【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。更多还原

【Abstract】 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)under UV light, which the results were consistent with clinical diagnostic results.It can be concluded that the POCT fluorescent microsphere immunochromatographic detection method for ALV established provides a simpler method for the detection of Avian leukosis virus and facilitates clinical detection for primary veterinarians.更多还原