两步快速纯化鸡产蛋下降综合症病毒方法的研究及标准抗原、标准血清的制备

发布时间：2018-03-24 00:17

本文选题：鸡产蛋下降综合症病毒　切入点：纯化　出处：《扬州大学》2015年硕士论文

【摘要】：鸡产蛋下降综合症(Egg Drop Syndrome-76,简称EDS-76)是由腺病毒感染引起种鸡和蛋鸡产蛋量急剧下降,同时伴有无壳蛋、薄壳蛋、软壳蛋、畸形蛋的一种病毒性疾病[2]。该病是世界范围内导致鸡产蛋率下降的重要原因之一。本研究在蔗糖密度梯度离心的基础上建立了一种快速纯化EDSV的新方法,制备了一批EDS-76抗鸡血清,以此为生物学材料,进一步标定EDS-76的抗原和抗血清,获得了标准抗原和标准阳性血清,为EDSV的诊断和防治提供了有效的方法和材料。1.鸡产蛋下降综合症病毒纯化方法的研究本研究将传统的蔗糖密度梯度离心与两步法快速纯化方法进行了比对分析。电镜比对结果显示：新建立的两步法快速纯化方法可以得到大量形态大小一致的球形病毒粒子,直径70-80nm,符合腺病毒粒子的典型特征；而传统纯化方法得到的病毒粒子则数量稀少,壳粒边缘有缺损,形态模糊。血凝性试验比对结果显示：传统方法得到的病毒HA效价为216,而新建立的两步法快速纯化方法得到的病毒HA效价为219,血凝效价较高。PCR鉴定结果显示：用引物I、11分别进行PCR扩增,发现两种纯化方法得到的病毒均可扩增出大小为918bp及2733bp的两条特异性条带。综合分析提示：新建立的两步法纯化方法比起传统方法更加省时省力,且病毒纯化效果更加显著,这为后期标准抗原的制备奠定了基础。2.鸡产蛋下降综合症标准抗原的制备将纯化的病毒抗原进行了PCR纯异性分析、Western-blot反应谱分析、琼扩试验分析以及病毒灭活条件测定。结果表明本研究纯化的病毒无CAV、REV、ALV、MDV、IBV、 GPV、IBDV、ILTV等外源性病毒,无菌检验、支原体检验合格,具有很高的纯粹性,且该病毒与经典AV-127种毒具有相同的Western-blot反应谱,同时该病毒还可以用于琼扩试验诊断。用甲醛和β-丙内酯分别灭活该纯化病毒,制备标准抗原,并进行比较,最后确定灭活条件为0.5%β-丙内酯作用60h。将纯化的病毒用PBS缓冲液稀释至效价为214,按上述灭活条件对病毒进行灭活,加入1%BSA保护剂,分装,冻干。对抗原的质量进行验证,同时检查物理性状、均一性、稳定性、保质期等进行鉴定,最后获得了高质量的标准抗原。3.鸡产蛋下降综合症标准血清的制备采取自然感染的途径对SPF鸡进行攻毒,6免之后制得效价为215的抗鸡血清。在EDSV感染的CEF上进行IFA效价检测,血清的IFA效价达1：1000。特异性鉴定发现该血清不与NDV、AIV、ALV-J、ALV-A、GPV、REV、MDV、IBV、CAV、TMUV、REOV等外源病毒反应,而只与EDSV反应。将制备的多抗血清,加入0.01%硫柳汞,分装,冻干。测净重,计算并分析分装差异系数CV值,并进行物理性状的检查以及无菌检验、支原体检验、均一性、稳定性、保质期等特性的鉴定,最终获得了性能良好的标准血清。
[Abstract]:Egg Drop Syndrome-76 (EDS-76) is caused by adenovirus infection to cause a sharp decrease in egg production in broilers and laying hens, accompanied by egg-free, thin-shell and soft-shell eggs. A viral disease of abnormal egg [2]. This disease is one of the important causes of the decrease of laying rate in the world. A new method for rapid purification of EDSV was established based on sucrose density gradient centrifugation. A batch of EDS-76 antisera were prepared and used as biological materials to further calibrate the antigens and antisera of EDS-76. The standard antigens and standard positive sera were obtained. This study compared the traditional sucrose density gradient centrifugation with two-step rapid purification method. The results of electron microscope comparison showed that a large number of spherical virus particles with uniform morphology and size could be obtained by the new two-step rapid purification method. The diameter of the virus particles is 70-80 nm, which is consistent with the typical characteristics of the adenovirus particles, while the number of the virus particles obtained by the traditional purification method is very small, and the edges of the shell particles are defective. The results of hemagglutination test showed that the HA titer obtained by the traditional method was 216, while the HA titer obtained by the new two-step rapid purification method was 219. The results of PCR showed that the HA titer of the virus was higher. Primer Ign11 was amplified by PCR. It was found that the two purification methods could amplify two specific bands of 918bp and 2733bp. The comprehensive analysis indicated that the new two-step purification method was more time-saving and labor-saving than the traditional method, and the effect of virus purification was more remarkable. This laid a foundation for the preparation of standard antigen in late stage. The preparation of standard antigen of laying down syndrome of chicken made purified virus antigen by PCR homozygous analysis and Western-blot analysis. The results showed that the purified viruses had high purity and high purity. The results showed that the purified viruses, such as IBV, GPVN IBDVV ILTV and so on, passed the tests of aseptic test and mycoplasma, and had high purity. The virus has the same Western-blot reaction spectrum as the classical AV-127 virus, and the virus can also be used for the diagnosis of agarose dilatation. The purified virus was inactivated with formaldehyde and 尾 -propiolactone, and the standard antigen was prepared and compared. The inactivation condition was determined as 0.5% 尾 -propiolactone for 60 h. The purified virus was diluted with PBS buffer to the titer of 214.The virus was inactivated according to the above inactivation conditions, and the virus was inactivated by adding 1%BSA protectant, packing, freeze-drying. The quality of antigen was verified. At the same time, check physical properties, uniformity, stability, shelf life, etc., Finally, the high quality standard antigen .3.The preparation of standard serum for laying down syndrome of chicken. The antiserum of SPF chicken was prepared by the way of natural infection. The titer of anti-chicken serum was 215. The titer of IFA was detected on CEF infected with EDSV. The IFA titer of the serum was 1: 1000. The specific identification showed that the serum did not react with exogenous viruses such as NDV AIVA, ALV-JV, ALV-AV, IFA, and so on, but only with EDSV. The prepared polyantibody serum was added 0.01% thiomersal, partitioned, lyophilized. The net weight was measured, and the CV value of the difference coefficient of partition was calculated and analyzed. The physical properties and aseptic test, mycoplasma test, homogeneity, stability, shelf life and other characteristics of the identification, finally obtained a good performance of the standard serum.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31

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