猪圆环病毒3型Cap蛋白单克隆抗体的制备及其在免疫组化试验中的初步应用
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摘要
为制备猪圆环病毒3型(PCV3)Cap蛋白单克隆抗体(MAb),并初步应用于感染细胞或组织样品中PCV3抗原的间接免疫荧光试验(IFA)或免疫组化(IHC)检测,本研究将PCV3 Cap蛋白的去核定位信号肽基因克隆于原核表达载体pET-28a中,经IPTG诱导表达了具有免疫原性的融合蛋白。以表达的重组蛋白免疫BALB/c小鼠,共获得8株能够稳定分泌针对PCV3 Cap蛋白的MAb杂交瘤细胞株。Western blot与IFA试验结果显示,8株MAbs均能够与真核表达的重组Cap蛋白反应。选取3株杂交瘤细胞制备3株MAbs,小鼠腹水效价分别为1∶409 600、1∶204 800和1∶409 600。利用3株MAbs对自然感染PCV3临床病猪的腹股沟淋巴结进行IHC检测,结果显示3株MAbs的腹水均能够与感染PCV3的临床样品发生特异性的免疫反应。本研究制备的MAbs具有较好的特异性,为深入研究Cap蛋白的结构和PCV3快速诊断奠定了基础。
To develop monoclonal antibody(MAb) against capsid protein(Cap) of porcine circovirus 3(PCV3), the cap gene encoding the Cap of PCV3 was cloned into the expression vector pET-28a for expression in E.coli. The expressed recombinant Cap was used to immunize BALB/c mice, eight hybridomas which stably secreted MAbs against the Cap of PCV3 were obtained,and all the 8 MAbs were able to react with the PCV3 verified by western blot and immunofluorescence assay. Then 3 hybridomas were selected to prepare ascites, and titers of these ascites were 1∶409,600, 1∶204,800 and 1∶409,600, respectively. The results of immunohistochemistry showed that the ascites of the 3 MAbs had positive reactivity with the PCV3 in lymph node tissue clinical samples of pigs infected with PCV3. In conclusion, the MAbs developed in this study were specific to PCV3, which laid a foundation for the further study of the structure of PCV3 Cap and development of the rapid diagnosis method for PCV3 infection in pigs.
To develop monoclonal antibody(MAb) against capsid protein(Cap) of porcine circovirus 3(PCV3), the cap gene encoding the Cap of PCV3 was cloned into the expression vector pET-28a for expression in E.coli. The expressed recombinant Cap was used to immunize BALB/c mice, eight hybridomas which stably secreted MAbs against the Cap of PCV3 were obtained,and all the 8 MAbs were able to react with the PCV3 verified by western blot and immunofluorescence assay. Then 3 hybridomas were selected to prepare ascites, and titers of these ascites were 1∶409,600, 1∶204,800 and 1∶409,600, respectively. The results of immunohistochemistry showed that the ascites of the 3 MAbs had positive reactivity with the PCV3 in lymph node tissue clinical samples of pigs infected with PCV3. In conclusion, the MAbs developed in this study were specific to PCV3, which laid a foundation for the further study of the structure of PCV3 Cap and development of the rapid diagnosis method for PCV3 infection in pigs.
引文
[1]Palinski R,Pineyro P,Shang Peng-cheng,et al.A Novel porcine circovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure[J].J Virol,2017,91(1):e01879-16.
[2]Ku Xu-gang,Chen Fang-zhou,Li Pan-pan,et al.Identification and genetic characterization of porcine circovirus type 3 in China[J].Transbound Emerg Dis,2017,64(3):703-708.
[3]Shen Han-qin,Liu Xiang-cong,Zhang Peng-fei,et al.Genome characterization of a porcine circovirus type 3 in South China[J].Transbound Emerg Dis,2018,65(1):264-266.
[4]Phan T G,Giannitti F,Rossow S,et al.Detection of a novel circovirus PCV3 in pigs with cardiac and multi-systemic inflammation[J].Virol J,2016,13(1):184.
[5]黄立平,刘长明,危艳武,等.抗猪圆环病毒2型Cap蛋白中和性单克隆抗体的制备及鉴定[J].中国预防兽医学报,2009,31(2):132-136.
[6]贺会利,李军,潘艳,等.广西首例猪圆环病毒3型的发现及其衣壳蛋白序列分析[J].南方农业学报,2017,48(8):1499-1503.
[7]Sun Jiu-meng,Wei Li,Lu Zong-ji,et al.Retrospective study of porcine circovirus 3 infection in China[J].Transbound Emerg Dis,2018,65(3):607-613.
[8]Zhang Jia-xin,Liu Zhen-guo,Zou Ya-wen,et al.First molecular detection of porcine circovirus type 3 in dogs in China[J].Virus Genes,2017,54(1):1-5.
[9]Mahe D,Truong C A C,Le C P,et al.Differential recognition of ORF2 protein from type 1 and type 2 porcine circoviruses and identification of immunorelevant epitopes[J].J Gen Virol,2000,81(7):1815-1824.
[10]Fenaux M,Opriessnig T,Halbur P G,et al.Immunogenicity and pathogenicity of chimeric infectious DNA clones of pathogenic porcine circovirus type 2(PCV2)and nonpathogenic PCV1 in weanling pigs[J].J Virol,2003,77(20):11232-11243.
[11]Nawagitgul P,Harms P A,Morozov I,et al.Modified indirect porcine circovirus(PCV)type 2-based and recombinant capsid protein(ORF2)-based enzyme-linked immunosorbent assays for detection of antibodies to PCV[J].Clin Diagn Lab Immunol,2002,9(1):33-40.
[12]Zhao Dong,Wang Xiao-bo,Gao Qing-qing,et al.Retrospective survey and phylogenetic analysis of porcine circovirus type 3 in Jiangsu province,China,2008 to 2017[J].Arch Virol,2018,163(9):2531-2538.
[2]Ku Xu-gang,Chen Fang-zhou,Li Pan-pan,et al.Identification and genetic characterization of porcine circovirus type 3 in China[J].Transbound Emerg Dis,2017,64(3):703-708.
[3]Shen Han-qin,Liu Xiang-cong,Zhang Peng-fei,et al.Genome characterization of a porcine circovirus type 3 in South China[J].Transbound Emerg Dis,2018,65(1):264-266.
[4]Phan T G,Giannitti F,Rossow S,et al.Detection of a novel circovirus PCV3 in pigs with cardiac and multi-systemic inflammation[J].Virol J,2016,13(1):184.
[5]黄立平,刘长明,危艳武,等.抗猪圆环病毒2型Cap蛋白中和性单克隆抗体的制备及鉴定[J].中国预防兽医学报,2009,31(2):132-136.
[6]贺会利,李军,潘艳,等.广西首例猪圆环病毒3型的发现及其衣壳蛋白序列分析[J].南方农业学报,2017,48(8):1499-1503.
[7]Sun Jiu-meng,Wei Li,Lu Zong-ji,et al.Retrospective study of porcine circovirus 3 infection in China[J].Transbound Emerg Dis,2018,65(3):607-613.
[8]Zhang Jia-xin,Liu Zhen-guo,Zou Ya-wen,et al.First molecular detection of porcine circovirus type 3 in dogs in China[J].Virus Genes,2017,54(1):1-5.
[9]Mahe D,Truong C A C,Le C P,et al.Differential recognition of ORF2 protein from type 1 and type 2 porcine circoviruses and identification of immunorelevant epitopes[J].J Gen Virol,2000,81(7):1815-1824.
[10]Fenaux M,Opriessnig T,Halbur P G,et al.Immunogenicity and pathogenicity of chimeric infectious DNA clones of pathogenic porcine circovirus type 2(PCV2)and nonpathogenic PCV1 in weanling pigs[J].J Virol,2003,77(20):11232-11243.
[11]Nawagitgul P,Harms P A,Morozov I,et al.Modified indirect porcine circovirus(PCV)type 2-based and recombinant capsid protein(ORF2)-based enzyme-linked immunosorbent assays for detection of antibodies to PCV[J].Clin Diagn Lab Immunol,2002,9(1):33-40.
[12]Zhao Dong,Wang Xiao-bo,Gao Qing-qing,et al.Retrospective survey and phylogenetic analysis of porcine circovirus type 3 in Jiangsu province,China,2008 to 2017[J].Arch Virol,2018,163(9):2531-2538.