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目的:结缔组织生长因子(Connective tissue growth factor,CTGF)含有4个不同功能结构域,构建表达带有3×Flag标签的全长基因及5个缺失突变体的重组腺病毒,以研究CTGF各结构域的功能和作用机制。方法:通过聚合酶链反应(Polymerasechain reaction,PCR)等克隆方法制备目标质粒DNA,然后通过腺病毒穿梭载体与骨架载体的重组、包装构建全长CTGF和缺失突变体重组腺病毒。用anti-Flag抗体作为一抗做Western blot检测目的基因表达情况。结果:通过腺病毒基因组DNA PCR确认有CTGF全长和缺失突变体的存在;Western blot法验证了CTGF全长和缺失突变体的表达。结论:成功构建了6个表达CTGF全长和缺失突变体的重组腺病毒,为进一步研究CTGF的结构域功能奠定了基础。
OBJECTIVE: CTGF contains four different functional domains and constructs a recombinant adenovirus expressing the full-length gene with 3 × Flag and 5 deletion mutants to study the effects of various structures of CTGF Domain function and mechanism. Methods: The target plasmid DNA was prepared by cloning method such as polymerase chain reaction (PCR). Then the full length CTGF and the recombinant adenovirus lacking the mutant were constructed by recombination of adenovirus shuttle vector and backbone vector. Anti-Flag antibody was used as primary antibody to detect the expression of the target gene by Western blot. Results: The CTGF full-length and deletion mutants were confirmed by PCR with adenovirus genomic DNA. The expression of CTGF full-length and deletion mutants was verified by Western blot. CONCLUSION: Six recombinant adenovirus expressing CTGF full-length and deletion mutants were successfully constructed, which laid the foundation for further study on the domain function of CTGF.