鸡传染性贫血病毒VP3基因的原核表达及抗血清制备
|
摘要
为了研究鸡传染性贫血病毒(Chicken anemia virus,CIAV)VP3蛋白与病毒自身蛋白之间的相互作用,需对该蛋白进行原核表达制备相应兔源抗血清,试验通过提取CIAV M9905株基因组DNA,PCR扩增出VP3基因,构建重组质粒pET30a(+)-VP3,然后转化入大肠杆菌BL21(DE3)感受态细胞中,进一步通过IPTG诱导表达后用Ni-NTA亲和柱纯化获得融合蛋白,经SDS-PAGE分析后对融合蛋白进行乳化,免疫新西兰兔制备抗血清,并对其进行ELISA效价测定、Western-blot及IFA检测。结果表明:SDS-PAGE分析证实获得高纯度的重组蛋白VP3,大小约为22 ku,且其在大肠杆菌中主要以包涵体的形式存在;ELISA法检测制备的兔源抗VP3血清效价在1∶6 400以上;经Western-blot及IFA检测证实,VP3兔源血清可特异性结合Vero细胞表达的VP3蛋白。说明试验成功表达出CIAV VP3蛋白,制备的兔抗血清具有较好的特异性。
In order to research the interaction between Chicken anemia virus(CIAV) VP3 protein and virus proteins,the VP3 protein needed to be expressed prokaryoticly and its anti-rabbit sera were prepared. The genomic DNA of CIAV M9905 strain was extracted in this experiment,then the VP3 gene was amplified by PCR,and recombinant prokaryotic expression vector pET30 a(+)-VP3 was constructed,which was transformed into Escherichia coli BL21(DE3) competent cells. After IPTG induction and the purification by the immobilized Ni-NTA resin,the fusion protein VP3 was obtained. After the SDS-PAGE analysis,the fusion protein VP3 was emulsified and New Zealand rabbits were immunized for the preparation of anti-VP3 sera. Anti-VP3 sera were determined by indirect ELISA for titer detection,by Western-blot and IFA methods. The results showed that the SDS-PAGE analysis comfirmed that the recombinant VP3 with high purity was obtained,with a molecular weight of 22 ku,and existed mainly in the form of inclusion body in Escherichia coli. The prepared rabbit anti-VP3 sera showed a titer greater than 1∶6 400 using ELISA method. Western-blot and IFA results confirmed that the sera could specifically bind to the VP3 protein expressed in Vero cells. The results suggested that the CIAV VP3 protein was successfully expressed in this experiment,and the prepared rabbit antisera had good specificity.
In order to research the interaction between Chicken anemia virus(CIAV) VP3 protein and virus proteins,the VP3 protein needed to be expressed prokaryoticly and its anti-rabbit sera were prepared. The genomic DNA of CIAV M9905 strain was extracted in this experiment,then the VP3 gene was amplified by PCR,and recombinant prokaryotic expression vector pET30 a(+)-VP3 was constructed,which was transformed into Escherichia coli BL21(DE3) competent cells. After IPTG induction and the purification by the immobilized Ni-NTA resin,the fusion protein VP3 was obtained. After the SDS-PAGE analysis,the fusion protein VP3 was emulsified and New Zealand rabbits were immunized for the preparation of anti-VP3 sera. Anti-VP3 sera were determined by indirect ELISA for titer detection,by Western-blot and IFA methods. The results showed that the SDS-PAGE analysis comfirmed that the recombinant VP3 with high purity was obtained,with a molecular weight of 22 ku,and existed mainly in the form of inclusion body in Escherichia coli. The prepared rabbit anti-VP3 sera showed a titer greater than 1∶6 400 using ELISA method. Western-blot and IFA results confirmed that the sera could specifically bind to the VP3 protein expressed in Vero cells. The results suggested that the CIAV VP3 protein was successfully expressed in this experiment,and the prepared rabbit antisera had good specificity.
引文
[1] NATESAN S,KATARIA J M,DHAMA K,et al.Biobogical and molecular characterization of Chicken anaemia virus isolates of Indian origin [J].Virus Res,2006,118(1/2):78-86.
[2] GUELEN L,PATERSON H,GAKEN J,et al.TAT-apoptin is efficiently delivered and induces apoptosis in cancer cells [J].Oncogenet,2004,23(5):1153-1165.
[3] DANEN-VAN OORSCHOT A A,FISCHER D F,GRIMBERGEN J M,et al.Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells [J].Proc Natl Acad Sci USA,1997,94(11):5843-5847.
[4] ZHANG L,ZHAO H,CUI Z,et al.A peptide derived from apoptin inhibits glioma growth [J].Oncotarget,2017,8(19):31119-31132.
[5] RUIZ-MARTINEZ S,CASTRO J,VILANOVA M,et al.A truncated apoptin protein variant selectively kills cancer cells [J].Invest New Drugs,2017,35(3):260-268.
[6] WANG Y,SONG X,GAO H,et al.C-terminal region of apoptin affects Chicken anemia virus replication and virulence [J].Virol J,2017,14(1):38.
[7] POON I K,ORO C,DIAS M M,et al.Apoptin nuclear accumulation is modulated by a CRM1-recognized nuclear export signal that is active in normal bur not in tumor cells [J].Cancer Res,2005,65(16):7059-7064.
[8] DANEN-VAN OORSCHOT A A,VOSKAMP P,SEELEN M C,et al.Human death effector domain-associated factor interacts with the viral apoptosis agonist apoptin and exerts tumor-preferential cell killing [J].Cell Death Differ,2004,11(5):564-573.
[9] LELIVELD S R,DAME R T,MOMMAAS M A,et al.Apoptin protein multimers form distinct higher-order nucleoprotein complexes with DNA [J].Nucleic Acids Res,2003,31(16):4805-4813.
[10] TEODORO J G,HEILMAN D W,PARKER A E,et al.The viral protein apoptin associates with the anaphase-promoting complex to induce G2/M arrest and apoptosis in the absence of p53 [J].Genes Dev,2004,18(16):1952-1957.
[11] DOUGLAS A J,PHENIX K,MAWHINNEY K A,et al.Identification of a 24 kDa protein expressed by Chicken anaemia virus [J].J Gen Virol,1995,76 (Pt7):1557-1562.
[12] CHENG J H,SHEU S C,LIEN Y Y,et al.Identification of the NLS and NES motifs of VP2 from Chicken anemia virus and the interaction of VP2 with mini-chromosome maintenance protein 3 [J].BMC Vet Res,2012,8:15.
[13] KAFFASHI A,PAGEL C N,NOORMOHAMMADI A H,et al.Evidence of apoptosis induced by viral protein 2 of Chicken anaemia virus [J].Arch Virol,2015,160 (10):2557-2563.
[14] SUN F,PAN W,GAO H,et al.Identification of the interaction and interaction domains of Chicken anemia virus VP2 and VP3 proteins [J].Virology,2018,513:188-194.
[15] 李殿申,崔玉海.来源于鸡贫血病毒的凋亡素研究进展[J].生物工程杂志,2005(B04):19-22.
[16] ROHN J,ZHANG Y H,AALBERS R I,et al.A tumor specific kinase activity regulates the viral death protein apoptin[J].Biol Chem,2002,277 (52):50820-50827.
[17] 陆桂丽,王笑梅,高玉龙,等.鸡传染性贫血病毒vp3基因的原核表达及其免疫学活性分析[J].中国预防兽医学报,2006,28 (6):650-652.
[18] 傅文彬,石璇,赵健,等.凋亡蛋白质的原核表达与纯化[J].食品与药品,2008,10 (3):7-11.
[2] GUELEN L,PATERSON H,GAKEN J,et al.TAT-apoptin is efficiently delivered and induces apoptosis in cancer cells [J].Oncogenet,2004,23(5):1153-1165.
[3] DANEN-VAN OORSCHOT A A,FISCHER D F,GRIMBERGEN J M,et al.Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells [J].Proc Natl Acad Sci USA,1997,94(11):5843-5847.
[4] ZHANG L,ZHAO H,CUI Z,et al.A peptide derived from apoptin inhibits glioma growth [J].Oncotarget,2017,8(19):31119-31132.
[5] RUIZ-MARTINEZ S,CASTRO J,VILANOVA M,et al.A truncated apoptin protein variant selectively kills cancer cells [J].Invest New Drugs,2017,35(3):260-268.
[6] WANG Y,SONG X,GAO H,et al.C-terminal region of apoptin affects Chicken anemia virus replication and virulence [J].Virol J,2017,14(1):38.
[7] POON I K,ORO C,DIAS M M,et al.Apoptin nuclear accumulation is modulated by a CRM1-recognized nuclear export signal that is active in normal bur not in tumor cells [J].Cancer Res,2005,65(16):7059-7064.
[8] DANEN-VAN OORSCHOT A A,VOSKAMP P,SEELEN M C,et al.Human death effector domain-associated factor interacts with the viral apoptosis agonist apoptin and exerts tumor-preferential cell killing [J].Cell Death Differ,2004,11(5):564-573.
[9] LELIVELD S R,DAME R T,MOMMAAS M A,et al.Apoptin protein multimers form distinct higher-order nucleoprotein complexes with DNA [J].Nucleic Acids Res,2003,31(16):4805-4813.
[10] TEODORO J G,HEILMAN D W,PARKER A E,et al.The viral protein apoptin associates with the anaphase-promoting complex to induce G2/M arrest and apoptosis in the absence of p53 [J].Genes Dev,2004,18(16):1952-1957.
[11] DOUGLAS A J,PHENIX K,MAWHINNEY K A,et al.Identification of a 24 kDa protein expressed by Chicken anaemia virus [J].J Gen Virol,1995,76 (Pt7):1557-1562.
[12] CHENG J H,SHEU S C,LIEN Y Y,et al.Identification of the NLS and NES motifs of VP2 from Chicken anemia virus and the interaction of VP2 with mini-chromosome maintenance protein 3 [J].BMC Vet Res,2012,8:15.
[13] KAFFASHI A,PAGEL C N,NOORMOHAMMADI A H,et al.Evidence of apoptosis induced by viral protein 2 of Chicken anaemia virus [J].Arch Virol,2015,160 (10):2557-2563.
[14] SUN F,PAN W,GAO H,et al.Identification of the interaction and interaction domains of Chicken anemia virus VP2 and VP3 proteins [J].Virology,2018,513:188-194.
[15] 李殿申,崔玉海.来源于鸡贫血病毒的凋亡素研究进展[J].生物工程杂志,2005(B04):19-22.
[16] ROHN J,ZHANG Y H,AALBERS R I,et al.A tumor specific kinase activity regulates the viral death protein apoptin[J].Biol Chem,2002,277 (52):50820-50827.
[17] 陆桂丽,王笑梅,高玉龙,等.鸡传染性贫血病毒vp3基因的原核表达及其免疫学活性分析[J].中国预防兽医学报,2006,28 (6):650-652.
[18] 傅文彬,石璇,赵健,等.凋亡蛋白质的原核表达与纯化[J].食品与药品,2008,10 (3):7-11.