新疆马驽巴贝斯虫地方虫株靶基因Bc48克
发布时间：2018-10-23 06:44
【摘要】：马驽巴贝斯虫病是由驽巴贝斯虫(Babesia caballi)寄生于驴、骡、马和斑马等马属动物红细胞内的主要以发热、贫血、黄疸的一种蜱传原虫病;是全球性流行、对马属动物危害较大的B类疾病。该病的发生、流行与其地方虫株致病力和其传播媒介的分布等密切相关,一般呈地方性流行。本文在分离、获得驽巴贝斯虫新疆地方虫株Bc-Y的基础上,借助外源基因扩增技术获得靶基因Bc48的不同片段,构建重组原核表达质粒、获得高效可溶性重组蛋白、以其为包被抗原研发rELISA检测试剂盒,应用于地方疑似区该病的检测、监控及综合防控,从而减少该病的感染率和马匹、媒介蜱携带率病原率等,促进特色马业的健康发展。(1)采用血液涂片与PCR方法相结合,对新疆昭苏县及和静县部分马驽巴贝斯虫病进行了流行病学调查,并分离地方流行虫株、测序、与GenBank中的序列进行比较,系统发育关系与他国驽巴贝斯虫属的种聚类;通过保虫实验顺利完成了新疆马驽巴贝斯虫Bc-Y虫株(伊犁地区流行虫株)的分离、测序。结果发现,血液涂片检查阳性率为8%,PCR检查阳性率为21.31%,在所检查的实验点中马驽巴贝斯虫病感染率最高可达到64.29%;测序结果与标准虫株同源性为96.7%-99.8%,分歧度0.2%-3.3%。(2)根据GenBank中已发表的马驽巴贝斯虫Bc48基因序列,设计合成4对引物,分别扩增Bc48基因的全长基因(QCL)、截短区域(A)(B)(C)片段从而构建四种重组原核表达质粒。结果显示:成功地扩增了Bc48基因的QCL、A、B、C四个片段,构建了pGEX-4T-1-QCL、GA、GB、GC四种重组原核表达质粒;通过测序后表明已克隆的Bc48序列及翻译氨基酸序列与其它国家的虫株序列进行比较,同源性可达99%;并成功表达出2种融合蛋白,筛选出GST-GB为高表达量蛋白,Western blotting结果显示纯化的融合蛋白具有良好的反应原性,可作为诊断抗原。(3)将已纯化的GST-GB蛋白作为包被抗原,通过优化其反应条件后,成功地建立了检测马驽巴贝斯虫抗体的rELISA方法;并进行重复性、特异性、敏感性等评测试验,并应用已建立的方法检测马血清样品(N=250)。结果显示:rELISA最佳抗原包被浓度为0.8μg/mL,包被时间为4℃过夜;3%脱脂乳为最佳封闭液;血清和酶标二抗的稀释度分别为1:50和1:10000,封闭时间、血清作用及酶标抗体孵育时间均为1 h;底物作用时间为15 min;其临界值为0.221。rELISA试验无交叉反应其变异系数均小于10%;检测伊犁昭苏及巴州和静地区马驽巴贝斯虫总感染率为28.4%,其中100份样品结果符合率为94.1%。该参数可为我区马驽巴贝斯虫病的防治奠定基础。
[Abstract]:Cabal Babes's disease is a tick-borne protozoa that is parasitized by Babes caballi (Babesia caballi) in the erythrocytes of horses, such as donkeys, mules, horses and zebras, with fever, anemia, and jaundice; it is a global epidemic. B disease, which is harmful to equine. The occurrence and prevalence of the disease are closely related to the pathogenicity of local insect strains and the distribution of its transmission vectors. In this paper, on the basis of isolation and isolation of Bc-Y from Xinjiang native strain of Babes caballi, different fragments of target gene Bc48 were obtained by using exogenous gene amplification technique, and the recombinant prokaryotic expression plasmid was constructed, and the highly soluble recombinant protein was obtained. The rELISA kit was used as the coating antigen to detect, monitor and control the disease in local suspected areas, so as to reduce the infection rate of the disease, the pathogen rate of horse and vector ticks, etc. To promote the healthy development of the special horse industry. (1) using blood smear and PCR method, the epidemiological investigation of some horse caballi Babes's disease in Zhaosu County and Hejing County of Xinjiang was carried out, and local endemic strains were isolated and sequenced. Compared with the sequence in GenBank, phylogenetic relationship was compared with the species cluster of the genus Babes in other countries, and the isolation and sequencing of the Bc-Y strain of Babes caballi in Xinjiang (endemic strain in Yili area) was successfully completed through conservation experiments. And it turns out, The positive rate of blood smear was 21.31. The highest infection rate of Babes's disease in horse was 64.29. The result of sequencing was 96.7-99.8 with the standard strain, and the divergence was 0.2- 3.3%. (2) according to the published results in GenBank, the infection rate of Babes's disease in horses could reach 64.29. (2) according to the published results in GenBank. Bc48 gene sequence of Babes caballi, Four recombinant prokaryotic expression plasmids were constructed by designing and synthesizing four pairs of primers to amplify the truncated (A) (B) (C) fragment of full-length (QCL), of Bc48 gene. The results showed that the four QCL,A,B,C fragments of Bc48 gene were amplified successfully and four recombinant prokaryotic expression plasmids of pGEX-4T-1-QCL,GA,GB,GC were constructed. The results showed that the cloned Bc48 sequence and translated amino acid sequence were compared with other insect strains. Two kinds of fusion proteins were successfully expressed and GST-GB was selected as high expression protein, Western blotting. The results showed that the purified fusion protein had good reactivity and could be used as diagnostic antigen. (3) the purified GST-GB protein was used as coating antigen. After optimizing the reaction conditions, a rELISA method was successfully established for the detection of the antibody against Babes caballi, and the reproducibility, specificity and sensitivity were evaluated, and the established method was used for the detection of horse serum samples. The results showed that the best antigen coating concentration of rELISA was 0.8 渭 g / mL, the encapsulation time was 4 鈩，
本文编号：2288337
[Abstract]:Cabal Babes's disease is a tick-borne protozoa that is parasitized by Babes caballi (Babesia caballi) in the erythrocytes of horses, such as donkeys, mules, horses and zebras, with fever, anemia, and jaundice; it is a global epidemic. B disease, which is harmful to equine. The occurrence and prevalence of the disease are closely related to the pathogenicity of local insect strains and the distribution of its transmission vectors. In this paper, on the basis of isolation and isolation of Bc-Y from Xinjiang native strain of Babes caballi, different fragments of target gene Bc48 were obtained by using exogenous gene amplification technique, and the recombinant prokaryotic expression plasmid was constructed, and the highly soluble recombinant protein was obtained. The rELISA kit was used as the coating antigen to detect, monitor and control the disease in local suspected areas, so as to reduce the infection rate of the disease, the pathogen rate of horse and vector ticks, etc. To promote the healthy development of the special horse industry. (1) using blood smear and PCR method, the epidemiological investigation of some horse caballi Babes's disease in Zhaosu County and Hejing County of Xinjiang was carried out, and local endemic strains were isolated and sequenced. Compared with the sequence in GenBank, phylogenetic relationship was compared with the species cluster of the genus Babes in other countries, and the isolation and sequencing of the Bc-Y strain of Babes caballi in Xinjiang (endemic strain in Yili area) was successfully completed through conservation experiments. And it turns out, The positive rate of blood smear was 21.31. The highest infection rate of Babes's disease in horse was 64.29. The result of sequencing was 96.7-99.8 with the standard strain, and the divergence was 0.2- 3.3%. (2) according to the published results in GenBank, the infection rate of Babes's disease in horses could reach 64.29. (2) according to the published results in GenBank. Bc48 gene sequence of Babes caballi, Four recombinant prokaryotic expression plasmids were constructed by designing and synthesizing four pairs of primers to amplify the truncated (A) (B) (C) fragment of full-length (QCL), of Bc48 gene. The results showed that the four QCL,A,B,C fragments of Bc48 gene were amplified successfully and four recombinant prokaryotic expression plasmids of pGEX-4T-1-QCL,GA,GB,GC were constructed. The results showed that the cloned Bc48 sequence and translated amino acid sequence were compared with other insect strains. Two kinds of fusion proteins were successfully expressed and GST-GB was selected as high expression protein, Western blotting. The results showed that the purified fusion protein had good reactivity and could be used as diagnostic antigen. (3) the purified GST-GB protein was used as coating antigen. After optimizing the reaction conditions, a rELISA method was successfully established for the detection of the antibody against Babes caballi, and the reproducibility, specificity and sensitivity were evaluated, and the established method was used for the detection of horse serum samples. The results showed that the best antigen coating concentration of rELISA was 0.8 渭 g / mL, the encapsulation time was 4 鈩，
本文编号：2288337
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