摘要
采用噬菌体随机12肽库经健康牛血清反复多次预淘选之后,以临床分离的布鲁氏菌阳性混合血清IgG为靶分子,筛选布鲁氏菌抗原表位。3轮生物淘选后,挑取所有得到的噬菌体克隆,提取单链DNA进行测序。运用间接ELISA方法、竞争ELISA方法及Western-bloting方法分别对测序得到的不同噬菌体克隆的亲和力、特异性及反应原性进行鉴定。最终得到两个亲和力高且特异性好的噬菌体克隆,分别命名为P2和P14。
将特异性噬菌体P2和P14扩增、纯化,两者等量混合后的溶液稀释至适当浓度作为检测抗原直接包被酶标板,以HRP-山羊抗牛IgG作为二抗,建立牛布鲁氏菌抗体间接ELISA检测方法,对检测方法中的各种反应条件进行了优化,并使用该方法对临床采集血清样本进行了检测。结果显示,该方法特异性好,灵敏度高,与传统虎红平板凝集试验检测方法符合率达到96.7%。
以胶体金标记G蛋白制备金标垫,特异性混合噬菌体克隆包被NC膜作为检测线,采用间接法原理组装了布鲁氏菌抗体快速检测免疫层析胶体金试纸条,并对照本实验室建立的间接ELISA方法和虎红平板凝集试验方法对临床血清样品进行了检测,检测符合率分别为96.7%和93.3%,为检测试纸条的进一步研究和应用打下良好的基础。
Brucellosis also called undulant fever is a highly contagious zoonosis around the world which caused by Brucella spp. Brucella are gram-negative facultative intracellular bacterial pathogens that pose threats to livestock, wild animals, people and marine mammals and so on. Animals were Infected usually showed heat waves, infertility and abortion and other Reproductive disorders. Brucellosis had caused serious harm to human health and enormous economic losses to the development of animal husbandry, which attracted the worldwide attention. At present there was not a single method can be applied to diagnose brucellosis at any case. Bacteriological detection methods were time-consuming, laborious, needed exclusive culture medium and had a high-risk to laboratories. Bacteriological detection methods had been limited enormously in the clinical detection. Traditional immunological detection methods had also existed too high false-positive and false-negative phenomenas to use in the clinical detection, and a single detection method could not agree the diagnosis requirement. Therefore, Searching an novel diagnostic antigen and establishing a specific, sensitive, rapid and accurate diagnostic methods for monitoring brucellosis have a great significance in our country. In this study, the peptides that bind specifically to the purified polyclonal serum IgG of Brucellosis were screened using phage display random peptides library. Specific antigens of Brucella were preparated and used in an indirect ELISA method and immuno-colloidal gold method, The preliminary test results of serum samples using the mathod proved that both the indirect ELISA method and the colloidal gold test dipstick had good sensitivity and specificity which can be used for diagnosis of brucellosis and epidemiological investigation.
Phage peptide library technology has proven to be a powerful technology for selecting polypeptides with desired biological and physicochemical properties from huge molecular libraries. In this study, the specific peptides against Brucella were panned from a 12-phage display peptide library using purified polyclonal serum IgG of brucellosis. After 3 rounds of panning, the enrichment of specific phage mimic peptides were obtained and 16 different peptide sequences from all phage clones sequenced were got. The affinity and specificity of 16 different phage peptide sequences were identificated by indirect ELISA and competitive inhibition ELISA. Finally, Brucella-specific epitopes P2 and P14 were obtained and an indirect ELISA methods for detecting Brucella antibody was developed. Serum samples were detected using this method. The reaction conditions were optimized. The results show that: the optimal concentration of mixed-phage antigen is 1011pfu/ml; the best-coated condition is 4℃for 12h; the optimum conditions of blocking is using 1% BSA for 45min at 37℃; the best dilution of serum at 1:100 and incubated for 60min; the best reacting time of HRP-goat anti-bovine IgG is 45min; the best time of OPD chromogenic substrate is 10min. The indirect ELISA method is specificity, sensitivity and good repeatability. A total of 120 serum samples were deteced by the indirect ELISA, And the result was compared to the RBT. The detection rate of the indirect ELISA is high and the results are in good consistent with traditional methods at a rate of 96.7%.The indirect ELISA provide a sensitive, specific, rapid and accurate method for surveillance and epidemiological investigations of Brucellosis.
In this study, colloidal gold immunochromatography assay was also used in detection, The gold-labeled protein G was prepared as the gold pad, the specific phage clones were coated NC membrane as the test line, the indirect method was used to assemble a colloidal gold dipstick for detecting Brucella antibodies rapidly. Serum samples were detected using this dipstick, And the result was compared to the indirect ELISA method and RBT. The results indicate that: the colloidal gold-labeled probe was prepared Successfully; the best coating pH of colloidal gold was 9.0, the best coating density of protein G were 7.2μg/ml; the best test coating concentration of specific phage was 1014pfu/ml; the optimal concentration of goat anti-rabbit IgG was 1.25mg/ml; the highest serum dilution at 1:100; the dipstick did’t reacted with E. coli O157, Yersinia enterocolitica O9 and Salmonella positive serum; the dipstick’s repeatability was well. The detecting results of serum samples by the dipstick showed that the sensitivity of dipstick was high, Contrasting with the indirect ELISA and RBT, the coincidence rate were 96.7% and 93.3%.The dipstick provided a serological methods of detecting Brucellosis in filed to primary sector.
引文
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