人SPRED2基因重组腺病毒载体的制备及其对K562细胞ERK通路的影响

Preparation of Recombinant Adenovirus Vectors Harbouring the Human SPRED2 Gene and its Effects on ERK Pathway in K562 Cells

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 establish plasmids and recombinant adenovirus vectors harbouring the human SPRED2 gene,investigate the effects of Spred2 over-expression on ERK signalling pathway in K562 cells,and provide basis for future researches on the effects of Spred2 in hematopoietic cells.Methods： cDNA from HepG2 cells were used as templates for Spred2 full-length CDS cloning by RT-PCR,the Spred2 full-length CDS were subcloned to pCDNA3.0 and pshuttle-CMV vectors to establish pCDNA3.0-Spred2 and pshuttle-CMV-Spred2 recombinant plasmids,linearized pshuttle-CMV-Spred2 and adenovirus backbone plasmids Adf11p were trans-ferred to BJ5183 competent cells for homologous recombination and produced recombinant plasmids Adf11p-Spred2.Adf11p-Spred2 plasmids were linearized and transferred to HEK293 cells for virus packaging.Virus particles were amplified in HEK293 cells and puri-fied by CsCl density gradient centrifugation.TCID50 methods were used for virus titer detection.K562 cells were infected with Adf11p-Spred2 and control virus at 100 MOI（multiplicity of infection）.Spred2 expression and ERK activity were determined by West-ern blot assays.Results： The results of enzymatic digestion,DNA sequencing and Western blot assays showed that,the vectors were suc-cessfully established and over-expressed in K562 cells;Spred2 over-expression intensively inhibited the ERK activity.Conclusion： Recombinant adenovirus harbouring human SPRED2 gene with high infection efficiency in K562 cells were successfully established,adenovirus mediated Spred2 over-expression significantly inhibited the ERK signaling pathway.