兔抗肠道病毒71型截短VP1抗体的制备及鉴定
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摘要
目的制备兔抗肠道病毒71型(EV71)截短VP1多克隆抗体,为EV71基础研究奠定基础。方法 RT-PCR扩增EV71VP1-N160基因(480 bp),以p GEX-6p-1为表达载体,构建重组表达质粒p GEX-6p-1-VP1-N160,转化大肠埃希菌BL21(DE3),IPTG诱导表达GST-VP1-N160融合蛋白,并对其进行纯化。以纯化的GST-VP1-N160融合蛋白作为免疫原,经背部皮下免疫新西兰大耳白兔,制备多克隆抗体,ELISA检测多克隆抗体效价,间接免疫荧光法和免疫印迹检测抗体特异性。结果经双酶切鉴定,表明重组表达质粒p GEX-6p-1-VP1-N160构建正确;GST-VP1-N160融合蛋白相对分子质量为35 000~55 000,主要为不可溶性包涵体形式,其在大肠杆菌中高效表达;纯化后目的蛋白纯度约为95%,蛋白质量浓度1.9 mg/m L;免疫家兔后,免疫血清效价可达1012,抗VP1-N160多克隆抗体可以识别EV71原核表达的重组蛋白及天然病毒抗原中的VP1,特异性好。结论成功制备了抗EV71截短VP1多克隆抗体。
Objective To generate rabbit polyclonal antibody against truncated VP1 of enterovirus 71( EV71) and to provide a new approach for the fundamental research on EV71. Methods N-terminal fragment( 480 bp) of EV71 VP1,designated as VP1-N160,was amplified by RT-PCR and inserted into E.coli BL21( DE3) to construct a recombinant expression vector named as p GEX-6p-1-VP1-N160. After expression induced by IPTG and purification,the fusion protein GSTVP1-N160 was used to immunize New Zealand white rabbit by hypodermic injection in preparation of polyclonal antibody.The titer of the polyclonal antibody was measured by ELISA and specificity was determined by IFA and WB. Results The result of enzymatic double digestion showed that the recombinant plasmid p GEX-6p-1-VP1-N160 was correctly constructed.GST-VP1-N160 fusion protein with a molecular mass of 35 000 ~ 55 000 was expressed at high level in E. coli and it was mainly in a form as insoluble inclusion bodies. The purity of the purified protein was about 95%,and the protein concentration was 1.9 mg / m L. After immunizing rabbit,the ELISA titer of the polyclonal antibody obtained is in 1012 dilution for original antisera. The results of WB and IFA indicated that the polyclonal antibody against VP1-N160 could react specifically with prokaryotically expressed GST-VP1-N160,and react with the natural VP1 in EV71 as well. Conclusion The polyclonal antibody against truncated VP1 of EV71 was successfully obtained.
Objective To generate rabbit polyclonal antibody against truncated VP1 of enterovirus 71( EV71) and to provide a new approach for the fundamental research on EV71. Methods N-terminal fragment( 480 bp) of EV71 VP1,designated as VP1-N160,was amplified by RT-PCR and inserted into E.coli BL21( DE3) to construct a recombinant expression vector named as p GEX-6p-1-VP1-N160. After expression induced by IPTG and purification,the fusion protein GSTVP1-N160 was used to immunize New Zealand white rabbit by hypodermic injection in preparation of polyclonal antibody.The titer of the polyclonal antibody was measured by ELISA and specificity was determined by IFA and WB. Results The result of enzymatic double digestion showed that the recombinant plasmid p GEX-6p-1-VP1-N160 was correctly constructed.GST-VP1-N160 fusion protein with a molecular mass of 35 000 ~ 55 000 was expressed at high level in E. coli and it was mainly in a form as insoluble inclusion bodies. The purity of the purified protein was about 95%,and the protein concentration was 1.9 mg / m L. After immunizing rabbit,the ELISA titer of the polyclonal antibody obtained is in 1012 dilution for original antisera. The results of WB and IFA indicated that the polyclonal antibody against VP1-N160 could react specifically with prokaryotically expressed GST-VP1-N160,and react with the natural VP1 in EV71 as well. Conclusion The polyclonal antibody against truncated VP1 of EV71 was successfully obtained.
引文
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[13]Liu CC,Chou AH,Lien SP,et al.Identification and characterization of a cross-neutralization epitope of enterovirus 71[J].Vaccine,2011,29(26):4362-4372.
[14]Xu L,He D,Yang L,et al.A broadly cross-protective vaccine presenting the neighboring epitopes within the VP1 GH loop and VP2 EF loop of enterovirus 71[J].Sci Rep,2015,5:12973-12984.
[15]Foo DG,Alonso S,Phoon MC,et al.Identification of neutralizing linear epitopes from the VP1 capsid protein of enterovirus 71using synthetic peptides[J].Virus Res,2007,125(1):61-68.
[2]Pathinayake PS,Hsu AC,Wark PA.Innate immunity and immune evasion by enterovirus 71[J].Viruses,2015,7(12):6613-6630.
[3]Knipe DM,Rowley PM.Fields Virology[M].6th ed.Philadelphia,2013:453-489.
[4]Chen HF,Chang MH,Chiang BL,et al.Oral immunization of mice using transgenic tomato fruit expressing VP1 protein from enterovirus 71[J].Vaccine,2006,24(15):2944-2951.
[5]Chen HL,Huang JY,Chu TW,et al.Expression of VP1 protein in the milk of transgenic mice:a potential oral vaccine protects against enterovirus 71 infection[J].Vaccine,2008,26(23):2882-2889.
[6]Chiu CH,Chu C,He CC,et al.Protection of neonatal mice from lethal enterovirus 71 infection by maternal immunization with attenuated Salmonella enterica serovar Typhimurium expressing VP1 of enterovirus 71[J].Microbes Infect,2006,8(7):1671-1678.
[7]Meng T,Kolpe AB,Kiener TK,et al.Display of VP1 on the surface of baculovirus and its immunogenicity against heterologous human enterovirus 71 strains in mice[J].PLo S One,2011,6(7):e21757-e21768.
[8]Liu SL,Pan H,Liu P,et al.Comparative epidemiology and virology of fatal and nonfatal cases of hand,foot and mouth disease in mainland China from 2008 to 2014[J].Rev Med Virol,2015,25(2):115-128.
[9]Klein MH.EV71 vaccines:a first step towards multivalent hand,foot and mouth disease vaccines[J].Expert Rev Vaccines,2015,14(3):337-340.
[10]Shi M,Zhou Y,Cao LM,et al,Expression of enterovirus 71capsid protein VP1 in Escherichia coli and its clinical application[J].Brazilian Journal of Microbiology,2013,44(4):1215-1222.
[11]Xu J,Wang SX,G WH,et al.Expression and immunogenicity of novel subunit enterovirus 71 VP1 antigens[J].Biochem Biophys Res Commun,2012,420(4):755-761.
[12]Zhang J,Dong M,Jiang B,et al.Antigenic characteristics of the complete and truncated capsid protein VP1 of enterovirus 71[J].Virus Res,2012,167(2):337-342.
[13]Liu CC,Chou AH,Lien SP,et al.Identification and characterization of a cross-neutralization epitope of enterovirus 71[J].Vaccine,2011,29(26):4362-4372.
[14]Xu L,He D,Yang L,et al.A broadly cross-protective vaccine presenting the neighboring epitopes within the VP1 GH loop and VP2 EF loop of enterovirus 71[J].Sci Rep,2015,5:12973-12984.
[15]Foo DG,Alonso S,Phoon MC,et al.Identification of neutralizing linear epitopes from the VP1 capsid protein of enterovirus 71using synthetic peptides[J].Virus Res,2007,125(1):61-68.