本文選題：雞產(chǎn)蛋下降綜合癥病毒　切入點(diǎn)：純化　出處：《揚(yáng)州大學(xué)》2015年碩士論文

【摘要】：雞產(chǎn)蛋下降綜合癥(Egg Drop Syndrome-76,簡(jiǎn)稱EDS-76)是由腺病毒感染引起種雞和蛋雞產(chǎn)蛋量急劇下降,同時(shí)伴有無(wú)殼蛋、薄殼蛋、軟殼蛋、畸形蛋的一種病毒性疾病[2]。該病是世界范圍內(nèi)導(dǎo)致雞產(chǎn)蛋率下降的重要原因之一。本研究在蔗糖密度梯度離心的基礎(chǔ)上建立了一種快速純化EDSV的新方法,制備了一批EDS-76抗雞血清,以此為生物學(xué)材料,進(jìn)一步標(biāo)定EDS-76的抗原和抗血清,獲得了標(biāo)準(zhǔn)抗原和標(biāo)準(zhǔn)陽(yáng)性血清,為EDSV的診斷和防治提供了有效的方法和材料。1.雞產(chǎn)蛋下降綜合癥病毒純化方法的研究本研究將傳統(tǒng)的蔗糖密度梯度離心與兩步法快速純化方法進(jìn)行了比對(duì)分析。電鏡比對(duì)結(jié)果顯示：新建立的兩步法快速純化方法可以得到大量形態(tài)大小一致的球形病毒粒子,直徑70-80nm,符合腺病毒粒子的典型特征；而傳統(tǒng)純化方法得到的病毒粒子則數(shù)量稀少,殼粒邊緣有缺損,形態(tài)模糊。血凝性試驗(yàn)比對(duì)結(jié)果顯示：傳統(tǒng)方法得到的病毒HA效價(jià)為216,而新建立的兩步法快速純化方法得到的病毒HA效價(jià)為219,血凝效價(jià)較高。PCR鑒定結(jié)果顯示：用引物I、11分別進(jìn)行PCR擴(kuò)增,發(fā)現(xiàn)兩種純化方法得到的病毒均可擴(kuò)增出大小為918bp及2733bp的兩條特異性條帶。綜合分析提示：新建立的兩步法純化方法比起傳統(tǒng)方法更加省時(shí)省力,且病毒純化效果更加顯著,這為后期標(biāo)準(zhǔn)抗原的制備奠定了基礎(chǔ)。2.雞產(chǎn)蛋下降綜合癥標(biāo)準(zhǔn)抗原的制備將純化的病毒抗原進(jìn)行了PCR純異性分析、Western-blot反應(yīng)譜分析、瓊擴(kuò)試驗(yàn)分析以及病毒滅活條件測(cè)定。結(jié)果表明本研究純化的病毒無(wú)CAV、REV、ALV、MDV、IBV、 GPV、IBDV、ILTV等外源性病毒,無(wú)菌檢驗(yàn)、支原體檢驗(yàn)合格,具有很高的純粹性,且該病毒與經(jīng)典AV-127種毒具有相同的Western-blot反應(yīng)譜,同時(shí)該病毒還可以用于瓊擴(kuò)試驗(yàn)診斷。用甲醛和β-丙內(nèi)酯分別滅活該純化病毒,制備標(biāo)準(zhǔn)抗原,并進(jìn)行比較,最后確定滅活條件為0.5%β-丙內(nèi)酯作用60h。將純化的病毒用PBS緩沖液稀釋至效價(jià)為214,按上述滅活條件對(duì)病毒進(jìn)行滅活,加入1%BSA保護(hù)劑,分裝,凍干。對(duì)抗原的質(zhì)量進(jìn)行驗(yàn)證,同時(shí)檢查物理性狀、均一性、穩(wěn)定性、保質(zhì)期等進(jìn)行鑒定,最后獲得了高質(zhì)量的標(biāo)準(zhǔn)抗原。3.雞產(chǎn)蛋下降綜合癥標(biāo)準(zhǔn)血清的制備采取自然感染的途徑對(duì)SPF雞進(jìn)行攻毒,6免之后制得效價(jià)為215的抗雞血清。在EDSV感染的CEF上進(jìn)行IFA效價(jià)檢測(cè),血清的IFA效價(jià)達(dá)1：1000。特異性鑒定發(fā)現(xiàn)該血清不與NDV、AIV、ALV-J、ALV-A、GPV、REV、MDV、IBV、CAV、TMUV、REOV等外源病毒反應(yīng),而只與EDSV反應(yīng)。將制備的多抗血清,加入0.01%硫柳汞,分裝,凍干。測(cè)凈重,計(jì)算并分析分裝差異系數(shù)CV值,并進(jìn)行物理性狀的檢查以及無(wú)菌檢驗(yàn)、支原體檢驗(yàn)、均一性、穩(wěn)定性、保質(zhì)期等特性的鑒定,最終獲得了性能良好的標(biāo)準(zhǔn)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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.31

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