173(12): 7647-7653.
5. 　Alvarez-Vallina L, Hawkins RE. Antigen-specific targeting of CD28-mediated T cell co-stimulation using chimeric single-chain antibody variable fragment-CD28 receptors [J]. Eur J Immunol, 1996; 26(10): 2304-2309.
6. 　Hombach A, Heuser C, Marquardt T, et al. CD4+ T cells engrafted with a recombinant immunoreceptor efficiently lyse target cells in a MHC antigen- and Fas-independent fashion [J]. J Immunol, 2001; 167(2): 1090-1096.
7. 　Hombach A, Sent D, Schneider C, et al. T-cell activation by recombinant receptors: CD28 costimulation is required for interleukin 2 secretion and receptor-mediated T-cell proliferation but does not affect receptor-mediated target cell lysis [J]. Cancer Res, 2001; 61(5): 1976-1982.
8. 　Heuser C, Hombach A, Lsch C, et al. T-cell activation by recombinant immunoreceptors: impact of the intracellular signalling domain on the stability of receptor expression and antigen-specific activation of grafted T cells [J]. Gene Ther, 2003; 10(17): 1408-1419.
9. 　Hombach AA, Schildgen V, Heuser C, et al. T cell activation by antibody-like immunoreceptors: the position of the binding epitope within the target molecule determines the efficiency of activation of redirected T cells [J]. J Immunol, 2007; 178(7): 4650-4657.
10. 　Alonso-Camino V, Sánchez-Martín D, Compte M, et al. Lymphocyte display: a novel antibody selection platform based on T cell activation [J]. PLoS One, 2009; 4(9): e7174.
11. 　Compte M, Cuesta AM, Sánchez-Martín D, et al. Tumor immunotherapy using gene-modified human mesenchymal stem cells loaded into synthetic extracellular matrix scaffolds [J]. Stem Cells, 2009; 27(3): 753-760.
12. 　徐宏勇, 徐立, 李开宗, 等. 癌胚抗原嵌合锚定T细胞对胃癌的体内外抑癌效应观察 [J]. 中华外科杂志， 2008； 46(2): 148-149.
13. 　冯百岁, 姚雪华, 赵国强. 幽门螺杆菌细胞毒素相关基因A绿色荧光蛋白真核表达载体的构建 [J]. 中华医学杂志， 2007; 87(8): 570-572.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Construction and Identification of Fluorescent Protein Expression Vector with Chimeric Molecule in T Lymphocytes

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