一种基于重组截短Gc_(aa407～614)蛋白的赤羽病病毒抗体间接ELISA方法的建立
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摘要
为建立一种检测赤羽病病毒(Akabane virus,AKAV)抗体的ELISA方法,对Gc蛋白从407位至614位氨基酸的编码序列,经密码子优化后进行基因合成,然后克隆至重组表达载体PET-32a(+),转化大肠杆菌BL21(DE3)感受态细胞,用1 mmol/L IPTG在37℃进行诱导表达。用Ni~(+2)-NTA树脂亲和层析方法纯化重组蛋白(trGc_(aa407~614)),以Western blotting检测其抗原性;用纯化trGc_(aa407~614)建立间接ELISA方法(trGc_(aa407~614)-ELISA),并评价该方法的特异性、敏感性和重复性。结果显示:重组蛋白trGc_(aa407~614)以包涵体形式在大肠杆菌中高效表达,经Ni~(+2)-NTA树脂亲和层析纯化后的浓度2.4 mg/mL,纯度为94%,对山羊抗AKAV阳性血清呈现较好的抗原反应性;所建立的trGc_(aa407~614)-ELISA方法的最佳抗原包被浓度为4.0μg/mL,血清稀释度为1:80,血清阴阳性的OD450临界值为0.318;该方法对AKAV阳性血清呈特异性反应,组内试验和组间试验的变异系数均小于10%。用该方法对273份进口牛血清进行AKAV抗体检测,发现10份血清为阳性。总之,本研究建立的trGc_(aa407~614)-ELISA方法为血清样品中赤羽病病毒抗体的检测提供了有效手段。
In order to establish an indirect enzyme-linked immunosorbent assay(ELISA)for detection of antibodies against Akabane virus(AKAV),the sequence encoding 407-614 amino acids of the Gc proteins was selected,after optimizing the codon,the gene was amplified and then cloned into recombinant expression vector pET-32 a(+)to transform into competent cells of Escherichia coli(E.coli)BL21(DE3)and to induce the expression of protein at 37 ℃ by use of 1 mmol/L IPTG. The recombinant protein trGc_(aa407-614) was puri?ed by Ni~(2+)-NTA resin af?nity chromatography,and its antigenicity was detected by Western-blot. Furthermore,an indirect ELISA method(trGc_(aa407-614)-ELISA)was established using the purified trGc_(aa407-614) to evaluate as the coating antigens,and its specificity,sensitivity and repeatability was assessed. The results showed that the recombinant protein was expressed effectively in E.coli in the form of inclusion,with the concentration of 2.4 mg/mL and the purity of 94%,indicating a good antigenic reactivity when reacting with anti-AKAV positive serum in goat. For the trGc_(aa407-614)-ELISA,the optimum antigen concentration was 4.0 μg/mL with the serum dilution of 1:80,and the OD450 critical value of serum negative-positive was 0.318. Speci?c reaction was shown in the AKAV positive serum through the method,and the variable coef?cients of the intra-assay and inter-assay were less than 10%. 10 serums were detected to be positive when 273 imported bovine serums were tested for AKAV antibody. In conclusion,an effective method was provided for detection of antibodies against Akabane virus in serum samples by the recombinant protein trGc_(aa407-614)-ELISA established in this study.
In order to establish an indirect enzyme-linked immunosorbent assay(ELISA)for detection of antibodies against Akabane virus(AKAV),the sequence encoding 407-614 amino acids of the Gc proteins was selected,after optimizing the codon,the gene was amplified and then cloned into recombinant expression vector pET-32 a(+)to transform into competent cells of Escherichia coli(E.coli)BL21(DE3)and to induce the expression of protein at 37 ℃ by use of 1 mmol/L IPTG. The recombinant protein trGc_(aa407-614) was puri?ed by Ni~(2+)-NTA resin af?nity chromatography,and its antigenicity was detected by Western-blot. Furthermore,an indirect ELISA method(trGc_(aa407-614)-ELISA)was established using the purified trGc_(aa407-614) to evaluate as the coating antigens,and its specificity,sensitivity and repeatability was assessed. The results showed that the recombinant protein was expressed effectively in E.coli in the form of inclusion,with the concentration of 2.4 mg/mL and the purity of 94%,indicating a good antigenic reactivity when reacting with anti-AKAV positive serum in goat. For the trGc_(aa407-614)-ELISA,the optimum antigen concentration was 4.0 μg/mL with the serum dilution of 1:80,and the OD450 critical value of serum negative-positive was 0.318. Speci?c reaction was shown in the AKAV positive serum through the method,and the variable coef?cients of the intra-assay and inter-assay were less than 10%. 10 serums were detected to be positive when 273 imported bovine serums were tested for AKAV antibody. In conclusion,an effective method was provided for detection of antibodies against Akabane virus in serum samples by the recombinant protein trGc_(aa407-614)-ELISA established in this study.
引文
[1] KIRKLAND P D. Akabane virus infection[J]. Revue scientifique et technique, 2015, 34(2):403-410.
[2] KATO T, YANASE T, SUZUKI M, et al. Monitoring for bovine arboviruses in the most southwestern islands in Japan between 1994 and 2014[J]. BMC veterinary research, 2016, 12(1):125.
[3] OLUWAYELU D O, AIKI-RAJI C O, UMEH E C,et al. Serological investigation of Akabane virus infection in cattle and sheep in Nigeria[J]. Advances in virology,2016. DOI:10.1155/2016/2936082.
[4] AN D J, YOON S H, JEONG W, et al. Genetic analysis of Akabane virus isolates from cattle in Korea[J].Veterinary microbiology, 2010, 140(1/2):49-55.
[5] HORIKITA T, YOSHINAGA S, OKATANI A T,et al. Loss of milk yield due to Akabane disease in dairy cows[J]. Journal of veterinary medical science, 2005,67(3):287-290.
[6] WANG J, BLASDELL K R, YIN H, et al. A largescale serological survey of Akabane virus infection in cattle, yak, sheep and goats in China[J]. Veterinary microbiology, 2017, 207:7-12.
[7] JUN Q, QINGLING M, ZAICHAO Z, et al. A serological survey of Akabane virus infection in cattleand sheep in northwest China[J]. Trop animal health production, 2012, 44(8):1817-1820.
[8] ELLIOTT R M. Emerging viruses:the Bunyaviridae[J].Molecular medicine, 1997, 3(9):572-577.
[9] YANASE T, YOSHIDA K, OHASHI S, et al.Sequence analysis of the medium RNA segment of three Simbu serogroup viruses, Akabane, Aino, and Peaton viruses[J]. Virus research, 2003, 93(1):63-69.
[10] OGINO M,YOSHIMATSU K, EBIHARA H,et al. Cell fusion activities of Hantaan virus envelope glycoproteins[J]. Journal of virology, 2004, 78(19):10776-10782.
[11] LOZACH P Y, MANCINI R, BITTO D, et al. Entry of bunyaviruses into mammalian cells[J]. Cell host microbe, 2010, 7(6):488-499.
[12]徐树兰,童铁钢,白宇,等.赤羽病病毒囊膜糖蛋白G1基因的截短表达、纯化及间接ELISA诊断方法的建立[J].农业生物技术学报,2008,16(4):562-567.
[13]徐树兰,李少英,辛九庆,等.赤羽病病毒核衣壳蛋白抗体间接ELISA检测方法的建立[J].中国兽医科学,2006, 36(11):868-875.
[14]花群义,杨云庆,董俊,等.赤羽病病毒N基因硫氧还蛋白融合表达载体的构建与表达[J].中国兽医科技,2003, 33(10):7-14.
[15] CIFUENTES-MUNOZ N, SALAZAR-QUIROZ N,TISCHLER N D. et al. Hantavirus Gn and Gc envelope glycoproteins:key structural units for virus cell entry and virus assembly[J]. Viruses, 2014, 6(4):1801-1822.
[16] KITTELBERGER R, MCFADDEN A M, KIRKLAND P D,et al. Evaluation of two commercial enzyme-linked immunosorbent assay kits for the detection of serum antibodies against Akabane virus in cattle[J]. Journal of veterinary diagnostic investigation, 2013, 25(5):645-648.
[17] TSUDA T, YOSHIDA K, YANASE T, et al.Competitive enzyme-linked immunosorbent assay for the detection of the antibodies specific to akabane virus[J].Journal of veterinary diagnostic investigation, 2004, 16(6):571-576.
[18] OLUWAYELU D, WERNIKE K, ADEBIYI A, et al.Neutralizing antibodies against Simbu serogroup viruses in cattle and sheep, Nigeria, 2012-2014[J]. BMC veterinary research, 2018, 14(1):277.
[2] KATO T, YANASE T, SUZUKI M, et al. Monitoring for bovine arboviruses in the most southwestern islands in Japan between 1994 and 2014[J]. BMC veterinary research, 2016, 12(1):125.
[3] OLUWAYELU D O, AIKI-RAJI C O, UMEH E C,et al. Serological investigation of Akabane virus infection in cattle and sheep in Nigeria[J]. Advances in virology,2016. DOI:10.1155/2016/2936082.
[4] AN D J, YOON S H, JEONG W, et al. Genetic analysis of Akabane virus isolates from cattle in Korea[J].Veterinary microbiology, 2010, 140(1/2):49-55.
[5] HORIKITA T, YOSHINAGA S, OKATANI A T,et al. Loss of milk yield due to Akabane disease in dairy cows[J]. Journal of veterinary medical science, 2005,67(3):287-290.
[6] WANG J, BLASDELL K R, YIN H, et al. A largescale serological survey of Akabane virus infection in cattle, yak, sheep and goats in China[J]. Veterinary microbiology, 2017, 207:7-12.
[7] JUN Q, QINGLING M, ZAICHAO Z, et al. A serological survey of Akabane virus infection in cattleand sheep in northwest China[J]. Trop animal health production, 2012, 44(8):1817-1820.
[8] ELLIOTT R M. Emerging viruses:the Bunyaviridae[J].Molecular medicine, 1997, 3(9):572-577.
[9] YANASE T, YOSHIDA K, OHASHI S, et al.Sequence analysis of the medium RNA segment of three Simbu serogroup viruses, Akabane, Aino, and Peaton viruses[J]. Virus research, 2003, 93(1):63-69.
[10] OGINO M,YOSHIMATSU K, EBIHARA H,et al. Cell fusion activities of Hantaan virus envelope glycoproteins[J]. Journal of virology, 2004, 78(19):10776-10782.
[11] LOZACH P Y, MANCINI R, BITTO D, et al. Entry of bunyaviruses into mammalian cells[J]. Cell host microbe, 2010, 7(6):488-499.
[12]徐树兰,童铁钢,白宇,等.赤羽病病毒囊膜糖蛋白G1基因的截短表达、纯化及间接ELISA诊断方法的建立[J].农业生物技术学报,2008,16(4):562-567.
[13]徐树兰,李少英,辛九庆,等.赤羽病病毒核衣壳蛋白抗体间接ELISA检测方法的建立[J].中国兽医科学,2006, 36(11):868-875.
[14]花群义,杨云庆,董俊,等.赤羽病病毒N基因硫氧还蛋白融合表达载体的构建与表达[J].中国兽医科技,2003, 33(10):7-14.
[15] CIFUENTES-MUNOZ N, SALAZAR-QUIROZ N,TISCHLER N D. et al. Hantavirus Gn and Gc envelope glycoproteins:key structural units for virus cell entry and virus assembly[J]. Viruses, 2014, 6(4):1801-1822.
[16] KITTELBERGER R, MCFADDEN A M, KIRKLAND P D,et al. Evaluation of two commercial enzyme-linked immunosorbent assay kits for the detection of serum antibodies against Akabane virus in cattle[J]. Journal of veterinary diagnostic investigation, 2013, 25(5):645-648.
[17] TSUDA T, YOSHIDA K, YANASE T, et al.Competitive enzyme-linked immunosorbent assay for the detection of the antibodies specific to akabane virus[J].Journal of veterinary diagnostic investigation, 2004, 16(6):571-576.
[18] OLUWAYELU D, WERNIKE K, ADEBIYI A, et al.Neutralizing antibodies against Simbu serogroup viruses in cattle and sheep, Nigeria, 2012-2014[J]. BMC veterinary research, 2018, 14(1):277.