检测抗伪狂犬病毒抗体胶体金免疫层析方法的建立及初步评价

发布时间：2018-04-26 00:28

本文选题：伪狂犬 + 抗体检测　；参考：《中国农业科学院》2015年硕士论文

【摘要】：猪伪狂犬病是由伪狂犬病毒引起的一种以仔猪致命性脑炎、育肥猪呼吸道症状以及母猪的流产为主要临床表现的猪的传染病,该病给许多国家的养猪业造成巨大的经济损失。虽然,基因缺失疫苗免疫与g E ELISA抗体检测试剂盒的联合应用帮助许多国家完成了对该病的净化,但是,由于野猪是该病病原的宿主库,仍然会造成临近猪群的感染,同时,感染后的猪群作为隐性带毒者,也会成为长期的潜伏传染源。因此,对于该病的预防仍然十分重要。目前,国际上广泛使用g E基因缺失疫苗免疫猪群取得了良好的预防效果,且疫苗免疫与野毒感染后,动物血清中是否会检测到抗伪狂犬病毒g E蛋白抗体已经被作为鉴别诊断常用的方法。PRV-g E ELISA商品化的试剂盒就常常被用来进行DIVA诊断。尽管如此,ELISA方法由于需要特定的仪器、有一定技术培训的人员在实验室中才可以完成,无法由无技术培训的饲养人员或兽医直接简便的在现地做出快速的诊断。因此,本研究通过胶体金免疫层析试验建立了抗伪狂犬病毒g E抗体的DIVA快速、简便的检测方法,并进行了初步评价。本实验通过扩增包含g E蛋白重要表位的部分基因,经测序,克隆于原核表达质粒p ET-32a,于大肠杆菌表达菌BL21中诱导、表达并纯化,获得表达蛋白,Western-blot结果表明,表达蛋白可以与抗PRV阳性血清特异结合,将纯化后的g E表达蛋白与商品化的猪的Ig G分别作为检测线(T线)和质控线(C线)喷点于硝酸纤维素膜上,以胶体金颗粒标记金黄色葡萄球菌蛋白A(SPA)作为示踪剂,建立了检测猪血清中抗PRV-g E蛋白抗体的的胶体金免疫层析方法。判定标准为:1)待检的血清中含有抗PRV-g E抗体,可与金标SPA结合形成Ig G-SPA复合物,经层析膜流经T线和C线时,Ig G-SPA复合物就会和T线上的PRV-g E蛋白以及C线上的猪的Ig G发生特异性结合,形成两条红色的线,判定为阳性结果;2)若待检血清中不含有抗PRV-g E抗体,则只有质控线出现;3)若出现质控线未形成红色条带时,则该方法检测结果无效。经过探索该胶体金得最佳反应条件,我们最终确定C线包被猪的Ig G、浓度为2mg/ml,T线浓度为2mg/ml,金标SPA最佳标记p H为8.0、浓度为0.1mg/ml,血清最佳稀释倍数为1:50倍稀释。继而,对本实验建立的检测方法分别进行了特异性、敏感性、重复性和稳定性以及符合率的检验。结果表明,该方法特异性良好,只在检测伪狂犬病毒血清时出现阳性反应,对其他病毒血清检测为阴性例如猪繁殖与呼吸综合征病毒、猪圆环病毒、猪肺支原体、猪流行性腹泻与传染性胃肠炎病毒等;对同一批样品进行批内重复试验与批间重复试验,该方法检测均为同一结果,并且在4℃可稳定储存长达4个月之久不改变其特性;与爱德士伪狂犬g E抗体检测试剂盒对比,该方法敏感性略高,该方法特异性和敏感性分别为81.6%和90.7%,并且两者之间有良好的符合性(Kappa=0.7289)。我们又进行了猪的攻毒实验,分别用建立的胶体金免疫层析方法和爱德士ELISA方法对不同的4组(每组7只)猪即非免疫攻毒组、非免疫非攻毒组、免疫攻毒组、免疫非攻毒组猪进行g E抗体检测,监测时间为45天,发现猪只开始出现g E抗体阳性的时间均为第9天、11天、9天、13天,也表明这两种方法有良好的一致性。综上所述,本实验建立了检测抗伪狂犬病毒抗体胶体金免疫层析方法,为检测伪狂犬野毒提供了一种快速、简便的方法。
[Abstract]:Porcine pseudorabies is a pig's infectious disease caused by the pseudorabies virus, a piglet fatal encephalitis, the respiratory symptoms of the fattening pig and the abortion of the sow as the main clinical manifestation. The disease causes huge economic losses to the pig industry in many countries. Although the gene deletion vaccine is combined with the G E ELISA antibody test kit It has been used to help many countries to complete the purification of the disease, but because the wild boar is the host Library of the pathogen, it will still cause the infection of the adjacent pigs, and the infected pigs, as recessive and poisonous, will also become a long-term latent source of infection. Therefore, the prevention of the disease is still very important. At present, the G E base is widely used in the world. As a result of the good preventive effect of vaccinated pigs, and the detection of anti pseudorabies virus G E antibodies in animal sera after immunization and wild virus infection, the.PRV-g E ELISA commercialized kit is often used for the diagnosis of DIVA. However, the ELISA method has been used. People who need specific instruments and some technical training can be completed in the laboratory, unable to make rapid and simple diagnosis directly and conveniently in the present field. Therefore, this study established a fast and simple DIVA detection prescription for the anti pseudocanine virus G E antibody by colloidal gold immunochromatography test. By amplification of some genes containing important epitopes of G E protein, this experiment was sequenced and cloned into the prokaryotic expression plasmid P ET-32a, induced in the Escherichia coli expressing bacteria BL21, expressed and purified, and obtained the expression protein. The result of Western-blot showed that the expression protein could be specifically combined with anti PRV positive serum and purified. The G E expression protein and the commercial pig's Ig G were sprayed on the nitrocellulose membrane as the detection line (T line) and the quality control line (C line), and the colloidal gold granules labeled Staphylococcus aureus protein A (SPA) was used as a tracer. The colloidal gold immunochromatography method for detecting the anti PRV-g E protein antibody in pig serum was established. The criterion was as follows: 1) The test serum contains anti PRV-g E antibody and can combine with gold standard SPA to form Ig G-SPA complex. When the chromatography membrane flows through the T line and the C line, the Ig G-SPA complex combines with the PRV-g E protein on the T line and the pig in the T line to form a specific combination, forming two red lines, determined as positive results; 2) if the serum is not contained in the tested serum. The only quality control line appeared; 3) if the quality control line did not form a red strip, the result of the method was invalid. After exploring the best reaction condition of the colloid gold, we finally determined that the C line was Ig G, the concentration was 2mg/ml, the concentration of T line was 2mg/ml, the best marker of the gold standard SPA P H was 8, the concentration was 0.1mg/ml, the best dilution times the serum times. The test methods established by this experiment were specific, sensitive, reproducible, and stable, and the coincidence rate was tested. The results showed that the method had good specificity, was only positive in the detection of pseudorabies virus sera and negative for other virus sera, such as porcine reproductive and respiratory syndrome. Syndrome virus, porcine circovirus, Mycoplasma pneumoniae, porcine epidemic diarrhea and infectious gastroenteritis virus and so on. The same batch of repeated test and interbatch repeat test were carried out on the same batch of samples. The test was the same result, and it was stable for up to 4 months at 4 centigrade without changing its characteristics; the anti physical examination of G E of edus pseudorabies was tested. The sensitivity of the method was slightly higher, and the specificity and sensitivity of the method were 81.6% and 90.7% respectively, and there were good conformance between the two (Kappa=0.7289). We also carried out a pig's attack test. We used the colloidal gold immunochromatography and EDIS ELISA method for the non immune attack of 4 different groups (7 pigs in each group), respectively. Group, non immunization non attack group, immune attack group, immune non attack group pigs were tested for G E antibody, and the monitoring time was 45 days. It was found that the time of G E antibody positive was ninth days, 11 days, 9 days and 13 days, which also showed that the two methods had good consistency. In the summary, the experiment established the detection of anti pseudorabies antibody colloid Gold immunochromatography assay provides a fast and simple method for detecting pseudorabies virus.

【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.4

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