本文選題：口瘡病毒　切入點：VEGF基因　出處：《中國獸醫(yī)科學(xué)》2017年02期

【摘要】：為了對口瘡病毒(ORFV)VEGF基因進行原核表達(dá)和亞細(xì)胞定位研究,PCR擴增VEGF基因,并將其分別克隆至原核表達(dá)載體p ET-32a(+)和真核表達(dá)載體p EGFP-C3中。將原核表達(dá)重組質(zhì)粒轉(zhuǎn)化至E.coli Rosseta感受態(tài)細(xì)胞中,經(jīng)IPTG誘導(dǎo)表達(dá)目的蛋白。將表達(dá)產(chǎn)物純化后免疫BALB/c小鼠,制備多克隆抗體血清,并利用瓊脂擴散試驗檢測其效價。將真核表達(dá)重組質(zhì)粒轉(zhuǎn)染BHK21細(xì)胞,利用激光共聚焦觀察VEGF蛋白在BHK21細(xì)胞中的亞細(xì)胞定位。結(jié)果表明:VEGF蛋白在大腸桿菌中獲得了高效表達(dá),主要以包涵體形式表達(dá),蛋白分子質(zhì)量大小約為33 ku。Western-blot結(jié)果表明,VEGF蛋白具有良好的反應(yīng)原性,制備的多克隆抗體效價達(dá)1∶8。VEGF蛋白在轉(zhuǎn)染BHK21細(xì)胞后主要在細(xì)胞質(zhì)中表達(dá),且能夠與制備的多抗血清反應(yīng)。本研究為進一步研究該蛋白的功能奠定了一定的基礎(chǔ)。
[Abstract]:In order to investigate the prokaryotic expression and subcellular localization of VEGF gene of oral ulcera virus, VEGF gene was amplified and cloned into prokaryotic expression vector pET-32a () and eukaryotic expression vector p EGFP-C3, respectively.The prokaryotic expression plasmid was transformed into E.coli Rosseta competent cells, and the target protein was induced by IPTG.The BALB/c mice were immunized with the purified product, and the polyclonal antibody serum was prepared, and the titer was detected by Agar diffusion test.The eukaryotic expression plasmid was transfected into BHK21 cells and the subcellular localization of VEGF protein in BHK21 cells was observed by confocal laser.The results showed that the protein was highly expressed in E. coli, mainly in the form of inclusion body. The molecular weight of the protein was about 33 ku.Western-blot.The polyclonal antibody titer of the prepared 1:8.VEGF protein was mainly expressed in the cytoplasm of BHK21 cells after transfection and could react with the prepared polyantiserum.This study laid a foundation for further study of the function of the protein.
【作者單位】： 安徽農(nóng)業(yè)大學(xué)動物科技學(xué)院;
【基金】：國家自然科學(xué)基金項目(31602063) 安徽省自然科學(xué)基金項目(1508085QC60) 安徽省科技重大專項(16030801106) 安徽農(nóng)業(yè)大學(xué)穩(wěn)定和引進人才科研資助項目(yj2015-16) 國家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系專項基金項目(CARS-39)
【分類號】：S852.65
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本文編號：1709041