Development of Indirect ELISA for Detecting Novel Duck Reovirus Using σB and σC Proteins as Coating Antigens
-
摘要: 以纯化的新型鸭呼肠孤病毒（NDRV）NP03株重组σB和σC蛋白作为包被抗原，对反应条件进行优化，建立检测NDRV抗体的间接ELISA方法。优化后的最佳条件为：σB蛋白包被浓度为12.5 μg·mL-1；σC蛋白包被浓度为6.25 μg·mL-1；封闭液为15% FBS；血清稀释度为1：80；酶标二抗稀释度为1：400；底物37℃显色5 min。该方法对MDRV、DHV、MPV和MD-GPV阳性血清均无交叉反应，具有良好的特异性。该方法与NDRV全病毒间接ELISA相比较，符合率高达92.5%。本研究进一步丰富了NDRV抗体的检测方法。Abstract: An indirect ELISA was developed using the purified recombinant novel duck reovirus(NDRV)σB and σC proteins as coating antigens. The optimized conditions for the methodology were determined to include:concentrations of σB at 12.5 μg·mL-1 and σC at 6.25 μg·mL-1,a blocking buffer of 15% FBS, serum samples being diluted 80 times, goat anti-duck HRP-IgG being diluted 400 times,and substrate incubation at 37℃ for 5 min. The assay was found to be specific without any cross reaction with antibodies of MDRV, DHV, MPV, or MD-GPV. The coincidence rate between the indirect ELISA coated with the recombinant σB and σC proteins and that coated with NDRVwas 92.5%. It appeared that the newly developed ELISA exhibited satisfactory sensitivity and specificity on measurements,and could be an alternative means for detecting anti-NDRV antibodies.
-
Key words:
- novel duck reovirus /
- prokaryotic expression /
- σB protein /
- σC protein /
- indirect ELISA
-
图 1 纯化的σB和σC蛋白的SDS-PAGE及western blot分析
M为蛋白预染Marker；1~2为σB纯化蛋白的SDS-PAGE分析；3为σB纯化蛋白的western blot分析；4~5为σC纯化蛋白的SDS-PAGE分析；6为σC纯化蛋白的western blot分析。
Figure 1. SDS-PAGE and western blot analyses on purified σB and σC proteins
表 1 最佳抗原包被浓度及血清最佳稀释度的方阵滴定结果
Table 1. Optimal concentrations for coating antigens and serum dilution as determined by checkerboard titration method
血清稀释倍数 血清 蛋白 抗原质量浓度/(μg·mL-1) 50 25 12.5 6.25 3.125 1∶20 阳性 σB 1.742 1.738 1.730 1.721 1.693 阴性 0.267 0.267 0.260 0.255 0.212 阳性 σC 1.813 1.816 1.810 1.748 1.743 阴性 0.285 0.258 0.250 0.224 0.198 1∶40 阳性 σB 1.711 1.703 1.718 1.682 1.653 阴性 0.233 0.227 0.200 0.193 0.187 阳性 σC 1.769 1.773 1.737 1.692 1.737 阴性 0.212 0.194 0.188 0.166 0.158 1∶80 阳性 σB 1.698 1.691 1.664 1.594 1.487 阴性 0.188 0.176 0.162 0.157 0.148 阳性 σC 1.720 1.714 1.697 1.688 1.635 阴性 0.167 0.162 0.153 0.145 0.142 1∶160 阳性 σB 1.603 1.589 1.496 1.327 1.385 阴性 0.158 0.162 0.151 0.139 0.140 阳性 σC 1.607 1.524 1.470 1.415 1.430 阴性 0.142 0.137 0.130 0.126 0.126 1∶320 阳性 σB 1.305 1.312 1.217 1.153 1.008 阴性 0.148 0.138 0.121 0.121 0.110 阳性 σC 1.420 1.393 1.405 1.225 1.347 阴性 0.131 0.122 0.123 0.120 0.121 
下载: 导出CSV
表 2 间接ELISA检测方法的反应条件的优化
Table 2. Optimized conditions for indirect ELISA
项目 σB蛋白 σC蛋白 封闭液 待检血清 羊抗鸭IgG-HRP 最佳质量浓度或稀释度 12.5 μg·mL-1 6.25 μg·mL-1 15% FBS 1∶80 1∶400 最佳反应条件 4℃/12 h 4℃/12 h 37℃/3 h 37℃/60 min 37℃/45 min
下载: 导出CSV
表 3 间接ELISA特异性试验
Table 3. Specificity test on indirect ELISA
血清 OD490nm 值 S/P 值 结果 MDRV 0.242 0.039 阴性 DHV 0.230 0.031 阴性 MPV 0.191 0.006 阴性 MD-GPV 0.208 0.017 阴性 NDRV(+) 1.730 NDRV(-) 0.182
下载: 导出CSV
表 4 间接ELISA重复性试验
Table 4. Repeatability test on indirect ELISA
血清 批内重复性试验 批间重复性试验 ${\bar{X}}$±SD CV/% ${\bar{X}}$±SD CV/% 1 1.429±0.012 5.21 1.442±0.008 4.78 2 0.641±0.006 3.17 0.625±0.008 6.43 3 1.337±0.010 6.06 1.387±0.010 5.10 4 0.801±0.018 4.14 0.803±0.016 6.25 5 1.511±0.011 2.93 1.523±0.010 4.97 6 1.007±0.007 3.76 1.127±0.007 3.21 7 1.454±0.008 5.21 1.429±0.018 6.78 8 0.403±0.010 6.20 0.415±0.014 6.78
下载: 导出CSV
表 5 σB-σC-ELISA和NDRV-ELISA方法的比较试验
Table 5. Comparison between σB-σC-ELISA and NDRV-ELISA
项目 阳性 阴性 阳性率/% 符合率/% σB-σC-ELISA 31/80 49/80 38.75 92.5 NDRV-ELISA 33/80 47/80 41.25
下载: 导出CSV
-
[1] 陈少莺,陈仕龙,林锋强,等. 一种新的鸭病(暂名鸭出血性坏死性肝炎)病原学研究初报[J]. 中国农学通报,2009,25(16):28-31. http://www.baidu.com/link?url=x8-uIq0bctilTlTwLT1XIkMXTncI5vXqH-jTjs-iiZ6zCFQzZvq92rlcVwGn3RoI05LNh3ESw-XC3PlVHGGF-fDmEHVNzhdibScr8b2PFRaErGQyW6ULfFnOqLOQPHyjkj2U-sS18VfzmIYh1uTQ-W7h25HEUsIo_jFP9GYC0efoq5qAqssHrJAa3Hs5W17xmwgqqY3Pg5U70tUZlt2qufRLk2pSQVs0s3mFRdS_vcHPLzZtSV5XwE89Sb3EXbd5qO50tQFqs1-ZvKsFd8WbtNT5lHVmQvb69T9VaL8PVLgdn-vit7LZQm0Erz1jshDfrGzVeWqOKQ-GCdqixi8hG_kMkifg4rlNAGcLE6MUp6zTufPuS-46GHraI3RAqkbAl7E2IvpQM2LywUbTR3zeLn7Z6UkeureCN3VRCQ_aRq-BncpcKPfuv0N2BeAktWURpXG4FpfeGpFwye_F5Wn5USyPbxByg5mrN6MEjiTADrFtwwE5kztdEmoydOs4xjvDij8ip0Ymz3h-7FtWT3fCbxSLWvKwl1Fe44T5jWT8k4S&wd=&eqid=adb44b48000326b000000005582689d5 [2] 陈少莺,陈仕龙,林锋强,等. 新型鸭呼肠孤病毒的分离与鉴定[J]. 病毒学报,2012,28(3):224-230. http://www.cnki.com.cn/Article/CJFDTOTAL-BDXB201203008.htm [3] 王劭,陈少莺,陈仕龙,等. 新型鸭呼肠孤病毒NP03株S3基因序列分析[J]. 农业生物技术学报,2010,18(3):567-572. http://www.cnki.com.cn/Article/CJFDTOTAL-NYSB201003028.htm [4] ZHANG Y, GUO D C, LIU M, et al. Characterization of the σB-encoding genes of muscovy duck reovirus:σC-σB-ELISA for antibodies against duck reovirus in ducks[J]. Vet Microbiol, 2007, 121(3-4):231-241. doi: 10.1016/j.vetmic.2006.12.008 [5] 王锦祥,程晓霞,陈少莺,等. 新型鸭呼肠孤病毒σC基因原核表达及其抗原性的初步研究[J]. 福建农业学报,2014,29(4):310-313. http://www.fjnyxb.cn/CN/abstract/abstract2406.shtml [6] ZHU Y Q, LI Y F, BI Z L, et al. Protective immune responses in ducklings induced by a suicidal DNA vaccine of the sigma C gene of nocel duck reovirus[J]. Vet Immunol Immunopathol, 2015, 165(1-2):88-92. doi: 10.1016/j.vetimm.2015.03.001 [7] 王劭,陈少莺,陈仕龙,等. 新型鸭呼肠孤病毒RT-PCR方法的建立与应用[J]. 农业生物技术学报,2011,19(2):388-392. [8] 袁远华,吴志新,王俊峰,等. 新型鸭呼肠孤病毒SYBR Green I实时荧光定量RT-PCR检测方法的建立[J]. 中国预防兽医学报,2013,35(9):738-741. [9] 王锦祥,程晓霞,陈少莺,等. 新型鸭呼肠孤病毒σC蛋白间接ELISA检测方法的建立[J]. 中国预防兽医学报,2015,37(9):687-690. http://www.cnki.com.cn/Article/CJFDTOTAL-ZGXQ201509009.htm [10] YUN T, CHEN H P, YU B, et al. Development and application of an indirect ELISA for the detection of antibodies to novel duck reovirus[J]. J Virol Methods, 2015, 220:55-59. doi: 10.1016/j.jviromet.2015.04.012 [11] 董井泉,张蕾,马波,等. 1型鸭肝炎病毒VP1蛋白优势抗原区的鉴定及抗体检测ELISA方法的建立[J]. 中国预防兽医学报,2014,36(7):542-546. http://www.cnki.com.cn/Article/CJFDTOTAL-ZGXQ201407010.htm [12] ZHU Y Q, LI C F, BI Z L, et al. Molecular characterization of a novel reovirus isolated from Pekin ducklings in China[J]. Arch Virol, 2015, 160:365-369. doi: 10.1007/s00705-014-2241-x [13] 袁远华,王俊峰,吴志新,等. 1株番鸭源新型鸭呼肠孤病毒(QY株)生物学鉴定[J]. 中国兽医学报,2013,33(8):1174-1178. -
点击查看大图
计量
- 文章访问数: 1577
- HTML全文浏览量: 188
- PDF下载量: 289
- 被引次数: 0
