展示KDR胞外区VEGF结合域的T4噬菌体对肺癌细胞增殖侵袭的抑制作用  

T4 Bacteriophage Displaying VEGF-binding Domains of KDR Inhibits the Proliferation and Invasion of Lung Cancer Cells

作　　者：李嵚 何琳[1] 惠林萍[1] 赵晨阳[1] 于涛[1] 

机构地区：[1]中国医科大学附属四院中心实验室,沈阳110032

出　　处：《中国生物工程杂志》2013年第10期14-20,共7页China Biotechnology

基　　金：国家自然科学基金(81001028);辽宁省教育厅科学技术研究一般项目(L2010625)资助项目

摘　　要：目的:构建展示KDR的T4噬菌体并在细胞水平上检测其抑制肿瘤细胞增殖侵袭的作用及机制。方法:利用SOE-PCR的方法将KDR膜外区D2和D3与T4噬菌体小衣壳蛋白SOC基因融合并克隆到pET28b表达载体中,经IPTG诱导表达并纯化后,利用体外展示技术将重组KDR-SOC蛋白展示在T4噬菌体表面,得到T4-KDR噬菌体。MTT法检测T4-KDR噬菌体对肺癌A549细胞的增殖抑制作用,并采用Transwell法检测其对VEGF促侵袭作用的影响。RT-PCR法检测T4-KDR对MMP-2和MMP-9 mRNA水平的影响,并通过Western blot方法检测其对VEGF诱导ERK1/2磷酸化及其下游CyclinD1,p27,Bcl-2和Bax表达水平的影响。结果:KDR-SOC在大肠杆菌中稳定表达,展示KDR的T4-KDR噬菌体具有结合VEGF-165的能力,并显著抑制肿瘤细胞增殖和侵袭活性,降低MMP-2和MMP-9基因mRNA水平。Western blot结果表明T4-KDR噬菌体抑制VEGF诱导ERK1/2的磷酸化,降低CyclinD1及Bcl-2表达的同时,提高p27和Bax的表达。结论:展示KDR的T4噬菌体具有抑制肺癌细胞增殖和侵袭作用,其对MEK/ERK信号通路及其下游调控因子的影响是其作用的重要分子机制。Objective： To display VEGF-binding domains of KDR onto T4 bacteriophage using a well established in vitro assembly strategy, and investigate its anti-proliferative and anti-metastatic effects in A549 lung cancer cells. Methods： SOE-PCR was used to fuse D2 and D3 domain of KDR to the small outer capsid protein （SOC） of bacteriophage T4. The kdr-soc fused gene was then cloned into pET28b to allow the IPTG induction and overexpression in E.coli. The purified protein was assembled onto the T4 capsid using the in vitro assembly strategy. MTT and Transwell assay were then conducted to evaluate the actions of T4-KDR on the proliferation and VEGF-induced invasion of A549 lung cancer cells. Changes of the MMP-2 and -9 mRNA levels were also analyzed using RT-PCR. Western blot was conducted to investigate the levels of ERK1/2, CyclinD1, p27, Bcl-2 and Bax. Result： KDR-SOC fusion protein were overexpressed and purified from E.coli. About 818 copies of KDR-SOC were arrayed onto the T4 phage via SOC-capsid interactions, and retained the binding capability with VEGF-165. A549 cells treated with T4-KDR exhibited a lower proliferation rate and a less VEGF-induced invasion, together with reduced mRNA levels of both MMP-2 and MMP-9. Western blot revealed that T4-KDR treatment decreased phosphorylation level of ERK1/2, as well as the downstream CyclinD1 and Bcl-2 levels, but the expression level of p27 and Bax were upregulated. Conclusion： T4-KDR inhibits proliferation and invasion by modulating the MEK/ERK signaling pathway in lung cancer A549 cells.

关 键 词：T4噬菌体 增殖 侵袭 血管内皮生长因子受体-2 肺癌 

分 类 号：Q819[生物学—生物工程]