禽白血病病毒p27蛋白多克隆抗体的制备及POCT检测方法的建立  

Preparation of Avian leukosis virus p27 Protein Polyclonal Antibody and Estab lishment of POCT Detection Method

作　　者：邵颖[1] 黄燕 杨艳 龚柳菲 宋祥军 涂健[1] 祁克宗[1] SHAO Ying;HUANG Yan;YANG Yan;GONG Liufei;SONG Xiangjun;TU Jian;QI Kezong(College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,China)

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036

出　　处：《华北农学报》2022年第6期201-209,共9页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金项目(31972642);安徽省高校协同创新项目(GXXT-2019-035);安徽省大学生创新创业训练计划项目(202110364043);安徽省大学生创新创业训练计划项目(202110364027)。

摘　　要：旨在制备禽白血病病毒p27蛋白多克隆抗体和建立一种简便准确的禽白血病病毒的检测方法,通过扩增禽白血病病毒p27目的基因,构建pGEX-6p-1-p27、pET-32a-p27蛋白原核表达载体。将蛋白分别免疫BALB/c小鼠和新西兰白兔,制备鼠源多克隆抗体和兔源多克隆抗体,制备鼠源多克隆抗体偶联荧光微球,利用三维喷点平台将鼠源多克隆抗体IgG喷涂到样品垫;用划膜仪将纯化后的兔源多克隆抗体IgG和山羊抗兔抗体稀释液划到NC膜上,作为T线和C线,进行试纸条的组装,初步建立了即时检验荧光微球免疫层析检测ALV试纸法,并用POCT试纸条检测临床阳性样本。结果表明:成功构建pGEX-6p-1-p27、pET-32a-p27蛋白原核载体并诱导p27重组蛋白表达。将纯化后的p27融合蛋白与佐剂等容量混合后作为免疫原免疫BALB/c小鼠和新西兰白兔,成功制备出p27蛋白的鼠源和兔源多克隆抗体,通过血清效价以及Western Blot鉴定显示多抗的免疫原反应良好。荧光微球偶联鼠源多克隆抗体的最适pH值为6.2。POCT试纸条检测结果显示,ALV的阳性样品与荧光微球包被的鼠源多克隆抗体结合良好,T/C数据比其他样本高,且仅有阳性样本和荧光微球包被的鼠源多克隆抗体结合后,在紫外灯的照射下,C线(质控线)和T线(检测线)才均会有荧光条带,与临床诊断结果相符。试验建立的POCT荧光微球免疫层析检测ALV的方法,可为禽白血病病毒的检测提供一种更为简便的方法,为基层兽医临床检测提供便利。It aimed to develop a polyclonal antibody to Avian leukosis virus(ALV)and to establish a simple and accurate method for the detection of ALV.pGEX-6p-1-p27 and pET-32a-p27 protein vectors were constructed by amplifying the target gene of ALV p27.The proteins were immunized in BALB/c mice and New Zealand white rabbits,and mouse-derived polyclonal antibodies and rabbit-derived polyclonal antibodies were prepared.The mouse-derived polyclonal antibody coupled fluorescent microspheres were prepared,and the mouse-derived polyclonal antibody IgG was sprayed onto the sample pads using a three-dimensional spray point platform;the purified rabbit-derived polyclonal antibody IgG and goat anti-rabbit antibody dilution were scribed onto the NC membrane using a scribing instrument as T and C lines for the assembly of test strips,and a preliminary Point-of-care testing(POCT)immunochromatographic assay was established.The test strips were assembled and a preliminary point-of-care testing(POCT)method was developed for the detection of ALV by fluorescent microsphere immunochromatography.The results showed that the prokaryotic expression vectors for pGEX-6p-1-p27 and pET-32a-p27 were successfully constructed and the expression of p27 recombinant protein was induced.After mixing the purified p27 fusion protein with the adjuvant as an immunogen immune BALB/c mouse and a New Zealand white rabbit,a mouse-derived and rabbit-derived polyclonal antibody of the p27 protein was successfully prepared,and the immunogen response of the multi-antibody was shown to be good by serum potency and Western Blot identification.The optimal pH of the fluorescent microsphere-coupled murine polyclonal antibody was 6.2.POCT strips showed that positive samples for ALV bound well to the fluorescent microsphere-coated murine polyclonal antibody,with higher T/C data than other samples,and that positive samples bound to the fluorescent microsphere-coated polyclonal antibody showed fluorescent bands in the C line(quality control line)and T line(detection line)und

关 键 词：ALV P27 原核表达 多克隆抗体 荧光微球免疫层析 

分 类 号：S851.3[农业科学—预防兽医学]