本文選題：環(huán)形泰勒蟲　切入點(diǎn)：TaSP　出處：《河南農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：環(huán)形泰勒蟲(Theileria annulata)是一種可感染牛且危害嚴(yán)重的血液性原蟲,寄生在宿主的巨噬細(xì)胞、淋巴細(xì)胞和紅細(xì)胞,易引起被感染者的急性死亡。環(huán)形泰勒蟲病是一種世界范圍內(nèi)流行的疾病,嚴(yán)重危害各國的養(yǎng)牛業(yè)。在中國,感染牛的泰勒蟲主要是環(huán)形泰勒蟲,中華泰勒蟲和瑟氏泰勒蟲,其中又以環(huán)形泰勒蟲的危害最大,發(fā)病范圍最廣,在包括新疆、內(nèi)蒙古等13個(gè)省區(qū)均有感染環(huán)形泰勒蟲的病例的報(bào)道,給當(dāng)?shù)氐酿B(yǎng)牛業(yè)帶來嚴(yán)重影響,并且造成極大的經(jīng)濟(jì)損失。在我國,傳播環(huán)形泰勒蟲的媒介主要為璃眼蜱屬,其中大部分地區(qū)主要是殘緣璃眼蜱,新疆南部地區(qū)主要是小亞璃眼蜱。本研究以環(huán)形泰勒蟲的裂殖體膜表面蛋白(Theileria annulata sporozoites surface protein,TaSP)為研究對象,在前期研究的基礎(chǔ)上,通過優(yōu)化重組菌的表達(dá)條件,純化表達(dá)的重組TaSP蛋白,并以純化后的重組蛋白為抗原,建立牛環(huán)形泰勒蟲TaSP重組蛋白間接ELISA檢測方法。研究內(nèi)容如下:1.對已表達(dá)的TaSP(甘肅株)重組蛋白的菌液進(jìn)行PCR檢測,選取瓊脂糖凝膠電泳結(jié)果條帶大小約為400bp的PCR擴(kuò)增陽性結(jié)果的菌液送去測序。分析測序結(jié)果發(fā)現(xiàn)表達(dá)的目的基因大小為396bp,與已上傳蘇丹株和印度株相應(yīng)序列比對后發(fā)現(xiàn)同源性達(dá)97%。通過不同時(shí)段的誘導(dǎo)表達(dá),純化,對重組蛋白的反應(yīng)原性和特異性進(jìn)行Western-Blot檢測,結(jié)果表明重組TaSP蛋白與環(huán)形泰勒蟲標(biāo)準(zhǔn)陽性血清發(fā)生反應(yīng),不與環(huán)形泰勒蟲標(biāo)準(zhǔn)陰性血清發(fā)生反應(yīng),且不與中華泰勒蟲、牛巴貝斯蟲、雙芽巴貝斯蟲、大巴貝斯蟲、瑟氏泰勒蟲、呂氏泰勒蟲、尤氏泰勒蟲的標(biāo)準(zhǔn)陽性血清發(fā)生交叉反應(yīng)。2.以純化的TaSP重組蛋白為抗原,建立牛環(huán)形泰勒蟲的間接ELISA診斷方法。通過對反應(yīng)條件的優(yōu)化,確定了TaSP重組蛋白(即抗原)最適的包被濃度為3μg/ml;血清的最適稀釋比例為1:50;最適酶結(jié)合底物(原液)最佳稀釋比例為1:10000。通過驗(yàn)證,該檢測方法能與環(huán)形泰勒蟲陽性血清發(fā)生反應(yīng),而與牛巴貝斯蟲,雙芽巴貝斯蟲,瑟氏泰勒蟲,中華泰勒蟲,呂氏泰勒蟲,尤氏泰勒蟲陽性血清無交叉反應(yīng);對新疆省和甘肅省共140份野外樣品進(jìn)行ELISA和巢式PCR對比檢測,符合率為98.5%。因此,該方法特異性達(dá)到了100%,敏感性達(dá)到了95.7%。
[Abstract]:Theileria annulata is an infectious and seriously harmful blood protozoa that parasitizes macrophages, lymphocytes and red blood cells in the host. It is a worldwide epidemic disease that seriously harms cattle farming in various countries. In China, the main infectious species of Taylor's worms are the ringworm, the Chinese Taylor worm, and the Selenella. Among them, the ringworm is the most harmful and has the most extensive incidence. Cases of the infection have been reported in 13 provinces, including Xinjiang and Inner Mongolia, which have seriously affected the local cattle farming industry. And caused great economic losses. In our country, the main medium for transmission of Taylor's annulus is the genus Tereodes, most of which are mostly residual ticks. In this study, Theileria annulata sporozoites surface protein (TaSP), a merozoite membrane surface protein of Theileria annulata sporozoites surface, was studied in southern Xinjiang, and the expression conditions of recombinant bacteria were optimized on the basis of previous studies. The recombinant TaSP protein was purified and the purified recombinant protein was used as antigen to establish an indirect ELISA detection method for the recombinant TaSP protein of Taylor's cattle. The contents of the study were as follows: 1. The bacterial fluid of the expressed recombinant TaSP protein was detected by PCR. The result of agarose gel electrophoresis (agarose gel electrophoresis) showed that the size of the PCR amplification positive result was about 400bp, and the result of analysis and sequencing showed that the expressed target gene size was 396bp, which was compared with the corresponding sequence of the uploaded Sudan strain and Indian strain. It was found that the homology was 97%. The expression was induced by different periods of time. After purification, the reactivity and specificity of the recombinant protein were detected by Western-Blot. The results showed that the recombinant TaSP protein reacted with the standard positive serum, not with the standard negative serum, and with the Chinese Taylor's. The standard positive serums of bovine Babes, Babes, Babbs, Selenella, Taylor lui and Taylor uvulis were cross-reacted. 2. The purified TaSP recombinant protein was used as antigen. An indirect ELISA diagnostic method for Taylor ringworm was established. The reaction conditions were optimized. The optimal coating concentration of TaSP recombinant protein (antigen) is 3 渭 g / ml; the optimal dilution ratio of serum is 1: 50; the optimal dilution ratio of enzyme binding substrate (substrate) is 1: 10000. This method can react with the positive serum of Taylor's ring, but not with the positive serum of Babes's, Babes, Thessler's, Taylor's, Lui and Taylor's. A total of 140 field samples from Xinjiang and Gansu provinces were detected by ELISA and nested PCR. The coincidence rate was 98.50.Therefore, the specificity of this method was 100 and the sensitivity was 95.7.
【學(xué)位授予單位】：河南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.7

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