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目的克隆、真核表达SA11株轮状病毒VP3基因,对其致病机制进行了初步研究。方法用RT-PCR从SA11株总RNA中扩增出VP3基因,并克隆到真核表达载体pEGFP-C1上,构建重组表达载体pEGFP-C1/VP3,用荧光显微镜及Western blot检测VP3基因在真核细胞(293T细胞)的表达情况。以重组质粒pEGFP-C1/Rb94为对照,用流式、荧光显微镜、Western blot观察VP3蛋白对GFP基因表达的抑制作用。结果在293T细胞中检测到VP3基因的表达;pEGFP-C1/VP3组的流式荧光强度、Western blot的蛋白条带大小均明显弱于pEGFP-C1/Rb94对照组。结论成功构建了pEGFP-C1/VP3真核穿梭表达载体,在真核细胞中有效表达,实验结果提示VP3蛋白可能对宿主细胞大分子蛋白的表达有抑制作用,可能是轮状病毒的一种新的致病机制。
Objective To clone and eukaryotic expression of SA11 rotavirus VP3 gene and study its pathogenic mechanism. Methods VP3 gene was amplified from total RNA of SA11 strain by RT-PCR and cloned into eukaryotic expression vector pEGFP-C1 to construct recombinant expression vector pEGFP-C1 / VP3. VP3 gene was detected by fluorescence microscopy and Western blot in true Nuclear cells (293T cells) expression. The recombinant plasmid pEGFP-C1 / Rb94 as a control, flow cytometry, fluorescence microscopy, Western blot VP3 protein GFP gene expression inhibition. Results The expression of VP3 gene was detected in 293T cells. The fluorescence intensity of pEGFP-C1 / VP3 group and the protein band size of Western blot were weaker than that of pEGFP-C1 / Rb94 control group. Conclusion The eukaryotic expression vector pEGFP-C1 / VP3 was successfully constructed and expressed efficiently in eukaryotic cells. The results suggest that VP3 protein may inhibit the expression of host cell macromolecules and may be a new type of rotavirus Pathogenesis.