| 刘小云1 罗芳1 王华2 杨月峰2 张群伟2 王立生2.人SPRED2 基因重组腺病毒载体的制备及其对K562 细胞[J].现代生物医学进展英文版,2012,12(2):221-225. |
| 人SPRED2 基因重组腺病毒载体的制备及其对K562 细胞 |
| Preparation of Recombinant Adenovirus Vectors Harbouring the HumanSPRED2 Gene and its Effects on ERK Pathway in K562 Cells |
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| DOI： |
| 中文关键词: Spred2 重组腺病毒载体 ERK 通路 K562 细胞 |
| 英文关键词: SPRED2 Recombinant adenovirus vectors K562 cells ERK signal pathway |
| 基金项目: |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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 transferred
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 purified
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 Western
blot assays. Results: The results of enzymatic digestion, DNA sequencing and Western blot assays showed that, the vectors were successfully
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. |
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