广东小鹅瘟病毒血清学调查及VP3特异抗原片段的高效表达

发布时间：2018-04-14 12:42

本文选题：小鹅瘟病毒 + 血清学调查　；参考：《仲恺农业工程学院》2017年硕士论文

【摘要】：广东省是水禽养殖大省,然而小鹅瘟疫情仍不时发生并造成重大经济损失。针对小鹅瘟仍然缺乏高效的治疗药物和相关生物制品。针对小鹅瘟的防控主要是通过对产蛋前种鹅或种番鸭进行免疫,使雏鹅或雏番鸭获得母源抗体。所以对鹅和番鸭进行血清学调查可以有助于了解广东省小鹅瘟免疫水平的高低。本实验通过采集广东省内主要鹅养殖生产地的养殖场的种鹅、雏鹅、种番鸭和雏番鸭共529份血液样本,应用ELISA方法对部分样本进行检测,结果显示共有135份采集样品中小鹅瘟抗体呈阳性率,阳性率为25.52%。其中粤北地区的抗体阳性率只有27.94%;粤西地区的抗体阳性率为27.11%;粤东地区的抗体阳性率为17.74%;珠三角地区的抗体阳性率为25.75%。通过进一步分析表明,广东省内采集的370份鹅血清中共检测到70份样品阳性,鹅群中小鹅瘟抗体水平为18.92%,采集的159份番鸭血清中共检测65份样品为阳性,番鸭群中小鹅瘟抗体水平为40.88%。在311份种鹅(种番鸭)血清中共检测到95份样品阳性,种鹅(种番鸭)体内小鹅瘟抗体阳性率为41.21%;在218份雏鹅(雏番鸭)血清中共检测到40份样品阳性,雏鹅(雏番鸭)体内抗体阳性率为29.63%。针对目前市场小鹅瘟生物制品良莠不齐的现象,防控小鹅瘟病毒的相关生物制品的研发至关重要。本研究将小鹅瘟病毒颗粒表达抗原决定簇的主要成分VP3中的特异性抗原部分进行扩增,连接到NoV P-particle载体,构建并获得重组质粒,命名为P-particle-VP3。将构建的重组质粒转化至BL21感受态中并进行诱导表达,发现在加入的IPTG浓度为1m M/m L时表达效果最好,在IPTG浓度为1mM/mL时诱导5h时蛋白表达量达到最高峰。相关研究表明,NoV P-particle可作为新型的抗原呈递平台,能够提高抗原呈递效率,增强抗原的抗原性和免疫原性,有效加强生物机体的免疫应答。本研究构建的重组质粒和蛋白表达为后续高免血清的制备、ELISA检测试剂盒的开发和基因工程疫苗的研发提供物质基础和理论基础。
[Abstract]:Guangdong Province is a large province of waterfowl breeding, but goose plague still occurs from time to time and causes great economic losses.There is still a lack of effective therapeutic drugs and related biological products for Gosling plague.The prevention and control of Gosling plague is mainly through the immunization of goose or muscovy duck before laying eggs, so that the goose or young Muscovy duck can obtain maternal antibody.So the serological investigation of goose and muscovy duck can be helpful to understand the immune level of Gosling plague in Guangdong province.In this experiment, 529 blood samples were collected from breeding geese, goslings, muscovy ducks and young muscovy ducks in the main goose farms in Guangdong Province, and some samples were detected by ELISA method.The results showed that the positive rate of goose plague antibody in 135 samples was 25.52%.The positive rate of antibody was 27.94 in northern Guangdong, 27.11 in western Guangdong, 17.74in eastern Guangdong and 25.75g in Pearl River Delta.Further analysis showed that 70 samples were positive in 370 goose sera collected in Guangdong Province, 18.92 in goose flock and 65 in 159 Muscovy duck serums.The antibody level of Gosling plague in Muscovy Duck was 40.88.A total of 95 samples were positive in 311 goose (Muscovy duck) serums, the positive rate of goose plague antibody in breeding goose (Muscovy duck) was 41.21, and 40 samples were positive in 218 goose (muscovy duck) serum.The positive rate of antibody in Gosling (Muscovy Duck) was 29.63.It is very important to develop and control the related biological products of Gosling plague virus in view of the intermingled phenomena of Gosling plague biological products in the market at present.In this study, the specific antigen part of VP3, the main component of the antigen determinant of goose plague virus particles, was amplified and ligated to NoV P-particle vector. The recombinant plasmid was constructed and named P-Particle-VP3.The recombinant plasmid was transformed into the BL21 receptive state and induced to express. It was found that the expression effect was the best when the IPTG concentration was 1m m / mL, and the protein expression reached the peak when the IPTG concentration was 1mM/mL for 5 h.Related studies have shown that Nov P-particle can be used as a new antigen presentation platform, which can improve the efficiency of antigen presentation, enhance the antigenicity and immunogenicity of antigen, and enhance the immune response of organism.The expression of recombinant plasmids and proteins provided material and theoretical basis for the development of Elisa kit and the development of genetic engineering vaccine.
【学位授予单位】：仲恺农业工程学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.3

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