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

目的 　構(gòu)建含有由抗腫瘤相關(guān)糖蛋白72(anti-TAG72)單鏈抗體片段(single chain variable fragment,scFv)及CD28胞內(nèi)區(qū)及跨膜區(qū)的融合基因anti-TAG72 scFv-CD28的重組綠色熒光表達(dá)載體pEGFP-C3-anti-TAG72 scFv-CD28，并轉(zhuǎn)染獲得嵌合錨定T淋巴細(xì)胞。
方法 　將anti-TAG72單鏈抗體及CD28胞內(nèi)區(qū)及跨膜區(qū)基因的嵌合錨定融合分子克隆入綠色熒光蛋白真核表達(dá)載體pEGFP-C3，獲得pEGFP-C3-anti-TAG72 scFv-CD28表達(dá)載體。用卡那霉素篩選陽性克隆，經(jīng)酶切和測序鑒定。將上述重組質(zhì)粒pEGFP-C3-anti-TAG72 scFv-CD28以脂質(zhì)體法瞬時轉(zhuǎn)染外周血單核細(xì)胞(PBMC)，以獲得嵌合錨定T淋巴細(xì)胞，用熒光顯微鏡檢測嵌合分子anti-TAG72 scFv-CD28在PBMC中的表達(dá)，并檢驗嵌合錨定融合分子anti-TAG72 scFv-CD28的表達(dá)。
結(jié)果 　重組綠色熒光表達(dá)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(jié)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(zhuǎn)染后anti-TAG72 scFv-CD28片段及綠色熒光物質(zhì)表達(dá)于嵌合錨定T淋巴細(xì)胞胞膜表面及胞漿中，為綠色熒光顯色。
結(jié)論 　完成了綠色熒光表達(dá)載體pEGFP-C3-anti-TAG72 scFv-CD28的構(gòu)建，并成功在PBMC瞬時表達(dá)。

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(xì)胞的熒光表達(dá)及檢測. 中國普外基礎(chǔ)與臨床雜志, 2010, 17(10): 1046-1049. doi: 復(fù)制

1. 　徐宏勇, 徐立, 李開宗, 等. 靶向性腫瘤相關(guān)抗原TAG-72的scFv-CD28融合基因的真核表達(dá)載體的構(gòu)建 [J]. 中國普外基礎(chǔ)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(xì)胞表面受體基因的融合分子構(gòu)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(xì)胞致癌胚抗原陽性胃癌細(xì)胞的凋亡效應(yīng) [J]. 中華醫(yī)學(xué)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(xì)胞對胃癌的體內(nèi)外抑癌效應(yīng)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(xì)胞毒素相關(guān)基因A綠色熒光蛋白真核表達(dá)載體的構(gòu)建 [J]. 中華醫(yī)學(xué)雜志， 2007; 87(8): 570-572.
  1. 1. 　徐宏勇, 徐立, 李開宗, 等. 靶向性腫瘤相關(guān)抗原TAG-72的scFv-CD28融合基因的真核表達(dá)載體的構(gòu)建 [J]. 中國普外基礎(chǔ)與臨床雜志, 2002; 9(6): 398-401.
  2. 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(xì)胞表面受體基因的融合分子構(gòu)建 [J]. 中華消化雜志, 2004; 24(2): 75-77.
  3. 3. 　徐宏勇, 徐立, 高建宏, 等. 嵌合錨定T細(xì)胞致癌胚抗原陽性胃癌細(xì)胞的凋亡效應(yīng) [J]. 中華醫(yī)學(xué)雜志, 2007; 87(15): 1053-1057.
  4. 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. 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. 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. 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. 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. 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. 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. 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. 12. 　徐宏勇, 徐立, 李開宗, 等. 癌胚抗原嵌合錨定T細(xì)胞對胃癌的體內(nèi)外抑癌效應(yīng)觀察 [J]. 中華外科雜志， 2008； 46(2): 148-149.
  13. 13. 　馮百歲, 姚雪華, 趙國強. 幽門螺桿菌細(xì)胞毒素相關(guān)基因A綠色熒光蛋白真核表達(dá)載體的構(gòu)建 [J]. 中華醫(yī)學(xué)雜志， 2007; 87(8): 570-572.