非洲猪瘟病毒胶体金免疫层析试纸条的研制

发布时间：2018-02-11 20:40

本文关键词： 非洲猪瘟病毒抗体免疫金免疫层析试纸条　出处：《扬州大学》2015年硕士论文　论文类型：学位论文

【摘要】：非洲猪瘟(African swine fever, ASF)是由非洲猪瘟病毒(African swine fever virus, ASFV)引起的猪的一种急性、烈性、高度接触性传染性疾病。其临床特征主要为全身出血、呼吸障碍和神经症状,发病率和死亡率高,对养猪业的危害巨大。且目前尚无有效的疫苗用于该病的预防,主要通过检疫后扑杀来控制其爆发和流行。因此,建立科学有效的检测诊断方法对于非洲猪瘟的防范具有重要意义。为获得ASFV P72,融合蛋白,本研究利用前期研究中构建的重组菌株pET-30a-P72(BL21)接种到LB液体培养基,以IPTG进行诱导后,收集菌体并以超声波进行裂解,裂解物经SDS-PAGE分析,证明了该P72融合蛋白可以在大肠杆菌BL21中大量表达。通过免疫印记技术分析可知,该融合蛋白可以与鼠抗His标签单抗特异性反应,将表达的P72重组蛋白经His标签蛋白纯化试剂盒纯化后免疫新西兰大白兔制备多克隆抗体。复苏本实验室保存的抗ASFV的杂交瘤细胞株2D3,扩大培养后接种于10周龄以上经产BALB/c小鼠,采用体内腹水诱生法制备了含有抗ASFV单抗的腹水。将收集的ASFV抗体分别经饱和硫酸铵法、辛酸一硫酸铵联合沉淀法、protein G柱亲和层析法进行纯化,对纯化后的抗体经SDS-PAGE比较分析,最终选择纯化效果较好的protein G柱亲和层析法。纯化后的抗体经紫外分光光度仪测定浓度,并用已建立的间接ELISA法测定其效价,单抗为1：160000,多抗为1：51200。为制备ASFV胶体金免疫层析试纸条,研究中采用直径为30nm的胶体金颗粒标记纯化后的单抗2D3作为金标抗体,以纯化后的多抗固定于NC膜上作为检测线,其最佳浓度为1.47 mg/mL;将葡萄球菌A蛋白(SPA)固定于NC膜上作为质控线,其最佳浓度为1.0 mg/mL,组装成用于检测ASFV的胶体金免疫层析试纸条。以pET-30a和PGEX-6P-1为载体表达的ASFV P72蛋白(简称P3P蛋白和PXP蛋白)作为阳性样品,PBS缓冲液、生理盐水、蒸馏水等作为阴性样品来检测试纸条。结果显示阳性样品有明显的两个条带,而阴性样品只显示一个条带,且试纸条能检测到的P3P蛋白和PXP蛋白的最低浓度分别为45ng/mL和60ng/mL。
[Abstract]:African swine fever (swine) is an acute, severe and highly contagious disease in pigs caused by African swine fever virus (swine). Its clinical characteristics are mainly systemic hemorrhage, respiratory disorders and neurological symptoms, high morbidity and mortality. At present, there is no effective vaccine for the prevention of the disease, mainly by culling after quarantine to control its outbreak and epidemic. In order to obtain ASFV P72, fusion protein, the recombinant strain pET-30a-P72 BL21 was inoculated into LB liquid medium and induced by IPTG. The cell was collected and cracked by ultrasonic wave. SDS-PAGE analysis showed that the P72 fusion protein could be expressed in a large number of Escherichia coli BL21, and the results of immunological imprinting analysis showed that the P72 fusion protein could be expressed in a large amount in Escherichia coli BL21. The fusion protein can react specifically with mouse anti His tag monoclonal antibody. The expressed recombinant protein P72 was purified by His tag protein purification kit and then immunized with polyclonal antibodies from New Zealand white rabbits. The hybridoma cell line 2D3, which was preserved in our laboratory, was resuscitated and inoculated into BALB/c mice over 10 weeks of age. Ascites containing anti ASFV monoclonal antibody were prepared by in vivo ascites induction method. The collected ASFV antibodies were purified by saturated ammonium sulfate method, combined with octanoic acid-ammonium sulfate precipitation method and protein G column affinity chromatography. The purified antibodies were compared and analyzed by SDS-PAGE. The purified antibody was determined by UV spectrophotometry, and the titer of the purified antibody was determined by indirect ELISA method. In order to prepare ASFV colloidal gold immunochromatographic strip, the purified monoclonal antibody 2D3, labeled with colloidal gold particles of 30 nm in diameter, was used as the gold standard antibody, and the purified polyantibody was fixed on the NC membrane as the detection line, the monoclonal antibody was 1: 160000 and the polyantibody was 1: 51200.To prepare the ASFV colloidal gold immunochromatographic strip, the purified McAb 2D3 was used as the gold standard antibody. The optimum concentration is 1.47 mg / mL. Staphylococcal protein A SPAs are immobilized on the NC membrane as the quality control line. The best concentration was 1.0 mg / mL, and the colloidal gold immunochromatographic strip was assembled to detect ASFV. The ASFV P72 protein (P3P protein and PXP protein) expressed on pET-30a and PGEX-6P-1 was used as positive sample buffer solution and normal saline. The results showed that there were two obvious bands in the positive sample, but only one band in the negative sample. The lowest concentrations of P3P protein and PXP protein were 45 ng / mL and 60 ng / mL, respectively.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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