广西鸭甲型肝炎病毒的分离鉴定及DHAV-1卵黄抗体ELISA检测方法的建立
发布时间：2018-10-31 21:10
【摘要】：近年来,鸭病毒性肝炎(DVH)仍然在国内包括广西等多个省份流行,并且该病的主要病原鸭甲型肝炎病毒(DHAV)存在不同血清型。因此,有必要对广西近年流行的DHAV进行病毒分离鉴定,并针对主要流行的1型鸭甲型肝炎病毒(DHAV-1)建立一种监测免疫种鸭的抗体水平的间接ELISA方法,为今后病原研究及免疫抗体监测提供材料和技术。这对该病的流行病学研究与防控具有重要的现实意义。本研究从疑似DVH的临床病例采集12份病料,病料经处理后利用常规技术进行病毒的分离与鉴定,通过制备1型、3型兔单因子血清并用血清中和试验对分离毒株进行血清型鉴定。结果表明,从临床样品分离到7株病毒,分别命名为GXNN01、XNN02、GXGL01、GXLZ01、GXLZ02、GXNN03和GXGL02,病毒分离率为58.33%。血清中和试验确定GXNN01、GXNN02、 GXGL01、GXLZ01、GXLZ02为DHAV-1毒株,GXNN03、GXGL02为韩国新型DHAV(DHAV-3)毒株。本研究对分离病毒的主要结构蛋白VPl基因进行克隆、序列测定及分析后表明,5个DHAV-1分离株之间的VP1基因的核苷酸同源性为94.0%-99.0%,氨基酸同源性为93.3%-98.7%,与DHAV-1代表毒株(包括疫苗毒A66株)核苷酸同源性为91.0%-98.0%,氨基酸的同源性为91.2%-99.2%；2个DHAV-3之间的VP1核苷酸同源性为95.4%,氨基酸同源性为95.9%；与韩国新型即DHAV-3毒株(包括GD1株)的核苷酸同源性为90.4%-98.3%,氨基酸的同源性为91.3%-98.3%。本次分离毒株与台湾新型即DHAV-2的氨基酸同源性均低于20%。本研究针对目前广西主要流行DHAV-1,运用差速、超速离心法制备DHAV-1抗原,作为包被抗原,利用盐析法抽提纯化卵黄抗体,建立检测DHAV-1卵黄抗体的间接ELISA方法。通过ELISA反应条件的优化,得出最佳抗原包被浓度是6.78μg/mL,最佳酶标抗体(兔抗鸭IgG/HRP)工作浓度确定为0.31μg/mL,最佳样品抗体浓度确定为10.37μg/mL、敏感测定值为0.08μg/mL, OD450|临界值为0.233,建立了检测血清1型DHAV卵黄抗体的间接ELISA检测方法。该方法的特异性及重复性良好。用建立的方法对40份临床卵黄样品的检测,阳性检出率为80%。证明检测方法适用于血清1型DHAV卵黄抗体的检测,且操作简单、结果直观,适用于基层生产单位。本次研究共分离到7株DHAV毒株,包括5株DHAV-1毒株,2株DHAV-3毒株,并对它们的主要结构蛋白基因VPl进行遗传进化分析,结果显示均与DHAV-2毒株同源性低。并成功建立了DHAV-1卵黄抗体ELISA检测方法检测方法。研究成果将为今后广西DHAV流行病学研究以及种鸭DHAV-1免疫水平监测和疾病防控提供有用材料及技术支持。
[Abstract]:In recent years, duck viral hepatitis (DVH) is still prevalent in many provinces, including Guangxi, and the main pathogen of the disease, duck hepatitis A virus (DHAV), has different serotypes. Therefore, it is necessary to isolate and identify the DHAV prevalent in Guangxi in recent years, and to establish an indirect ELISA method to monitor the antibody level of duck hepatitis A virus type 1 (DHAV-1). To provide materials and techniques for pathogen research and immune antibody monitoring in the future. This is of great practical significance to the epidemiological study and prevention and control of the disease. In this study, 12 samples were collected from clinical cases suspected of DVH. After treatment, the virus was isolated and identified by routine technique, and type 1 was prepared. The serotype of the isolated strain was identified by serum neutralization test. The results showed that 7 strains of virus were isolated from clinical samples, named GXNN01,XNN02,GXGL01,GXLZ01,GXLZ02,GXNN03 and GXGL02, respectively. The isolation rate was 58.33. Serum neutralization test identified GXNN01,GXNN02, GXGL01,GXLZ01,GXLZ02 as DHAV-1 strain and GXNN03,GXGL02 as Korean new DHAV (DHAV-3) strain. In this study, the VPl gene of the main structural protein of the virus was cloned. The sequencing and analysis showed that the nucleotide homology of the VP1 gene among the five DHAV-1 isolates was 94.0-99.0. The amino acid homology was 93.3- 98.7, the nucleotide homology was 91.0-98.0 with DHAV-1 representative virus strain (including vaccine A66 strain), the homology of amino acid was 91.2-99.2; The nucleotide homology of VP1 and amino acid between two DHAV-3 were 95.4 and 95.9respectively. The nucleotide homology was 90.4-98.3 and the amino acid homology was 91.3- 98.3with DHAV-3 strain (including GD1 strain) in Korea. The amino acid homology of the isolated strain was lower than 20% with the new DHAV-2 in Taiwan. In this study, DHAV-1 antigens were prepared by differential and ultracentrifugation for the main prevalent DHAV-1, in Guangxi at present. As coating antigens, the yolk antibodies were extracted and purified by salting out method, and an indirect ELISA method for detection of DHAV-1 yolk antibodies was established. Through the optimization of ELISA reaction conditions, the best antigen coating concentration is 6.78 渭 g / mL, the best working concentration of rabbit anti-duck IgG/HRP is 0.31 渭 g / mL, and the best sample antibody concentration is 10.37 渭 g / mL. The sensitive value was 0.08 渭 g / mL and the critical value of OD450 was 0.233. An indirect ELISA method was established for the detection of serum type 1 DHAV yolk antibody. The method has good specificity and reproducibility. 40 clinical yolk samples were detected by the established method, and the positive rate was 80%. It was proved that the method was suitable for the detection of yolk antibody of serotype 1 DHAV, and the operation was simple, the result was intuitionistic, and it was suitable for basic production units. In this study, 7 DHAV strains were isolated, including 5 DHAV-1 strains and 2 DHAV-3 strains. The genetic evolution analysis of their main structural protein gene VPl showed that they all had low homology with DHAV-2 strain. A method for the detection of DHAV-1 yolk antibody by ELISA was established successfully. The results will provide useful materials and technical support for the epidemiological study of DHAV in Guangxi and the surveillance of DHAV-1 immune level and disease prevention and control of duck breeds in the future.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
本文编号：2303563
[Abstract]:In recent years, duck viral hepatitis (DVH) is still prevalent in many provinces, including Guangxi, and the main pathogen of the disease, duck hepatitis A virus (DHAV), has different serotypes. Therefore, it is necessary to isolate and identify the DHAV prevalent in Guangxi in recent years, and to establish an indirect ELISA method to monitor the antibody level of duck hepatitis A virus type 1 (DHAV-1). To provide materials and techniques for pathogen research and immune antibody monitoring in the future. This is of great practical significance to the epidemiological study and prevention and control of the disease. In this study, 12 samples were collected from clinical cases suspected of DVH. After treatment, the virus was isolated and identified by routine technique, and type 1 was prepared. The serotype of the isolated strain was identified by serum neutralization test. The results showed that 7 strains of virus were isolated from clinical samples, named GXNN01,XNN02,GXGL01,GXLZ01,GXLZ02,GXNN03 and GXGL02, respectively. The isolation rate was 58.33. Serum neutralization test identified GXNN01,GXNN02, GXGL01,GXLZ01,GXLZ02 as DHAV-1 strain and GXNN03,GXGL02 as Korean new DHAV (DHAV-3) strain. In this study, the VPl gene of the main structural protein of the virus was cloned. The sequencing and analysis showed that the nucleotide homology of the VP1 gene among the five DHAV-1 isolates was 94.0-99.0. The amino acid homology was 93.3- 98.7, the nucleotide homology was 91.0-98.0 with DHAV-1 representative virus strain (including vaccine A66 strain), the homology of amino acid was 91.2-99.2; The nucleotide homology of VP1 and amino acid between two DHAV-3 were 95.4 and 95.9respectively. The nucleotide homology was 90.4-98.3 and the amino acid homology was 91.3- 98.3with DHAV-3 strain (including GD1 strain) in Korea. The amino acid homology of the isolated strain was lower than 20% with the new DHAV-2 in Taiwan. In this study, DHAV-1 antigens were prepared by differential and ultracentrifugation for the main prevalent DHAV-1, in Guangxi at present. As coating antigens, the yolk antibodies were extracted and purified by salting out method, and an indirect ELISA method for detection of DHAV-1 yolk antibodies was established. Through the optimization of ELISA reaction conditions, the best antigen coating concentration is 6.78 渭 g / mL, the best working concentration of rabbit anti-duck IgG/HRP is 0.31 渭 g / mL, and the best sample antibody concentration is 10.37 渭 g / mL. The sensitive value was 0.08 渭 g / mL and the critical value of OD450 was 0.233. An indirect ELISA method was established for the detection of serum type 1 DHAV yolk antibody. The method has good specificity and reproducibility. 40 clinical yolk samples were detected by the established method, and the positive rate was 80%. It was proved that the method was suitable for the detection of yolk antibody of serotype 1 DHAV, and the operation was simple, the result was intuitionistic, and it was suitable for basic production units. In this study, 7 DHAV strains were isolated, including 5 DHAV-1 strains and 2 DHAV-3 strains. The genetic evolution analysis of their main structural protein gene VPl showed that they all had low homology with DHAV-2 strain. A method for the detection of DHAV-1 yolk antibody by ELISA was established successfully. The results will provide useful materials and technical support for the epidemiological study of DHAV in Guangxi and the surveillance of DHAV-1 immune level and disease prevention and control of duck breeds in the future.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
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