• 1.第四軍醫(yī)大學(xué)西京醫(yī)院肝膽外科（西安710032）;;
  • 2.第四軍醫(yī)大學(xué)西京醫(yī)院消化病研究所;;
  • 3.第四軍醫(yī)大學(xué)病理學(xué)教研室;

目的構(gòu)建含抗腫瘤相關(guān)抗原TAG72單鏈抗體及CD28胞內(nèi)區(qū)及跨膜區(qū)融合基因的哺乳細(xì)胞表達(dá)載體，為制備消化道腫瘤靶向性嵌合錨定T細(xì)胞，并探討其對消化道腫瘤的殺傷活性。方法應(yīng)用PCR法，將抗TAG72單鏈抗體(single chain variable fragment,scFv)克隆入哺乳細(xì)胞表達(dá)載體pcDNA3.0，在5′端及3′端分別引入相應(yīng)酶切位點(diǎn)； 用RTPCR法從正常人外周血T細(xì)胞克隆CD28胞內(nèi)區(qū)及跨膜區(qū)的cDNA，然后將其克隆于scFv的下游，并在5′端及3′端引入相應(yīng)的酶切位點(diǎn)。結(jié)果抗TAG-72 scFv cDNA片段為729 bp，與已知的序列相符； CD28胞內(nèi)區(qū)及跨膜區(qū)的cDNA片段為240 bp，與Genebank公布的序列一致。重組的表達(dá)載體經(jīng)酶切瓊脂糖電泳測序加以證實(shí)。結(jié)論完成了抗腫瘤相關(guān)抗原TAG72 scFv及CD28胞內(nèi)區(qū)及跨膜區(qū)融合基因scFvCD28pcDNA3.0的構(gòu)建，為制備消化道腫瘤靶向性嵌合錨定T細(xì)胞奠定了實(shí)驗(yàn)基礎(chǔ)。

引用本文： 徐宏勇,徐立,李開宗,竇科峰,付由池,劉彥仿. 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△. 中國普外基礎(chǔ)與臨床雜志, 2002, 9(6): 398-401. doi: 復(fù)制

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  1. 1. Xu M, Real FX, Welt S, et al. Expression of TAG72 in normal colon, transitional mucosa, and colon cancer [Ｊ]. Int J Cancer, 1989; 44(6)∶985.
  2. 2. Werther JL, RiveraMacMurray S, Bruckner H, et al. Mucinassociated sialosylTn antigen expression in gastric cancer correlates with an adverse outcome [Ｊ]. Br J Cancer,1994; 69(3)∶613.
  3. 3. Kjeldsen T, Clausen H, Hirohashi S, et al. Preparation and characterization of monoclonal antibodies directed to the tumorassociated Olinked sialosyl26 alphaNacetylgalactosaminyl (sialosylTn) epitope [Ｊ]. Cancer Res,1988; 48(8)∶2214.
  4. 4. Hombach A, Heuser C, Sircar R, et al. T cell targeting of TAG72+ tumor cells by a chimeric receptor with antibodylike specificity for a carbohydrate epitope [Ｊ]. Gastroenterology, 1997; 113(4)∶1163.
  5. 5. AlvarezVallina A, Hawkins RE. Antigenspecific targening of CD28 mediated T cell costimulation using chimeric singlechain antibody variable fragmentCD28 receptors [Ｊ]. Eur J Immunol,1996； 26(10)∶2304.
  6. 6. Hombach A, Wieczarkowiecz A, Marquardt T,et al. Tumorspecific T cell activation by recombinant immunoreceptors: CD3 zeta signaling and CD28 costimulation are simultaneously required for efficient IL2 secretion and can be integrated into one combined CD28/CD3 zeta signaling receptor molecule [Ｊ]. J Immunol， 2001; 167(11)∶6123.
  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.