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目的:构建并鉴定人白细胞介素16(IL-16)的重组腺病毒pAd-IL-16表达载体,为进一步研究IL-16功能奠定基础。方法:分离正常人外周血单个核细胞(PBMC),组胺刺激后提取总RNA,RT-PCR扩增IL-16基因,将其克隆到含有绿色荧光蛋白(GFP)标记的腺病毒穿梭质粒pAdTrack-CMV后,再与腺病毒骨架质粒pAdEasy-1共转化BJ5183细菌,获得重组腺病毒载体pAd-IL-16,转染HEK293T细胞包装病毒。荧光显微镜下观察细胞内绿色荧光,测定病毒效价,RT-PCR和Western blot分别分析细胞内IL-16 mRNA和蛋白质的表达。结果:经双酶切及基因鉴定,重组穿梭质粒和重组腺病毒质粒均见约400bp插入片段,测序结果和GenBank中的基因序列一致;重组病毒效价为2.8×108pfu/mL;重组病毒感染后,HEK293T细胞中IL-16 mRNA和蛋白质均有表达。结论:成功构建IL-16重组腺病毒载体,并可在HEK293T细胞中表达,为进一步研究IL-16的功能奠定了基础。
Objective: To construct and identify the recombinant adenovirus pAd-IL-16 expression vector of human interleukin-16 (IL-16), and lay the foundation for further study of IL-16 function. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers. Total RNA was extracted after stimulation with histamine. The IL-16 gene was amplified by RT-PCR and cloned into the shuttle vector pAdTrack containing green fluorescent protein (GFP) -CMV, and co-transformed into BJ5183 with the adenovirus backbone plasmid pAdEasy-1 to obtain the recombinant adenovirus vector pAd-IL-16 and transfected into HEK293T cell packaging virus. Fluorescence microscopy was used to observe the intracellular green fluorescence, and the virus titer was determined. The expression of IL-16 mRNA and protein were analyzed by RT-PCR and Western blot, respectively. Results: The recombinant shuttle plasmid and recombinant adenovirus plasmid were all about 400 bp in size. The sequencing result was consistent with that in GenBank. The recombinant virus titer was 2.8 × 108pfu / mL. After recombinant virus infection , IL-16 mRNA and protein were expressed in HEK293T cells. Conclusion: The IL-16 recombinant adenovirus vector was successfully constructed and expressed in HEK293T cells, which laid the foundation for further study on the function of IL-16.