论文部分内容阅读
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 construct T4 phage displaying KDR and to detect its effect on tumor cell proliferation and invasion at the cellular level. Methods: The SOE-PCR method was used to fuse the outer capsid protein D2 and D3 of the KDR membrane with T4 phage coat protein SOC gene and cloned into pET28b expression vector. After induced by IPTG and purified, the recombinant KDR- SOC protein is displayed on the surface of T4 bacteriophage to obtain T4-KDR bacteriophage. The inhibitory effect of T4-KDR phage on the proliferation of A549 cells was detected by MTT assay. The effect of T4-KDR on the proliferation of A549 cells was detected by Transwell assay. The effect of T4-KDR on MMP-2 and MMP-9 mRNA expression was detected by RT-PCR. The expression of ERK1 / 2 phosphorylation and its downstream CyclinD1, p27, Bcl-2 and Bax Impact. RESULTS: KDR-SOC was stably expressed in E. coli. The KDR phage displayed the ability of binding to VEGF-165 and significantly inhibited tumor cell proliferation and invasion, and decreased the mRNA levels of MMP-2 and MMP-9. Western blot results showed that T4-KDR phage inhibition of VEGF induced ERK1 / 2 phosphorylation, decreased CyclinD1 and Bcl-2 expression, while increasing p27 and Bax expression. CONCLUSION: T4 phage displaying KDR can inhibit the proliferation and invasion of lung cancer cells. The effect of KDR on the MEK / ERK signaling pathway and downstream regulatory factors is an important molecular mechanism.