表达结核分枝杆菌CFP10基因的重组腺病毒对A549细胞炎性因子的影响

The Effects of a Recombinant Adenovirus Expressing CFP10 Gene of Mycobacterium tuberculosis on Inflammatory Factors to A549 Cells

通过构建表达结核分枝杆菌(Mycobacterium tuberculosis,Mtb)CFP10基因的重组腺病毒,探讨CFP10蛋白对A549细胞炎性因子表达的影响。采用酶切、连接的方法将CFP10编码基因插入到腺病毒穿梭质粒pShuttle-AdV4中,构建重组穿梭质粒pShuttle-AdV4-CFP10,重组穿梭质粒经测序验证后和骨架质粒pGP-Ad-Pac Vector经限制性内切酶PacⅠ酶切线性化后共转染至HEK 293A细胞中进行病毒包装,获得重组腺病毒AdV4-CFP10,经定量PCR和Western blot验证后进行病毒扩增,CsCl密度梯度离心纯化获得高纯度的重组腺病毒。AdV4-CFP10感染Ⅱ型肺泡上皮细胞A549,利用荧光定量PCR和ELISA技术检测A549细胞中细胞因子IL-1α、IL-6、IL-8和TNF-α等的表达水平,初步探究CFP10对A549细胞炎性因子分泌的影响。成功构建了表达结核分枝杆菌CFP10基因的重组腺病毒AdV4-CFP10,AdV4-CFP10转染至A549细胞后,与空病毒相比,过表达CFP10显著上调A549细胞炎性因子的分泌水平。为进一步深入研究结核分枝杆菌CFP10蛋白对A549细胞炎性反应的调控机制提供参考。

Adopting the construction of a recombinant adenovirus being able to express the CFP10 gene of Mycobacterium tuberculosis(Mtb)was investigated its effects on the expression of inflammatory factors in A549 cells.CFP10 gene was inserted into pShuttle-AdV4 vector of adenovirus to construct a recombinant vector pShuttle-AdV4-CFP10 adopting digestion and linkage.After confirmation of the recombinant vector by sequence analysis,both of pShuttle-AdV4-CFP10 and adenovirus backbone plasmid pGP-Ad-Pac Vector were digested linearizationally with restriction enzyme Pac I and co-transfected to HEK 293A cells to package recombinant adenovirus AdV4-CFP10.After proved by quantitative PCR and Western blot the AdV4-CFP10 were carried out virus amplification,then purified by CsC1 gradient centrifuge and obtained high pureness recombinant adenovirus.The AdV4-CFP10 was then infected into A549 alveolar epithelial cells,and the expression levels of cytokines IL-1α,IL-6,IL-8 and TNF-αin A549 cells were further detected by fluorescence q-PCR and ELISA to investigate the inflammatory factors effect on CFP10 on A549 cells.The results showed that the recombinant genetic adenovirus AdV4-CFP10 expressing CFP10 gene of Mtb was successfully constructed.After transfection of AdV4-CFP10 into A549 cells,over-expression of CFP10 significantly up-regulated the secretion levels of downstream inflammatory factors in A549 cells as compared with empty virus.This study had laid a foundation for further study on CFP10 protein of Mtb to A549 cell inflammatory response and the regulation mechanisms.