| 人sCD40L-Ig重组腺病毒载体的构建、表达及功能检测 |
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| 引用本文： | 李钊伦,田普训,薛武军.人sCD40L-Ig重组腺病毒载体的构建、表达及功能检测[J].细胞与分子免疫学杂志,2005,21(6):668-671. |
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| 作者姓名： | 李钊伦 田普训 薛武军 |
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| 作者单位： | 西安交通大学第一附属医院肾移植科,陕西,西安,710061 |
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| 基金项目： | 国家自然科学基金资助项目(No.30371416) |
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| 摘 要： | 目的:构建含有分泌型人胞外区CD40L融合蛋白(sCD40L-Ig)基因的重组腺病毒载体,确定其表达和功能学意义。方法:通过PCR获得人源IgGFc和sCD40L基因并予以连接,将其插入到腺病毒穿梭质粒pAdTrack-CMV中,构建重组质粒pAdTrack-sCD40L-Ig。将其与pAdEasy-1-BJ5183菌行同源重组后,用293细胞包装,通过观察绿色荧光蛋白(GFP)和Western blot分析等方法鉴定重组腺病毒,并进行双向混合淋巴细胞反应(MLR)以确定其功能。结果:所构建的sCD40L-Ig基因的重组腺病毒,经酶切和PCR鉴定正确。原代腺病毒的滴度达到2.69×1011pfu/L,并有相对分子质量(Mr)为61×103的目的蛋白表达。MLR显示,重组腺病毒对淋巴细胞的增殖有抑制作用。结论:成功地构建了含有sCD40L-Ig基因的重组腺病毒,并对MLR有抑制作用。
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| 关 键 词： | sCD40L融合蛋白 重组腺病毒 混合淋巴细胞培养 免疫耐受 |
| 文章编号： | 1007-8738（2005）06-0668-04 |
| 收稿时间： | 2005-09-12 |
| 修稿时间： | 2005-10-16 |
Construction of recombinant adenovirus expressing Scd40l-Ig |
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| LI Zhao-lun,TIAN Pu-xun,XUE Wu-jun.Construction of recombinant adenovirus expressing Scd40l-Ig[J].Journal of Cellular and Molecular Immunology,2005,21(6):668-671. |
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| Authors: | LI Zhao-lun TIAN Pu-xun XUE Wu-jun |
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| Abstract: | AIM: To construct adenovirus expressing secretory human CD40L-Ig (sCD40 ligand Ig fusion protein). METHODS: The genes encoding the extracellular domain of human CD40L and IgG Fc were amplified with PCR and inserted into shuttle vector pAdTrack-CMV which then was transformed into E.coli pAdEasy-1-BJ5183 to produce recombinant Ad plasmid. The recombinant plasmid was digested with Pac I and transfected into 293 cells to generate recombinant adenovirus. The recombinant adenovirus was then used in mixed lymphocytes reaction (MLR) to test its function. RESULTS: The recombinant adenovirus sCD40L-Ig was produced. It could inhibit the lymphocyte proliferation in MLR. CONCLUSION: The sCD40L-Ig adenovirus is prepared successfully and its inhibition of MLR is confirmed. |
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| Keywords: | sCD40 ligand Ig fusion protein recombination adenovirus mixed lymphocytes reaction immune tolerance |
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