本文選題：偽狂犬 + 抗體檢測　； 參考：《中國農業(yè)科學院》2015年碩士論文

【摘要】：豬偽狂犬病是由偽狂犬病毒引起的一種以仔豬致命性腦炎、育肥豬呼吸道癥狀以及母豬的流產為主要臨床表現的豬的傳染病,該病給許多國家的養(yǎng)豬業(yè)造成巨大的經濟損失。雖然,基因缺失疫苗免疫與g E ELISA抗體檢測試劑盒的聯(lián)合應用幫助許多國家完成了對該病的凈化,但是,由于野豬是該病病原的宿主庫,仍然會造成臨近豬群的感染,同時,感染后的豬群作為隱性帶毒者,也會成為長期的潛伏傳染源。因此,對于該病的預防仍然十分重要。目前,國際上廣泛使用g E基因缺失疫苗免疫豬群取得了良好的預防效果,且疫苗免疫與野毒感染后,動物血清中是否會檢測到抗偽狂犬病毒g E蛋白抗體已經被作為鑒別診斷常用的方法。PRV-g E ELISA商品化的試劑盒就常常被用來進行DIVA診斷。盡管如此,ELISA方法由于需要特定的儀器、有一定技術培訓的人員在實驗室中才可以完成,無法由無技術培訓的飼養(yǎng)人員或獸醫(yī)直接簡便的在現地做出快速的診斷。因此,本研究通過膠體金免疫層析試驗建立了抗偽狂犬病毒g E抗體的DIVA快速、簡便的檢測方法,并進行了初步評價。本實驗通過擴增包含g E蛋白重要表位的部分基因,經測序,克隆于原核表達質粒p ET-32a,于大腸桿菌表達菌BL21中誘導、表達并純化,獲得表達蛋白,Western-blot結果表明,表達蛋白可以與抗PRV陽性血清特異結合,將純化后的g E表達蛋白與商品化的豬的Ig G分別作為檢測線(T線)和質控線(C線)噴點于硝酸纖維素膜上,以膠體金顆粒標記金黃色葡萄球菌蛋白A(SPA)作為示蹤劑,建立了檢測豬血清中抗PRV-g E蛋白抗體的的膠體金免疫層析方法。判定標準為:1)待檢的血清中含有抗PRV-g E抗體,可與金標SPA結合形成Ig G-SPA復合物,經層析膜流經T線和C線時,Ig G-SPA復合物就會和T線上的PRV-g E蛋白以及C線上的豬的Ig G發(fā)生特異性結合,形成兩條紅色的線,判定為陽性結果;2)若待檢血清中不含有抗PRV-g E抗體,則只有質控線出現;3)若出現質控線未形成紅色條帶時,則該方法檢測結果無效。經過探索該膠體金得最佳反應條件,我們最終確定C線包被豬的Ig G、濃度為2mg/ml,T線濃度為2mg/ml,金標SPA最佳標記p H為8.0、濃度為0.1mg/ml,血清最佳稀釋倍數為1:50倍稀釋。繼而,對本實驗建立的檢測方法分別進行了特異性、敏感性、重復性和穩(wěn)定性以及符合率的檢驗。結果表明,該方法特異性良好,只在檢測偽狂犬病毒血清時出現陽性反應,對其他病毒血清檢測為陰性例如豬繁殖與呼吸綜合征病毒、豬圓環(huán)病毒、豬肺支原體、豬流行性腹瀉與傳染性胃腸炎病毒等;對同一批樣品進行批內重復試驗與批間重復試驗,該方法檢測均為同一結果,并且在4℃可穩(wěn)定儲存長達4個月之久不改變其特性;與愛德士偽狂犬g E抗體檢測試劑盒對比,該方法敏感性略高,該方法特異性和敏感性分別為81.6%和90.7%,并且兩者之間有良好的符合性(Kappa=0.7289)。我們又進行了豬的攻毒實驗,分別用建立的膠體金免疫層析方法和愛德士ELISA方法對不同的4組(每組7只)豬即非免疫攻毒組、非免疫非攻毒組、免疫攻毒組、免疫非攻毒組豬進行g E抗體檢測,監(jiān)測時間為45天,發(fā)現豬只開始出現g E抗體陽性的時間均為第9天、11天、9天、13天,也表明這兩種方法有良好的一致性。綜上所述,本實驗建立了檢測抗偽狂犬病毒抗體膠體金免疫層析方法,為檢測偽狂犬野毒提供了一種快速、簡便的方法。
[Abstract]:Porcine pseudorabies is a pig's infectious disease caused by the pseudorabies virus, a piglet fatal encephalitis, the respiratory symptoms of the fattening pig and the abortion of the sow as the main clinical manifestation. The disease causes huge economic losses to the pig industry in many countries. Although the gene deletion vaccine is combined with the G E ELISA antibody test kit It has been used to help many countries to complete the purification of the disease, but because the wild boar is the host Library of the pathogen, it will still cause the infection of the adjacent pigs, and the infected pigs, as recessive and poisonous, will also become a long-term latent source of infection. Therefore, the prevention of the disease is still very important. At present, the G E base is widely used in the world. As a result of the good preventive effect of vaccinated pigs, and the detection of anti pseudorabies virus G E antibodies in animal sera after immunization and wild virus infection, the.PRV-g E ELISA commercialized kit is often used for the diagnosis of DIVA. However, the ELISA method has been used. People who need specific instruments and some technical training can be completed in the laboratory, unable to make rapid and simple diagnosis directly and conveniently in the present field. Therefore, this study established a fast and simple DIVA detection prescription for the anti pseudocanine virus G E antibody by colloidal gold immunochromatography test. By amplification of some genes containing important epitopes of G E protein, this experiment was sequenced and cloned into the prokaryotic expression plasmid P ET-32a, induced in the Escherichia coli expressing bacteria BL21, expressed and purified, and obtained the expression protein. The result of Western-blot showed that the expression protein could be specifically combined with anti PRV positive serum and purified. The G E expression protein and the commercial pig's Ig G were sprayed on the nitrocellulose membrane as the detection line (T line) and the quality control line (C line), and the colloidal gold granules labeled Staphylococcus aureus protein A (SPA) was used as a tracer. The colloidal gold immunochromatography method for detecting the anti PRV-g E protein antibody in pig serum was established. The criterion was as follows: 1) The test serum contains anti PRV-g E antibody and can combine with gold standard SPA to form Ig G-SPA complex. When the chromatography membrane flows through the T line and the C line, the Ig G-SPA complex combines with the PRV-g E protein on the T line and the pig in the T line to form a specific combination, forming two red lines, determined as positive results; 2) if the serum is not contained in the tested serum. The only quality control line appeared; 3) if the quality control line did not form a red strip, the result of the method was invalid. After exploring the best reaction condition of the colloid gold, we finally determined that the C line was Ig G, the concentration was 2mg/ml, the concentration of T line was 2mg/ml, the best marker of the gold standard SPA P H was 8, the concentration was 0.1mg/ml, the best dilution times the serum times. The test methods established by this experiment were specific, sensitive, reproducible, and stable, and the coincidence rate was tested. The results showed that the method had good specificity, was only positive in the detection of pseudorabies virus sera and negative for other virus sera, such as porcine reproductive and respiratory syndrome. Syndrome virus, porcine circovirus, Mycoplasma pneumoniae, porcine epidemic diarrhea and infectious gastroenteritis virus and so on. The same batch of repeated test and interbatch repeat test were carried out on the same batch of samples. The test was the same result, and it was stable for up to 4 months at 4 centigrade without changing its characteristics; the anti physical examination of G E of edus pseudorabies was tested. The sensitivity of the method was slightly higher, and the specificity and sensitivity of the method were 81.6% and 90.7% respectively, and there were good conformance between the two (Kappa=0.7289). We also carried out a pig's attack test. We used the colloidal gold immunochromatography and EDIS ELISA method for the non immune attack of 4 different groups (7 pigs in each group), respectively. Group, non immunization non attack group, immune attack group, immune non attack group pigs were tested for G E antibody, and the monitoring time was 45 days. It was found that the time of G E antibody positive was ninth days, 11 days, 9 days and 13 days, which also showed that the two methods had good consistency. In the summary, the experiment established the detection of anti pseudorabies antibody colloid Gold immunochromatography assay provides a fast and simple method for detecting pseudorabies virus.

【學位授予單位】：中國農業(yè)科學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.4

【參考文獻】

相關期刊論文 前1條

1 李春華;王英;蔣鳳英;胡建華;;豬偽狂犬病研究進展[J];動物醫(yī)學進展;2008年03期

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本文編號：1803674