| 摘要: |
| 为制备非洲马瘟病毒VP7蛋白ELISA阳性血清标准样品，筛选一匹无常见马属动物疫病抗体的马作为靶动物，利用大肠杆菌原核表达系统制备的VP7蛋白为免疫原，将免疫原与MONTANIDE ISA35佐剂等体积混合乳化后，每两周免疫1次，共进行3次免疫，每周采集血清，检测其血清抗体ELISA效价。在血清效价高时大量采血分离血清，验证其无菌性，并将高效价血清进行冻干，参照《中华人民共和国兽药典》中的方法对该血清进行特异性检验、无菌检验、均一性评估和稳定性评估。结果表明：1）在免疫后第3周，可以检测到免疫马体内的非洲马瘟抗体。2）特异性结果显示马流感病毒、马疱疹病毒Ⅰ型、马疱疹病毒Ⅱ型、马疱疹病毒Ⅳ型、马动脉炎病毒、马传染性贫血病毒、马链球菌、马流产沙门菌、鼻疽伯克霍尔德氏菌、马驽巴贝斯虫和马泰勒虫抗体检测结果均为阴性。说明制备的标准血清背景干净，无常见马属动物疫病抗体。3）无菌检验结果显示酪胨琼脂培养基、胰酪大豆胨液体培养基和硫乙醇酸盐液体培养基上均无杂菌生长，说明制备的血清中无细菌和真菌等微生物污染。4）稳定性评估结果显示制备的血清在4、25和37 ℃环境下可稳定保存2个月，在-20和-80 ℃环境下可稳定保存6个月。综上，本研究成功制备了非洲马瘟病毒VP7蛋白ELISA阳性血清标准样品，该标准样品的制备为非洲马瘟的相关研究奠定理论基础。 |
| 关键词: 非洲马瘟 阳性血清 ELISA 标准样品 |
| DOI：10.11841/j.issn.1007-4333.2024.05.07 |
| 投稿时间：2023-08-24 |
| 基金项目:十四五国家重点研发计划（2021YFD1800500） |
|
| Preliminary preparation of African horse sickness virus VP7 protein ELISA positive serum standard |
|
YANG Yan1, HU Zhe1, GUO Kui1, ZHANG Zenan1, XUE Hong2, HUANG Xueying2, SHEN Dan2, WANG Xiaojun1
|
| (1.State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China;2.Guangzhou Animal Health Supervision Institute, Guangzhou 510440, China) |
| Abstract: |
| The study aimed to prepare a positive serum standard of VP7 protein for the ELISA of the African horse sickness virus， a horse without antibodies against common equine diseases was selected as the target animal， and VP7 protein expressed by the prokaryotic expression system of Escherichia coli was used as the immunogen. The immunogen was mixed with MONTANIDE ISA35 adjuvant in equal volume and emulsified， and then the horse was immunized once every 2 weeks for a total of 3 immunizations， and serum was collected to detect the serum antibody titer by ELISA. When the serum potency was highest， a large amount of blood was collected to isolate the serum and verify its sterility， and the high valence serum was lyophilized.The specificity， sterility， uniformity and stability of the serum were tested according to the methods in the Veterinary Pharmacopoeia of the People's Republic of China. The results showed that： 1） Antibodies against African horse sickness could be detected in the immunized horse in the third week after immunization. 2） The specificity results showed that antibodies against equine influenza virus， equine herpesvirus type I， equine herpesvirus type II， equine herpesvirus type IV， equine arteritis virus， equine infectious anemia virus， equine streptococcus， equine Salmonella abortus， Burkholderia pseudomallei， Babesia caballi and Theileria equi were all negative. The prepared standard serum had a clean background and no common equine pathogenic antibodies. 3）There was no growth of stray organisms on the case peptone agar medium， tryptic soy peptone liquid medium and thioglycolate liquid medium， indicating that the prepared serum was free from microbial contamination such as bacteria and fungi. 4）The stability test results showed that the prepared serum can be stably stored for 2 months at 4， 25 and 37 ℃ and for 6 months at -20 and -80 ℃ .In conclusion， this study successfully prepared positive serum standard of VP7 protein for the ELISA of the African horse sickness virus， which laid a theoretical foundation for related research on African horse sickness. |
| Key words: African horse sickness positive serum ELISA standard samples |
