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目的 构建人IL-10基因的重组腺病毒并进行体外表达和检测。方法从质粒pcDNA3IL-10的CMV启动子下游完整切下IL-10 cDNA的片段,将其定向插入Gateway载体,在克隆酶的作用下将阳性克隆质粒转入目的腺病毒载体。PacI酶线性化IL-10腺病毒DNA后阳离子质脂体转染293A细胞,获得人IL-10的复制缺陷型重组腺病毒。体外感染人胰腺癌细胞株Bxpc-3和大鼠胰腺细胞株AR-42J;Western blot检测人IL-10的表达。结果 成功地构建人IL-10重组腺病毒,病毒滴度达2×109PFU/mL。体外感染的细胞株均检测到IL-10的表达。结论 构建复制缺陷型重组腺病毒能够介导IL-10的基因表达,为细胞因子的抗炎冶疗奠定实验基础。
Objective To construct the recombinant adenovirus of human IL-10 gene and to express and detect it in vitro. Methods The fragment of IL-10 cDNA was cut down from the downstream of CMV promoter of plasmid pcDNA3IL-10 and inserted into the Gateway vector. The positive cloned plasmid was transferred into the target adenovirus vector under the action of the cloning enzyme. PacI linearized IL-10 adenovirus DNA after cationic liposomes transfected 293A cells to obtain human IL-10 replication-defective recombinant adenovirus. The human pancreatic cancer cell line Bxpc-3 and the rat pancreatic cell line AR-42J were infected in vitro. The expression of human IL-10 was detected by Western blot. Results Human IL-10 recombinant adenovirus was successfully constructed and the titer of virus was 2 × 109 PFU / mL. IL-10 expression was detected in all in vitro infected cell lines. Conclusion The construction of replication-deficient recombinant adenovirus can mediate the gene expression of IL-10 and lay an experimental foundation for the anti-inflammatory cytokines treatment.