【作者】 曹竹婷；

【导师】 简子健； 杨少华；

【作者基本信息】 新疆农业大学 ， 基础兽医， 2008， 硕士

【摘要】 本文在基础实验室条件下,通过普通高速离心机代替低温高速离心机、焦碳酸二乙酯(DEPC)处理水制胶、制胶板自制以及普通的电泳槽等改良措施,提取了含量较高的轮状病毒RNA,此法也适用于其他生物性病原的RNA抽提。虽然本法中有少量的RNA降解,但并不影响实验结果,这对于条件相对较薄弱的基层防疫、监督单位来说,仍具有较好的可行性和推广意义。从山东暴发犊牛腹泻的地区采集腹泻犊牛的粪便, RT-PCR检测出了特异性目的条带,SDS-PAGE检测出轮状病毒特异性的11条带。证实是A组的轮状病毒。以牛轮状病毒标准株NCDV作为抗原,以其制备的高免血清作为一抗,辣根过氧化物酶标记的羊抗牛IgG作为二抗,运用正交试验法结合方阵法建立了检测犊牛轮状病毒抗体的间接ELISA方法。本实验运用正交实验法结合方阵法确定间接ELISA最佳反应条件:包被抗原51.5 ng/孔,血清150倍稀释,酶标抗体2500稀释,对牛轮状病毒的检测灵敏度达36.78 ng。本次试验成功建立了检测牛轮状病毒抗体间接ELISA方法。旨在研制出经济有效的轮状病毒疫苗,减少养牛业的经济损失。首先对种子批毒进行TCID50、病毒对小鼠毒力、特异性试验、免疫原性试验、纯粹性检查。制备的疫苗进行无菌检验和安全检验结果符合标准后,分别免疫犊牛和怀孕牛。本试验制备的种子毒符合标准,研制出犊牛轮状病毒灭活疫苗,分别免疫犊牛和怀孕牛,对照组和免疫组的犊牛IgG抗体水平差异显著;而对照组和免疫组的怀孕牛乳清IgG抗体效价差异不显著。分析免疫怀孕牛失败的原因可能是疫苗免疫量少、免疫次数少、饲喂环境影响等原因。本实验证明了疫苗免疫犊牛的可行性,为犊牛轮状病毒性腹泻的防治奠定基础。更多还原

【Abstract】 Based on the basis of laboratory conditions, by an ordinary high-speed centrifuges instead of low-temperature high-speed centrifuges, Coke Diethyl (DEPC) water treatment system for plastic, rubber-made and the general improvement measures, such as electrophoresis tank, the extraction of higher rotavirus RNA, the law also applies to other sexually transmitted diseases of the original RNA extraction. Although this law in a small amount of RNA degradation, but does not affect the results, this relatively weak conditions for the grass-roots epidemic prevention and supervision units, still has good feasibility and significance of the promotion.This study was collection from calf diarrhea outbreak in Shandong. RT-PCR detected the specific purpose of the zone, SDS-PAGE detected rotavirus in 11 specific zones. A group confirmed that the rotavirus.Cattle NCDV as a standard strain of rotavirus antigen, its high for the preparation of an anti-serum as, horseradish peroxidase labeled goat anti-IgG as the anti-cattle, the use of orthogonal test with a phalanx of detecting calf Bovine rotavirus antibodies indirect ELISA method. The experimental use of orthogonal experiment with square indirect ELISA method to determine the best conditions: coating antigen 51.5 ng / hole, 150 times the serum dilution, dilution ELISA antibody 2500, the detection of bovine rotavirus sensitivity of 36.78 ng.This experiment were aimed to develop the most-effective rotavirus vaccine for reduce economic losses of cattle. The first was approved virus TCID50, virulence of the virus in mice, specific tests, immunogenicity trial, and purity check. Preparation of the vaccine sterile test and safety test results were meet the standards, were immune calves and pregnant cows, respectively. The trial of the virus to accord with the standard, bovine rotavirus vaccine were immune calves and pregnant cows, respectively .The control group and the immune group IgG antibody levels were significantly of calves; the control group and immune group of Pregnant cow’s milk-IgG antibody titers were not significantly different. Analyze the failure of immune pregnant cow were vaccine may be less, the less time of immune, the feeding environmental impact and other reasons. The experimental has proved that feasibility of bovine rotavirus vaccine immune calves and lied the foundation of prevention of rotavirus diarrhea.vaccine immune calves and laid the foundation of prevention of rotavirus diarrhea.更多还原