新疆馬駑巴貝斯蟲地方蟲株靶基因Bc48克
發(fā)布時間：2018-10-23 06:44
【摘要】：馬駑巴貝斯蟲病是由駑巴貝斯蟲(Babesia caballi)寄生于驢、騾、馬和斑馬等馬屬動物紅細胞內的主要以發(fā)熱、貧血、黃疸的一種蜱傳原蟲病;是全球性流行、對馬屬動物危害較大的B類疾病。該病的發(fā)生、流行與其地方蟲株致病力和其傳播媒介的分布等密切相關,一般呈地方性流行。本文在分離、獲得駑巴貝斯蟲新疆地方蟲株Bc-Y的基礎上,借助外源基因擴增技術獲得靶基因Bc48的不同片段,構建重組原核表達質粒、獲得高效可溶性重組蛋白、以其為包被抗原研發(fā)rELISA檢測試劑盒,應用于地方疑似區(qū)該病的檢測、監(jiān)控及綜合防控,從而減少該病的感染率和馬匹、媒介蜱攜帶率病原率等,促進特色馬業(yè)的健康發(fā)展。(1)采用血液涂片與PCR方法相結合,對新疆昭蘇縣及和靜縣部分馬駑巴貝斯蟲病進行了流行病學調查,并分離地方流行蟲株、測序、與GenBank中的序列進行比較,系統(tǒng)發(fā)育關系與他國駑巴貝斯蟲屬的種聚類;通過保蟲實驗順利完成了新疆馬駑巴貝斯蟲Bc-Y蟲株(伊犁地區(qū)流行蟲株)的分離、測序。結果發(fā)現,血液涂片檢查陽性率為8%,PCR檢查陽性率為21.31%,在所檢查的實驗點中馬駑巴貝斯蟲病感染率最高可達到64.29%;測序結果與標準蟲株同源性為96.7%-99.8%,分歧度0.2%-3.3%。(2)根據GenBank中已發(fā)表的馬駑巴貝斯蟲Bc48基因序列,設計合成4對引物,分別擴增Bc48基因的全長基因(QCL)、截短區(qū)域(A)(B)(C)片段從而構建四種重組原核表達質粒。結果顯示:成功地擴增了Bc48基因的QCL、A、B、C四個片段,構建了pGEX-4T-1-QCL、GA、GB、GC四種重組原核表達質粒;通過測序后表明已克隆的Bc48序列及翻譯氨基酸序列與其它國家的蟲株序列進行比較,同源性可達99%;并成功表達出2種融合蛋白,篩選出GST-GB為高表達量蛋白,Western blotting結果顯示純化的融合蛋白具有良好的反應原性,可作為診斷抗原。(3)將已純化的GST-GB蛋白作為包被抗原,通過優(yōu)化其反應條件后,成功地建立了檢測馬駑巴貝斯蟲抗體的rELISA方法;并進行重復性、特異性、敏感性等評測試驗,并應用已建立的方法檢測馬血清樣品(N=250)。結果顯示:rELISA最佳抗原包被濃度為0.8μg/mL,包被時間為4℃過夜;3%脫脂乳為最佳封閉液;血清和酶標二抗的稀釋度分別為1:50和1:10000,封閉時間、血清作用及酶標抗體孵育時間均為1 h;底物作用時間為15 min;其臨界值為0.221。rELISA試驗無交叉反應其變異系數均小于10%;檢測伊犁昭蘇及巴州和靜地區(qū)馬駑巴貝斯蟲總感染率為28.4%,其中100份樣品結果符合率為94.1%。該參數可為我區(qū)馬駑巴貝斯蟲病的防治奠定基礎。
[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，
本文編號：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，
本文編號：2288337
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