牛副流感病毒3型核衣壳蛋白的原核表达及间接ELISA方法的建立
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摘要
为建立以牛副流感病毒3型(BPIV3)核衣壳蛋白(NP)为包被抗原的间接ELISA方法,本研究扩增BPIV3的NP基因并克隆于原核表达载体,获得重组表达质粒p ET30-NP。将其转化至表达菌BL21(DE3),获得了可溶性表达的重组N蛋白,用镍柱在非变性条件下纯化后将其作为包被抗原,建立了检测BPIV3抗体的间接ELISA方法。特异性试验结果显示作为包被抗原的重组N蛋白仅与BPIV3阳性牛血清发生特异性反应,与牛传染性鼻气管炎病毒和牛病毒性腹泻病毒等牛的常见病原无血清学交叉反应,表明其特异性较强。牛BPIV3阳性血清在1:640倍稀释时按该方法检测仍为阳性,显示该方法具有较高的敏感性。重复性试验显示批内变异系数小于6%,批间变异系数小于12%,表明该方法具有较好的稳定性。对94份牛血清的比较试验显示,本研究建立的间接ELISA方法与病毒中和试验的符合率为98%。利用该方法检测了394份采自接种过BPIV3灭活苗牛场的血清样品和95份未接种疫苗牛场的血清样品,结果接种疫苗的牛血清均呈强阳性,而95份未接种疫苗牛血清的阳性率为80%。本研究建立的间接ELISA方法可以试用于国内BPIV3的流行病学调查和免疫监测,为国内BPIV3的防控提供技术支持。
In order to develop an indirect enzyme-linked immunosorbent assay(ELISA) for detection of antibodies to bovine parainfluenza virus type 3(BPIV3) using recombinant nucleocapisd protein(NP) as antigen, the NP gene amplified from the template cDNA of BPIV3 was cloned into the prokaryotic vector pET-30 a, resulting in recombinant expression plasmid pET30-NP.The pET30-NP was transformed into the E.coli strain BL21(DE3). The soluble recombinant NP was expressed and purified with a nickel column under non-denaturing conditions. Subsequently, the purified NP was used as coating antigen to develop an indirect ELISA for detecting antibodies to BPIV3. The specificity tests showed that the recombinant NP reacted specifically with positive serum of BPIV3, and had no serological cross-reaction with common pathogens of cattle, such as infectious bovine rhinotracheitisvirus and bovine viral diarrhea virus. The sensitivity analysis showed that the positive serum of BPIV3 was still positive at a dilution of 1 ∶640, indicating that the indirect ELISA has high sensitivity. The repeatability tests showed that the intra-batch coefficient of variation was less than 6% and the coefficient of variation between batches was less than 12%, indicating that the indirect ELISA has good reproducibility. The coincidence rate was 98% based on the results of the indirect ELISA method and the virus neutralization by testing serum samples of 94 bovine. The indirect ELISA was used to detect serum samples of 394 cattle immunized with BPIV3 inactivated vaccine and serum samples of 95 cattle without immunization. Results showed that all the serum samples collected from vaccinated cattle were strongly positive, while the positive rate of serum samples from 95 unvaccinated cattle was 80%. Taken together, the indirect ELISA developed in this study could be used for seroprevalence investigation and vaccination monitoring for BPIV3, providing technical support for the prevention and control of BPIV3 in China.
In order to develop an indirect enzyme-linked immunosorbent assay(ELISA) for detection of antibodies to bovine parainfluenza virus type 3(BPIV3) using recombinant nucleocapisd protein(NP) as antigen, the NP gene amplified from the template cDNA of BPIV3 was cloned into the prokaryotic vector pET-30 a, resulting in recombinant expression plasmid pET30-NP.The pET30-NP was transformed into the E.coli strain BL21(DE3). The soluble recombinant NP was expressed and purified with a nickel column under non-denaturing conditions. Subsequently, the purified NP was used as coating antigen to develop an indirect ELISA for detecting antibodies to BPIV3. The specificity tests showed that the recombinant NP reacted specifically with positive serum of BPIV3, and had no serological cross-reaction with common pathogens of cattle, such as infectious bovine rhinotracheitisvirus and bovine viral diarrhea virus. The sensitivity analysis showed that the positive serum of BPIV3 was still positive at a dilution of 1 ∶640, indicating that the indirect ELISA has high sensitivity. The repeatability tests showed that the intra-batch coefficient of variation was less than 6% and the coefficient of variation between batches was less than 12%, indicating that the indirect ELISA has good reproducibility. The coincidence rate was 98% based on the results of the indirect ELISA method and the virus neutralization by testing serum samples of 94 bovine. The indirect ELISA was used to detect serum samples of 394 cattle immunized with BPIV3 inactivated vaccine and serum samples of 95 cattle without immunization. Results showed that all the serum samples collected from vaccinated cattle were strongly positive, while the positive rate of serum samples from 95 unvaccinated cattle was 80%. Taken together, the indirect ELISA developed in this study could be used for seroprevalence investigation and vaccination monitoring for BPIV3, providing technical support for the prevention and control of BPIV3 in China.
引文
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[2] Muggli-Cockett N E, Cundiff L V, Gregory K E. Genetic analysis of bovine respiratory disease uin beef calves during the first year of life[J]. J Anim Sci, 1992, 70(7):2013-2019.
[3]霍志云,童钦,胡嘉欣,等.北方三省(区)牛副流感病毒3型的血清学调查[J].动物医学进展,2012,33(9):124-126.
[4]王海勇,童钦,王炜,等.我国牛副流感病毒3型血清学调查[J].中国预防兽医学报,2014, 36(2):154-156.
[5] Neill J D, Ridpath J F, Valayudhan B T. Identification and genome characterization of genotype B and genotype C bovine parainfluenza type 3 viruses isolated in the United States[J].BMC Vet Res, 2015, 11(1):112.
[6] Durbin A P, Siew J W, Murphy B R, et al. Minimum protein requirements for transcription and RNA replication of a minigenome of human parainfluenza virus type 3 and evaluation of the rule of six[J]. Virology, 1997, 234(1):74-83.
[7] Aguayo-Hiraldo P I, Arasaratnam R J, Tzannou I, et al. Characterizing the cellular immune response to Parainfluenza virus 3[J]. J Infect Dis, 2017, 216(2):153-161.
[8] Marcoppido G, Parrene V,Vila B. Antibodies to pathogenic livestock viruses in a wild vicune(vicugna vicugna)population in the argentinean andean altiplano[J]. J Wildlife Dis, 2010, 46(2):608-614.
[9] Morein B, Dieter Z. Parainfluenza-3 virus in cattle:mechanisms of infections and defence in the respiratory tract[J]. J Vet Med Sci, 1975, 12(3):40-41.
[10]王吉,付瑞,李晓波,等.牛副流感病毒3型实时荧光定量PCR检测方法的建立及在牛源样本检测中的应用[J].中国病毒病杂志,2018,8(5):393-399.
[11]祖立闯,朱远茂,王延辉,等.牛传染性鼻气管炎病毒重组gD蛋白间接ELISA方法的建立及应用[J].中国预防兽医学报,2008,30(7):537-543.
[12]林密,马军武,祁淑芸,等.O型口蹄疫病毒固相竞争ELISA抗体检测方法的建立[J].中国预防兽医学报,2008, 30(8):627-630.
[2] Muggli-Cockett N E, Cundiff L V, Gregory K E. Genetic analysis of bovine respiratory disease uin beef calves during the first year of life[J]. J Anim Sci, 1992, 70(7):2013-2019.
[3]霍志云,童钦,胡嘉欣,等.北方三省(区)牛副流感病毒3型的血清学调查[J].动物医学进展,2012,33(9):124-126.
[4]王海勇,童钦,王炜,等.我国牛副流感病毒3型血清学调查[J].中国预防兽医学报,2014, 36(2):154-156.
[5] Neill J D, Ridpath J F, Valayudhan B T. Identification and genome characterization of genotype B and genotype C bovine parainfluenza type 3 viruses isolated in the United States[J].BMC Vet Res, 2015, 11(1):112.
[6] Durbin A P, Siew J W, Murphy B R, et al. Minimum protein requirements for transcription and RNA replication of a minigenome of human parainfluenza virus type 3 and evaluation of the rule of six[J]. Virology, 1997, 234(1):74-83.
[7] Aguayo-Hiraldo P I, Arasaratnam R J, Tzannou I, et al. Characterizing the cellular immune response to Parainfluenza virus 3[J]. J Infect Dis, 2017, 216(2):153-161.
[8] Marcoppido G, Parrene V,Vila B. Antibodies to pathogenic livestock viruses in a wild vicune(vicugna vicugna)population in the argentinean andean altiplano[J]. J Wildlife Dis, 2010, 46(2):608-614.
[9] Morein B, Dieter Z. Parainfluenza-3 virus in cattle:mechanisms of infections and defence in the respiratory tract[J]. J Vet Med Sci, 1975, 12(3):40-41.
[10]王吉,付瑞,李晓波,等.牛副流感病毒3型实时荧光定量PCR检测方法的建立及在牛源样本检测中的应用[J].中国病毒病杂志,2018,8(5):393-399.
[11]祖立闯,朱远茂,王延辉,等.牛传染性鼻气管炎病毒重组gD蛋白间接ELISA方法的建立及应用[J].中国预防兽医学报,2008,30(7):537-543.
[12]林密,马军武,祁淑芸,等.O型口蹄疫病毒固相竞争ELISA抗体检测方法的建立[J].中国预防兽医学报,2008, 30(8):627-630.