嵌合錨定融合分子在T淋巴細胞的熒光表達及檢測_《中國普外基礎與臨床雜志》

作者：

徐宏勇 ¹ , 徐立 ² , 李開宗 ³ , 竇科峰 ³

1.解放軍第451醫院肝膽普外科(西安 710054);;
2.第四軍醫大學西京醫院消化病研究所（西安 710032）;;
3.第四軍醫大學西京醫院肝膽外科（西安 710032）;

關鍵詞：

單鏈抗體片段 CD28 pEGFP-C3載體腫瘤相關糖蛋白72 轉染

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目的　構建含有由抗腫瘤相關糖蛋白72(anti-TAG72)單鏈抗體片段(single chain variable fragment,scFv)及CD28胞內區及跨膜區的融合基因anti-TAG72 scFv-CD28的重組綠色熒光表達載體pEGFP-C3-anti-TAG72 scFv-CD28，并轉染獲得嵌合錨定T淋巴細胞。
方法　將anti-TAG72單鏈抗體及CD28胞內區及跨膜區基因的嵌合錨定融合分子克隆入綠色熒光蛋白真核表達載體pEGFP-C3，獲得pEGFP-C3-anti-TAG72 scFv-CD28表達載體。用卡那霉素篩選陽性克隆，經酶切和測序鑒定。將上述重組質粒pEGFP-C3-anti-TAG72 scFv-CD28以脂質體法瞬時轉染外周血單核細胞(PBMC)，以獲得嵌合錨定T淋巴細胞，用熒光顯微鏡檢測嵌合分子anti-TAG72 scFv-CD28在PBMC中的表達，并檢驗嵌合錨定融合分子anti-TAG72 scFv-CD28的表達。
結果　重組綠色熒光表達載體pEGFP-C3-anti-TAG72 scFv-CD28中anti-TAG72 scFv-CD28片段為1 002 bp，與已知的序列相符。酶切、測序結果表明，嵌合錨定融合分子基因片段anti-TAG72 scFv-CD28被正確插入pEGFP-C3載體；轉染后anti-TAG72 scFv-CD28片段及綠色熒光物質表達于嵌合錨定T淋巴細胞胞膜表面及胞漿中，為綠色熒光顯色。
結論　完成了綠色熒光表達載體pEGFP-C3-anti-TAG72 scFv-CD28的構建，并成功在PBMC瞬時表達。

引用本文： 徐宏勇,徐立,李開宗,竇科峰. 嵌合錨定融合分子在T淋巴細胞的熒光表達及檢測. 中國普外基礎與臨床雜志, 2010, 17(10): 1046-1049. doi: 復制

1.	徐宏勇, 徐立, 李開宗, 等. 靶向性腫瘤相關抗原TAG-72的scFv-CD28融合基因的真核表達載體的構建 [J]. 中國普外基礎與臨床雜志, 2002; 9(6): 398-401.
2.	徐立, 徐宏勇, 樊代明. 胃腸道腫瘤靶向抗癌胚抗原單鏈抗體與T淋巴細胞表面受體基因的融合分子構建 [J]. 中華消化雜志, 2004; 24(2): 75-77.
3.	徐宏勇, 徐立, 高建宏, 等. 嵌合錨定T細胞致癌胚抗原陽性胃癌細胞的凋亡效應 [J]. 中華醫學雜志, 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, 李開宗, 等. 癌胚抗原嵌合錨定T細胞對胃癌的體內外抑癌效應觀察 [J]. 中華外科雜志， 2008； 46(2): 148-149.
13.	馮百歲, 姚雪華, 趙國強. 幽門螺桿菌細胞毒素相關基因A綠色熒光蛋白真核表達載體的構建 [J]. 中華醫學雜志， 2007; 87(8): 570-572.

1. 　徐宏勇, 徐立, 李開宗, 等. 靶向性腫瘤相關抗原TAG-72的scFv-CD28融合基因的真核表達載體的構建 [J]. 中國普外基礎與臨床雜志, 2002; 9(6): 398-401.
2. 　徐立, 徐宏勇, 樊代明. 胃腸道腫瘤靶向抗癌胚抗原單鏈抗體與T淋巴細胞表面受體基因的融合分子構建 [J]. 中華消化雜志, 2004; 24(2): 75-77.
3. 　徐宏勇, 徐立, 高建宏, 等. 嵌合錨定T細胞致癌胚抗原陽性胃癌細胞的凋亡效應 [J]. 中華醫學雜志, 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, 李開宗, 等. 癌胚抗原嵌合錨定T細胞對胃癌的體內外抑癌效應觀察 [J]. 中華外科雜志， 2008； 46(2): 148-149.
13. 　馮百歲, 姚雪華, 趙國強. 幽門螺桿菌細胞毒素相關基因A綠色熒光蛋白真核表達載體的構建 [J]. 中華醫學雜志， 2007; 87(8): 570-572.

《中國普外基礎與臨床雜志》

嵌合錨定融合分子在T淋巴細胞的熒光表達及檢測

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《中國普外基礎與臨床雜志》

嵌合錨定融合分子在T淋巴細胞的熒光表達及檢測

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

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Content

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