抗犬瘟热病毒核蛋白单克隆抗体的制备及其线性抗原表位鉴定
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摘要
为制备犬瘟热病毒(CDV)单克隆抗体(MAb),本研究用腺病毒表达的CDV截短N蛋白(aa401~aa523)免疫BALB/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行融合并筛选到2株能够稳定分泌抗CDV N蛋白的MAb,命名为N1-C8和N1-C41。经间接ELISA测定N1-C8和N1-C41培养上清液及小鼠腹水效价分别为1∶1 024、1∶106和1∶512、1∶105。通过肽扫描的方法筛选出N1-C8的抗原表位为线性表位~(440)ENQGGDKYPIHFNDE454,但并未能够筛选出N1-C41的抗原表位,推测可能是因为其抗原表位为空间构象型。Western blot结果显示N1-C8和N1-C41两株MAb在识别CDV的不同毒力病毒株上有差异,可能与CDV强弱病毒株在N蛋白的差异变化有关。本研究两株MAb的制备为CDV不同毒力株的鉴别诊断提供了可能。
To prepare the monoclonal antibodies(MAb) against canine distemper virus(CDV), two hybridoma cell lines,named N1-C8 and N1-C41, secreting MAb against the CDV was prepared by fusion of the SP2/0 myeloma cells with spleen cells of BALB/c mice immunized by purified recombinant nucleocapsid(aa401-aa523) protein of CDV prepared from adenovirus expression system. These culture supernatant and mouse ascites titer of the MAbs were 1∶1024, 1∶106 and 1∶512, 1∶105 for N1-C8 and N1-C41 respectively by indrect ELISA. The linear epitope ~(440)ENQGGDKYPIHFNDE454 is the epitope of N1-C8, which was selected by peptide scanning, but the epitope of N1-C41 was not screened, and it was speculated that the epitope might be space conformation. Western blot results showed that there were differences of MAbs N1-C8 and N1-C41 in identifying different virulence strains of CDV, this phenomenon may be associated with virulent and avirulent CDV strains in N protein changes. These MAbs could have potential for developing differential diagnosis for different virulent strains of CDV.
To prepare the monoclonal antibodies(MAb) against canine distemper virus(CDV), two hybridoma cell lines,named N1-C8 and N1-C41, secreting MAb against the CDV was prepared by fusion of the SP2/0 myeloma cells with spleen cells of BALB/c mice immunized by purified recombinant nucleocapsid(aa401-aa523) protein of CDV prepared from adenovirus expression system. These culture supernatant and mouse ascites titer of the MAbs were 1∶1024, 1∶106 and 1∶512, 1∶105 for N1-C8 and N1-C41 respectively by indrect ELISA. The linear epitope ~(440)ENQGGDKYPIHFNDE454 is the epitope of N1-C8, which was selected by peptide scanning, but the epitope of N1-C41 was not screened, and it was speculated that the epitope might be space conformation. Western blot results showed that there were differences of MAbs N1-C8 and N1-C41 in identifying different virulence strains of CDV, this phenomenon may be associated with virulent and avirulent CDV strains in N protein changes. These MAbs could have potential for developing differential diagnosis for different virulent strains of CDV.
引文
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[2]Ulrich R,Puff C,Wewetzer K,et al.Transcriptional changes in canine distemper virus-induced demyelinating leukoencephalitis favor a biphasic mode of demyelination[J].PLo S One,2014,9(4):e95917
[3]Kameo Y,Nagao Y,Nishio Y,et al.Epizootic canine distemper virus infection among wild mammals[J].Vet Microbiol,2012,154(3-4):222-229.
[4]李梅荣,唐泰山,等.动物园大型猫科动物犬瘟热的免疫监测[J].中国病毒学,2006,21(4):368-370.
[5]Calderon M G,Remorini P,Periolo O,et al.Detection by RT-PCR and genetic characterrization of canine distemper virus from vaccinated and non-vaccinated dogs in Argentina[J].Vet Microbiol,2007,125(3-4):341-349.
[6]Zhao Jian-jun,Yan Xi-jun,Chai Xiu-li,et al.Phylogenetic analysis of the haemagglutinin gene of canine distemper virus strains detected from breeding foxes,raccoon dogs and minks in China[J].Vet Microbiol,2010,140(1-2):34-42.
[7]何洪彬,夏咸柱.犬瘟热的诊断及其预防免疫的研究进展[J].动物医学进展,2001,22(1):12-15.
[8]王铮,王铁成,冯娜.犬瘟热病毒单克隆抗体的制备及其生物学特性鉴定[J].动物医学进展,2014,35(3):73-77.
[9]王建科,程世鹏,闫喜军,等.抗水貂肠炎病毒单克隆抗体的制备及其生物学特性鉴定[J].中国兽医科学,2009,39(3):227-232.
[10]Hamburger D,Griot C,Zurbriggen A,et al.Loss of virulence of canine distemper virus is associated with a structural change recognized by a monoclonal antibody[J].Experientia,1991,47(8):842-845.
[11]Yoshida E,Iwatsuki K,Myashital N,et al.Molecular analysis of the nucleocapsid protein of recent isolates of canine distemper virus in Japan[J].Vet Microbiol,1998,59(2):237-244.
[12]Stettler M,Zurbriggen A.Nucleotide and deduced amino acid sequences of the nucleocapsid protein of the virulent A75/17-CDV strain of canine distemper virus[J].Vet Microbiol,1995,44(2):211-217.