【摘要】：近年來,鴨病毒性肝炎(DVH)仍然在國內(nèi)包括廣西等多個省份流行,并且該病的主要病原鴨甲型肝炎病毒(DHAV)存在不同血清型。因此,有必要對廣西近年流行的DHAV進行病毒分離鑒定,并針對主要流行的1型鴨甲型肝炎病毒(DHAV-1)建立一種監(jiān)測免疫種鴨的抗體水平的間接ELISA方法,為今后病原研究及免疫抗體監(jiān)測提供材料和技術。這對該病的流行病學研究與防控具有重要的現(xiàn)實意義。本研究從疑似DVH的臨床病例采集12份病料,病料經(jīng)處理后利用常規(guī)技術進行病毒的分離與鑒定,通過制備1型、3型兔單因子血清并用血清中和試驗對分離毒株進行血清型鑒定。結果表明,從臨床樣品分離到7株病毒,分別命名為GXNN01、XNN02、GXGL01、GXLZ01、GXLZ02、GXNN03和GXGL02,病毒分離率為58.33%。血清中和試驗確定GXNN01、GXNN02、 GXGL01、GXLZ01、GXLZ02為DHAV-1毒株,GXNN03、GXGL02為韓國新型DHAV(DHAV-3)毒株。本研究對分離病毒的主要結構蛋白VPl基因進行克隆、序列測定及分析后表明,5個DHAV-1分離株之間的VP1基因的核苷酸同源性為94.0%-99.0%,氨基酸同源性為93.3%-98.7%,與DHAV-1代表毒株(包括疫苗毒A66株)核苷酸同源性為91.0%-98.0%,氨基酸的同源性為91.2%-99.2%；2個DHAV-3之間的VP1核苷酸同源性為95.4%,氨基酸同源性為95.9%；與韓國新型即DHAV-3毒株(包括GD1株)的核苷酸同源性為90.4%-98.3%,氨基酸的同源性為91.3%-98.3%。本次分離毒株與臺灣新型即DHAV-2的氨基酸同源性均低于20%。本研究針對目前廣西主要流行DHAV-1,運用差速、超速離心法制備DHAV-1抗原,作為包被抗原,利用鹽析法抽提純化卵黃抗體,建立檢測DHAV-1卵黃抗體的間接ELISA方法。通過ELISA反應條件的優(yōu)化,得出最佳抗原包被濃度是6.78μg/mL,最佳酶標抗體(兔抗鴨IgG/HRP)工作濃度確定為0.31μg/mL,最佳樣品抗體濃度確定為10.37μg/mL、敏感測定值為0.08μg/mL, OD450|臨界值為0.233,建立了檢測血清1型DHAV卵黃抗體的間接ELISA檢測方法。該方法的特異性及重復性良好。用建立的方法對40份臨床卵黃樣品的檢測,陽性檢出率為80%。證明檢測方法適用于血清1型DHAV卵黃抗體的檢測,且操作簡單、結果直觀,適用于基層生產(chǎn)單位。本次研究共分離到7株DHAV毒株,包括5株DHAV-1毒株,2株DHAV-3毒株,并對它們的主要結構蛋白基因VPl進行遺傳進化分析,結果顯示均與DHAV-2毒株同源性低。并成功建立了DHAV-1卵黃抗體ELISA檢測方法檢測方法。研究成果將為今后廣西DHAV流行病學研究以及種鴨DHAV-1免疫水平監(jiān)測和疾病防控提供有用材料及技術支持。
[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.
【學位授予單位】：廣西大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.65

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