【摘要】：豬偽狂犬病在歐美一些發(fā)達(dá)國家已經(jīng)根除,在中國,目前只能通過疫苗接種進(jìn)行預(yù)防。自2011年末以來,華北大部分地區(qū)的已接種疫苗的豬場(chǎng)爆發(fā)了偽狂犬病,造成了嚴(yán)重的經(jīng)濟(jì)損失,而且,爆發(fā)偽狂犬病時(shí)沒有有效的藥物治療該種疾病,因此,研制有效的治療藥物對(duì)于控制豬偽狂犬病的蔓延至關(guān)重要。特異性卵黃抗體(IgY)在控制傳染性的細(xì)菌或病毒疾病方面正受到大量的關(guān)注,相對(duì)于哺乳動(dòng)物的IgG,IgY具有廉價(jià)、方便、高產(chǎn)和生物安全性好等優(yōu)勢(shì),但是,目前國內(nèi)外并沒有應(yīng)用抗豬偽狂犬病IgY的報(bào)道。本課題旨在通過制備偽狂犬病毒(PRV)滅活疫苗免疫蛋雞來得到較高水平的抗PRV的卵黃抗體,并對(duì)制備的IgY進(jìn)行體內(nèi)外效果的研究,為今后抗PRV卵黃抗體用于豬偽狂犬病的治療上提供科學(xué)依據(jù)。本課題主要研究內(nèi)容如下:1抗豬偽狂犬病毒(PRV)卵黃抗體的制備及間接ELISA檢測(cè)方法的建立選取24只40周齡健康的產(chǎn)蛋雞(三黃雞),隨機(jī)分成3組(A,B,C組),每組8只。用制備的滅活疫苗免疫3組雞群,免疫方案為:A組注射生理鹽水,B組使用滅活的PRV Bartha-K61疫苗株與白油佐劑乳化后免疫雞群,C組使用滅活的PRV Bartha-K61疫苗株與弗氏佐劑乳化后免疫雞群。首免后間隔4周后進(jìn)行第二次免疫,二免后間隔2周進(jìn)行第三次免疫,共免疫3次,3次免疫接種量分別為1mL,2mL,3mL。收集免疫雞群的雞蛋,無菌分離蛋黃,采用水稀釋、鹽析和超濾法方法從蛋黃中提取和純化IgY,SDS-PAGE檢測(cè)IgY。用全病毒包被酶標(biāo)板,純化的IgY作為一抗,建立了針對(duì)抗PRV卵黃抗體的間接ELISA檢測(cè)方法,用方陣滴定法確定各反應(yīng)液的工作濃度及作用時(shí)間,并對(duì)檢測(cè)方法的特異性、敏感性及重復(fù)性進(jìn)行了評(píng)估。用該方法檢測(cè)3組免疫雞群IgY水平。結(jié)果表明:通過分離、提取和純化各步驟,每10mL卵黃液可以得到IgY的濃度為4.6mg/mL;抗原最佳包被濃度為1:100,酶標(biāo)二抗工作濃度為1:4000,臨界值為0.170-0.200,建立的間接ELISA檢測(cè)方法特異性強(qiáng),敏感性高,重復(fù)性好;B組和C組都得到了較高水平的IgY,從整體來看,C組得到的IgY水平比B組要高。2抗PRV卵黃抗體的體內(nèi)外中和試驗(yàn)IgY的毒性試驗(yàn):將IgY倍比稀釋成不同的濃度,加入到96孔細(xì)胞培養(yǎng)板中,加入長勢(shì)良好的PK-15細(xì)胞,在熒光顯微鏡下觀察細(xì)胞病變(CPE)情況,結(jié)果顯示:IgY不會(huì)引起PK-15細(xì)胞產(chǎn)生CPE,具有較好的生物安全性。IgY細(xì)胞中和試驗(yàn):將IgY倍比稀釋成不同的濃度,加入到96孔細(xì)胞培養(yǎng)板中,加入200 TCID50的PRV在37℃中和1h后,再加入長勢(shì)良好的PK-15細(xì)胞繼續(xù)培養(yǎng),觀察細(xì)胞的病變情況,并提取細(xì)胞中病毒的DNA,用熒光定量PCR測(cè)定細(xì)胞中病毒的拷貝數(shù)。結(jié)果顯示:IgY對(duì)PK-15細(xì)胞的半數(shù)保護(hù)PD50為0.04,說明IgY對(duì)PRV具有較強(qiáng)的中和活性;當(dāng)IgY的濃度為575μg/mL時(shí),陽性對(duì)照組和IgY處理組細(xì)胞中病毒的拷貝數(shù)差異顯著,并且隨著IgY濃度的增加,細(xì)胞中病毒的拷貝數(shù)逐漸降低。IgY小鼠體內(nèi)中和試驗(yàn):將36只清潔級(jí)(Balb/c)小鼠隨機(jī)分成3組,每組12只。其中,陰性對(duì)照組:每只小鼠腹股溝皮下接種0.2mL生理鹽水;陽性對(duì)照組:每只小鼠腹股溝皮下接種0.2mL PRV LA病毒株(TCID50為107);試驗(yàn)組:將濃縮后的IgY(25.8mg/mL)與PRV病毒在37℃中和1h后,腹股溝皮下接種小鼠,0.2mL/只。于小鼠出現(xiàn)臨床癥狀后采取3組小鼠血液,提取血清中DNA,用PCR檢測(cè)小鼠有無發(fā)生病毒血癥。當(dāng)不再出現(xiàn)小鼠死亡后,分析小鼠的存活情況。剖殺所有小鼠,采集小鼠腦、肝臟、脾臟和腎臟,用PCR檢測(cè)組織中的病毒。IgY小鼠體內(nèi)中和試驗(yàn)表明:抗PRVIgY能為小鼠提供80%的保護(hù)率(8/10),能有效地抑制小鼠組織中PRV的增殖。
[Abstract]:* pseudorabies has been eradicated in some developed countries in Europe and America. In China, vaccination can only be prevented by vaccination. Since the end of 2011, pseudorabies has occurred in most of the farms in North China, causing serious economic losses, and there is no effective drug to treat the disease when the outbreak of pseudorabies. Therefore, the development of effective * therapeutic drugs is very important to control the spread of pseudorabies. The specific yolk antibody (IgY) is attracting a great deal of attention in controlling infectious bacteria or viral diseases. Compared with mammalian IgG, IgY has the advantages of low cost, convenience, high yield and good biosafety, but at present, it is not available at home and abroad. * a report on the application of IgY against swine pseudorabies. The aim of this study is to obtain a high level of yolk antibody against PRV by preparing pseudorabies virus (PRV) inactivated vaccine, and to study the in vitro and in vivo effects of the prepared IgY * so as to provide a scientific basis for the treatment of pseudorabies in the future. The research contents are as follows: 1 * preparation of yolk antibody against PRV and establishment of indirect ELISA detection method. 24 healthy 40 week old laying hens (three yellow chickens) were randomly divided into 3 groups (A, B, C group), 8 in each group. 3 groups of chickens were immunized with the inactivated vaccine, and the immunization regimen was: A group was injected with normal saline, and B group was inactivated PRV Bart. The chickens in group C were immunized with inactivated PRV Bartha-K61 vaccine strain and Freund's adjuvant after emulsification. The chickens in group C were immunized with inactivated PRV Bartha-K61 vaccine strain and Freund's adjuvant after the first immunization. The chickens were immunized twice after the first immunization. The chickens were immunized twice after the second immunization. The chickens were immunized three times with the dosage of 1 mL, 2 mL and 3 mL respectively. IgY was extracted and purified from egg yolk by water dilution, salting-out and ultrafiltration. IgY was detected by SDS-PAGE. An indirect ELISA method was developed for the detection of anti-PRV yolk antibody. The concentration and time of each reaction solution were determined by matrix titration. The specificity, sensitivity and reproducibility of the assay were evaluated. The IgY levels of three groups of immunized chickens were detected by this method. The results showed that the concentration of IgY was 4.6 mg/mL per 10 mL yolk solution, the optimum coating concentration of antigen was 1:100, the working concentration of ELISA was 1:4000, and the critical value was 0.170-0.200. The established indirect ELISA assay was highly specific, sensitive and reproducible; both group B and group C had higher levels of IgY than group B. On the whole, the level of IgY in group C was higher than that in group B. The results showed that IgY did not induce CPE in PK-15 cells, and had good biological safety. IgY cell neutralization test: IgY was diluted to different concentrations, added to 96-well cell culture plate, added 200 TCID50 PRV at 37 C and 1 h after adding. The results showed that half of the protective PD50 of IgY on PK-15 cells was 0.04, indicating that IgY had a strong neutralizing activity on PRV; when the concentration of IgY was 575 ug/mL, the positive pair was positive. In vivo neutralization test of IgY mice: 36 clean grade (Balb / c) mice were randomly divided into three groups, 12 mice in each group. Among them, negative control group: each mouse was subcutaneously inoculated with 0.2 mL normal saline; Sex control group: each mouse was subcutaneously inoculated with 0.2 mL PRV LA virus strain (TCID 50 107); experimental group: IgY (25.8 mg/mL) and PRV virus were concentrated in the groin of mice, inoculated subcutaneously with 0.2 mL/mouse after one hour at 37 C. After clinical symptoms of mice, blood samples were taken from three groups of mice, DNA was extracted from serum, and virus was detected by PCR. The survival of mice was analyzed when no more mice died. All mice were killed and the brain, liver, spleen and kidney of mice were collected. Viruses in tissues were detected by PCR. Neutralization test in IgY mice showed that anti-PRVIgY could provide 80% protection (8/10) for mice and inhibit the proliferation of PRV effectively.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.4

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本文編號(hào)：2220571