H3N2型豬流感病毒M2蛋白表達(dá)分析

王曉杜，陳培君，沈陽，邱亞峰，鄧緒芳，史子學(xué)，彭麗娜，羅金燕，劉超，馬志永

中國農(nóng)業(yè)科學(xué)院上海獸醫(yī)研究所，上海 200241

H3N2型豬流感病毒M2蛋白表達(dá)分析

王曉杜，陳培君，沈陽，邱亞峰，鄧緒芳，史子學(xué)，彭麗娜，羅金燕，劉超，馬志永

中國農(nóng)業(yè)科學(xué)院上海獸醫(yī)研究所，上海 200241

流感病毒的M2蛋白在流感病毒復(fù)制中起著重要作用，是抗流感病毒的靶標(biāo)分子。本研究以提取的病毒基因組RNA作為模板，RT-PCR擴(kuò)增H3N2亞型豬流感病毒M2基因，分別構(gòu)建了重組原核表達(dá)載體和重組真核表達(dá)載體，建立了M2蛋白的原核和真核表達(dá)系統(tǒng)。通過大腸桿菌表達(dá)系統(tǒng)，制備了M2重組蛋白，并免疫大鼠制備了多克隆抗體。Western blotting和間接免疫熒光方法檢測表明所制備的抗體能識別真核表達(dá)的M2蛋白和病毒感染細(xì)胞后表達(dá)M2蛋白，具有良好的特異性。重組M2真核表達(dá)載體轉(zhuǎn)染Vero細(xì)胞，表達(dá)的重組M2蛋白大小為20 kDa，定位于細(xì)胞漿中，與病毒感染細(xì)胞中的M2蛋白定位相同。Western blotting檢測表明M2蛋白在病毒感染細(xì)胞12 h后才能檢出，晚于NS1、NP和M1，屬于病毒復(fù)制的晚期蛋白，可作為病毒復(fù)制晚期的指示分子。本研究為弄清M2蛋白在病毒復(fù)制過程中的生物學(xué)功能奠定了基礎(chǔ)。

H3N2，豬流感病毒，M2蛋白，多克隆抗體

Abstract:M2 protein of influenza A virus is encoded by a spliced mRNA derived from RNA segment 7 and plays an important role in influenza virus replication.It is also a target molecule of anti-virus drugs.We extracted the viral genome RNAs from MDCK cells infected with swine influenza A virus(SIV)H3N2 subtype and amplified the SIV M2 gene by reverse transcriptase-polymerase chain reaction using the isloated viral genome RNAs as template.The amplified cDNA was cloned into a prokaryotic expression vector pET-28a(+)(designated pET-28a(+)-M2)and a eukaryotic expression vector p3xFLAG-CMV-7.1(designated p3xFLAG-CMV-7.1-M2), respectively.The resulted constructs were confirmed by restriction enzyme digestion and DNA sequencing analysis.We then transformed the plasmid pET-28a(+)-M2 into Escherichia coli BL21(DE3)strain and expressed it by adding 1 mmol/L of IPTG(isopropyl-β-D-thiogalactopyranoside).The recombinant M2 protein was purified from the induced bacterial cells using Ni2+affinity chromatography.Wistar rats were immunized with the purified M2 protein for producing polyclonal antibodies specific for it.Western blotting analysis and immunofluorescence analysis showed that the produced antibodies were capable of reacting with M2 protein expressed in p3xFLAG-CMV-7.1-M2-transfected cells as well as that synthesized in SIV-infected cells.We also transfected plasmid p3xFLAG-CMV-7.1-M2 into Vero cells and analyzed its subcellular localization by immunofluorescence.The M2 protein expressed in the Vero cells was 20 kDa in size and dominantly localized in the cytoplasm, showing a similar distribution to that in SIV-infected cells.Western blotting analysis of SIV-infected cells suggested that M2 was a late phase protein,which was detectable 12 h post-infection, later than NS1, NP and M1 proteins.It would be a potential molecular indicator of late phases replication of virus.Our results would be useful for studying the biological function of M2 protein in SIV replication.

Keywords:swine influenza virus, H3N2 subtype, M2 protein, gene expression

近年來流感病毒給人類帶來巨大災(zāi)難，特別是高致病性禽流感不僅給雞群帶來毀滅性打擊，也給人類帶來了生命的威脅。而豬作為人、禽流感的混合器，在獸醫(yī)公共衛(wèi)生方面具有重要的研究意義，豬流感病毒(Swine influenza virus，SIV)不僅給養(yǎng)豬業(yè)帶來重要影……