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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。
OBJECTIVE: To construct a recombinant adenovirus vector containing the secreted human extracellular CD40L fusion protein (sCD40L-Ig) gene and determine its expression and functional significance. Methods: The human IgGFc and sCD40L genes were obtained by PCR and inserted into the adenovirus shuttle plasmid pAdTrack-CMV to construct the recombinant plasmid pAdTrack-sCD40L-Ig. After homologous recombination with pAdEasy-1-BJ5183, the recombinant adenovirus was packaged in 293 cells, and the recombinant adenovirus was identified by observation of green fluorescent protein (GFP) and Western blot analysis. The mixed lymphocyte reaction (MLR) Determine its function. Results: The constructed recombinant adenovirus of sCD40L-Ig gene was identified by restriction enzyme digestion and PCR. The primary adenovirus titer reached 2.69 × 1011pfu / L, and the relative molecular mass (Mr) 61 × 103 of the target protein expression. MLR shows that the recombinant adenovirus has an inhibitory effect on the proliferation of lymphocytes. Conclusion: The recombinant adenovirus containing sCD40L-Ig gene has been successfully constructed and has an inhibitory effect on MLR.