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。
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. coil pAdEasy-l-BJS183 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.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2005年第6期668-671,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(No.30371416)
