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目的:构建携带SPRED2的质粒载体与重组腺病毒载体,并观察其在K562细胞的表达及对ERK信号通路的作用,为Spred2在造血细胞中的作用的研究奠定基础。方法:以HepG2细胞cDNA为模板,RT-PCR克隆SPRED2全长CDS序列,并亚克隆到pCDNA3.0和pshuttle-CMV质粒载体,构建携带SPRED2的真核表达载体pCDNA3.0-Spred2与穿梭载体pshuttle-CMV-Spred2;将线性化pshuttle-CMV-Spred2与腺病毒骨架质粒Adf11p在感受态细胞BJ5183中进行同源重组,产生重组质粒Adf11p-Spred2;后者经线性化后转染至HEK293细胞进行病毒包装;在HEK293细胞扩增病毒颗粒,以CsCl密度梯度离心法进行纯化,TCID50法测定病毒滴度;将病毒颗粒以100MOI感染K562细胞,Western blot检测Spred2过表达情况及Spred2对细胞ERK的影响。结果:经酶切、DNA测序、Western blot检测等方法鉴定,证明pCDNA3.0-Spred2与Adf11p-Spred2携带Spred2序列正确,能够在HEK293细胞、K562细胞正确表达,Spred2过表达能够显著抑制K562细胞ERK活性。结论:成功构建对K562细胞有高感染效率的SPRED2重组腺病毒载体,且Spred2对K562细胞ERK信号通路有显著抑制作用。
OBJECTIVE: To construct a plasmid vector and a recombinant adenovirus vector carrying SPRED2 and to observe its expression in K562 cells and its effect on ERK signaling pathway, which laid the foundation for the study of the role of Spred2 in hematopoietic cells. METHODS: The full-length CDS sequence of SPRED2 was cloned by RT-PCR and cloned into pCDNA3.0 and pshuttle-CMV plasmid vectors using HepG2 cDNA as a template. The eukaryotic expression vector pCDNA3.0-Spred2 carrying SPRED2 and the shuttle vector pshuttle CMV-Spred2. The homologous recombination of linearized pshuttle-CMV-Spred2 and adenovirus backbone plasmid Adf11p in competent cell BJ5183 resulted in the recombinant plasmid Adf11p-Spred2; the latter was linearized and transfected into HEK293 cells for virus The virus particles were amplified in HEK293 cells and purified by CsCl density gradient centrifugation. The virus titer was determined by TCID50 method. K562 cells were infected with 100 MOI of virus particles. The overexpression of Spred2 and the effect of Spred2 on ERK were detected by Western blot. Results: The results of restriction endonuclease digestion, DNA sequencing and Western blot showed that the Spred2 sequences of pCDNA3.0-Spred2 and Adf11p-Spred2 were correct and could be correctly expressed in HEK293 and K562 cells. Spred2 overexpression significantly inhibited ERK active. CONCLUSION: SPRED2 recombinant adenovirus vector with high infection efficiency to K562 cells was successfully constructed and Spred2 significantly inhibited the ERK signaling pathway in K562 cells.