抗猪伪狂犬病毒卵黄抗体的制备及其保护效力的研究

发布时间：2018-09-03 16:43

【摘要】：猪伪狂犬病在欧美一些发达国家已经根除,在中国,目前只能通过疫苗接种进行预防。自2011年末以来,华北大部分地区的已接种疫苗的猪场爆发了伪狂犬病,造成了严重的经济损失,而且,爆发伪狂犬病时没有有效的药物治疗该种疾病,因此,研制有效的治疗药物对于控制猪伪狂犬病的蔓延至关重要。特异性卵黄抗体(IgY)在控制传染性的细菌或病毒疾病方面正受到大量的关注,相对于哺乳动物的IgG,IgY具有廉价、方便、高产和生物安全性好等优势,但是,目前国内外并没有应用抗猪伪狂犬病IgY的报道。本课题旨在通过制备伪狂犬病毒(PRV)灭活疫苗免疫蛋鸡来得到较高水平的抗PRV的卵黄抗体,并对制备的IgY进行体内外效果的研究,为今后抗PRV卵黄抗体用于猪伪狂犬病的治疗上提供科学依据。本课题主要研究内容如下:1抗猪伪狂犬病毒(PRV)卵黄抗体的制备及间接ELISA检测方法的建立选取24只40周龄健康的产蛋鸡(三黄鸡),随机分成3组(A,B,C组),每组8只。用制备的灭活疫苗免疫3组鸡群,免疫方案为:A组注射生理盐水,B组使用灭活的PRV Bartha-K61疫苗株与白油佐剂乳化后免疫鸡群,C组使用灭活的PRV Bartha-K61疫苗株与弗氏佐剂乳化后免疫鸡群。首免后间隔4周后进行第二次免疫,二免后间隔2周进行第三次免疫,共免疫3次,3次免疫接种量分别为1mL,2mL,3mL。收集免疫鸡群的鸡蛋,无菌分离蛋黄,采用水稀释、盐析和超滤法方法从蛋黄中提取和纯化IgY,SDS-PAGE检测IgY。用全病毒包被酶标板,纯化的IgY作为一抗,建立了针对抗PRV卵黄抗体的间接ELISA检测方法,用方阵滴定法确定各反应液的工作浓度及作用时间,并对检测方法的特异性、敏感性及重复性进行了评估。用该方法检测3组免疫鸡群IgY水平。结果表明:通过分离、提取和纯化各步骤,每10mL卵黄液可以得到IgY的浓度为4.6mg/mL;抗原最佳包被浓度为1:100,酶标二抗工作浓度为1:4000,临界值为0.170-0.200,建立的间接ELISA检测方法特异性强,敏感性高,重复性好;B组和C组都得到了较高水平的IgY,从整体来看,C组得到的IgY水平比B组要高。2抗PRV卵黄抗体的体内外中和试验IgY的毒性试验:将IgY倍比稀释成不同的浓度,加入到96孔细胞培养板中,加入长势良好的PK-15细胞,在荧光显微镜下观察细胞病变(CPE)情况,结果显示:IgY不会引起PK-15细胞产生CPE,具有较好的生物安全性。IgY细胞中和试验:将IgY倍比稀释成不同的浓度,加入到96孔细胞培养板中,加入200 TCID50的PRV在37℃中和1h后,再加入长势良好的PK-15细胞继续培养,观察细胞的病变情况,并提取细胞中病毒的DNA,用荧光定量PCR测定细胞中病毒的拷贝数。结果显示:IgY对PK-15细胞的半数保护PD50为0.04,说明IgY对PRV具有较强的中和活性;当IgY的浓度为575μg/mL时,阳性对照组和IgY处理组细胞中病毒的拷贝数差异显著,并且随着IgY浓度的增加,细胞中病毒的拷贝数逐渐降低。IgY小鼠体内中和试验:将36只清洁级(Balb/c)小鼠随机分成3组,每组12只。其中,阴性对照组:每只小鼠腹股沟皮下接种0.2mL生理盐水;阳性对照组:每只小鼠腹股沟皮下接种0.2mL PRV LA病毒株(TCID50为107);试验组:将浓缩后的IgY(25.8mg/mL)与PRV病毒在37℃中和1h后,腹股沟皮下接种小鼠,0.2mL/只。于小鼠出现临床症状后采取3组小鼠血液,提取血清中DNA,用PCR检测小鼠有无发生病毒血症。当不再出现小鼠死亡后,分析小鼠的存活情况。剖杀所有小鼠,采集小鼠脑、肝脏、脾脏和肾脏,用PCR检测组织中的病毒。IgY小鼠体内中和试验表明:抗PRVIgY能为小鼠提供80%的保护率(8/10),能有效地抑制小鼠组织中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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.4

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