本文選題：環(huán)形泰勒蟲 + 抗原表位��； 參考：《新疆農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：牛環(huán)形泰勒蟲病(Tropical theileriasis)是由寄生于牛的紅細胞、淋巴細胞及巨噬細胞內(nèi)的環(huán)形泰勒蟲(Theileria annulata)引發(fā)的一種蜱傳急性、熱性血液原蟲病。本病除了會直接引起病畜死亡、使病牛體重和產(chǎn)奶量減少,而且病后殘存在家畜體內(nèi)的蟲體,還會導(dǎo)致家畜抵抗力下降,在其他疾病暴發(fā)時再次誘發(fā)此病,造成嚴重的經(jīng)濟損失,對畜牧業(yè)危害很大。環(huán)形泰勒蟲裂殖子表面抗原(The merozoite surface antigen,Tams1),裂殖體表面抗原(The schizonts surface antigen,Tasp)及子孢子表面抗原(The sporozoite antigen,Spag1)已被確認具有較好的抗原性,但鑒于寄生蟲抗原基因存在多態(tài)性及感染免疫的特點,以環(huán)形泰勒蟲單一抗原的生物制品在免疫時會產(chǎn)生不完全保護,做檢測抗原時存在敏感性與特異性不高等問題。本研究對牛環(huán)形泰勒蟲的三種表面抗原做進一步研究,旨在探索表面抗原基因串聯(lián)表達后的免疫原性,為今后泰勒焦蟲亞單位疫苗的研制奠定基礎(chǔ)。本實驗應(yīng)用生物信息學(xué)方法分析串聯(lián)表面抗原基因后所表達出的串聯(lián)蛋白Tams1-Spag1、Tasp-Spag1的主要特性,預(yù)測出串聯(lián)重組蛋白Tams1-Spag1、Tasp-Spag1具有良好的抗原性。在此基礎(chǔ)上,將成功構(gòu)建的重組融合表達質(zhì)粒p ET-28a-Tams1-Spag1、pET-28a-Tasp-Spag1轉(zhuǎn)化至E.coil BL21(DE3)感受態(tài)細胞中,經(jīng)IPTG誘導(dǎo)重組蛋白的表達并運用Western blot實驗檢測此串聯(lián)蛋白的反應(yīng)原性。在對重組蛋白進行初步純化后免疫小鼠,采取小鼠血清后檢測該血清中的抗體,從而鑒定此重組蛋白的免疫原性。生物信息學(xué)分析發(fā)現(xiàn),串聯(lián)重組蛋白Tams1-Spag1含有219個氨基酸屬于穩(wěn)定的非分泌型親水性蛋白。分別含有13個與11個可能的糖基化與磷酸化位點,存在兩個低復(fù)雜性的結(jié)構(gòu)域,一個NOT的結(jié)構(gòu)域�？赡苡�6個B細胞表位優(yōu)勢區(qū)段與10個T細胞表位優(yōu)勢區(qū)段,存在兩段交叉反應(yīng)性表位肽。串聯(lián)重組蛋白Tasp-Spag1具有236個氨基酸,屬于不穩(wěn)定的非分泌型、非跨膜親水蛋白,分別具有33個與21個可能的糖基化與磷酸化位點,有三段低復(fù)雜性的結(jié)構(gòu)域,并且含有Sorb、NL的同源區(qū)域,可能有10個B細胞表位優(yōu)勢區(qū)段與7個T細胞表位優(yōu)勢區(qū)段,具有兩段交叉反應(yīng)性表位肽。原核表達后通過SDS-PAGE與Western blot檢測顯示,得到大小與預(yù)期分子量相當?shù)哪康牡鞍?對表達條件進行優(yōu)化,發(fā)現(xiàn)此兩種蛋白的表達均以包涵體形式存在。串聯(lián)重組蛋白Tams1-Spag1在OD600=1時加入IPTG至終濃度為1.0 mmol/L,37℃過夜誘導(dǎo)時,其表達量最高。串聯(lián)重組蛋白Tasp-Spag1在OD600=1.2時加入IPTG至終濃度為0.9 mmol/L,37℃過夜誘導(dǎo)時,其蛋白的表達量最高。采用KCL染色切膠后電洗脫法對蛋白進行純化,通過Western blot檢測,表明此兩種串聯(lián)蛋白的生物活性沒有改變,仍具有良好的反應(yīng)原性。將純化的兩組蛋白與弗氏佐劑乳化后免疫小鼠,經(jīng)間接ELISA和Western Blot檢測此小鼠血清,結(jié)果表明兩組串聯(lián)蛋白均能夠誘導(dǎo)小鼠產(chǎn)生相應(yīng)抗體,且該抗體可以與牛環(huán)形泰勒焦蟲病血源細胞疫苗蛋白特異性反應(yīng)。本次試驗結(jié)果可以證明串聯(lián)重組蛋白Tams1-Spag1、Tasp-Spag1具有良好的反應(yīng)原性和一定的免疫原性。
[Abstract]:Bovine ring Taylor disease (Tropical theileriasis) is a tick transmitted acute, hot blood protozoon caused by the red blood cells parasitic on cattle, lymphocytes and macrophages, caused by the ring-shaped Taylor worm (Theileria annulata). This disease can cause the death of the sick animal directly, reduce the weight and milk production of the infected cattle, and remain in the domestic animal body after the disease. The internal insect body also leads to the decline of domestic animal resistance, causing the disease again when other diseases outbreaks, causing serious economic losses and is very harmful to animal husbandry. The merozoite surface antigen (Tams1), The schizonts surface antigen, Tasp, and subspore surface antigen (The schizonts surface antigen, Tasp). The sporozoite antigen, Spag1) has been confirmed to have good antigenicity, but in view of the polymorphism of the parasite antigen gene and the characteristics of infection immunity, the biological products with the single antigen of the ring Taylor worm can produce incomplete protection when immunized, and there are no high sensitivity and specificity in the detection of antigen. Three kinds of surface antigen of the form Taylor worm were further studied to explore the immunogenicity of the surface antigen gene after the series expression, and lay the foundation for the future development of the vaccine of the Taylor focal insect. This experiment used the Bioinformatics Method to analyze the series protein Tams1-Spag1, Tasp-Spag1, which was expressed by the series surface antigen gene. The recombinant fusion protein Tams1-Spag1 and Tasp-Spag1 had good antigenicity. On this basis, the recombinant fusion expression plasmid P ET-28a-Tams1-Spag1 was successfully constructed, and pET-28a-Tasp-Spag1 was transformed into E.coil BL21 (DE3) receptive cells. The recombinant protein was induced by IPTG to express the recombinant protein and detected this series with Western blot. The immunogenicity of the recombinant protein was identified after the recombinant protein was preliminarily purified and the antibody in the serum was detected by the mouse serum. The bioinformatics analysis showed that the series recombinant protein Tams1-Spag1 contained 219 amino acids in the stable non secretory hydrophilic protein. 13 and 11 possible glycosylation and phosphorylation sites, there are two low complex domains, one NOT domain. There may be 6 B cell epitopes and 10 T cell epitopes, two cross reactive epitopes. Series recombinant protein Tasp-Spag1 has 236 amino acids, which belong to the unstable non secretory type. Non transmembrane hydrophilic proteins, with 33 and 21 possible glycosylation and phosphorylation sites, have three low complex domains, and contain Sorb, NL homologous regions, and there may be 10 B cell epitopes and 7 T cell epitopes, with two segments of cross reactivity epitopes. Prokaryotic expression passes through SDS-PAGE and Weste RN blot detection showed that the target protein was equal to the expected molecular weight, and the expression conditions were optimized and the expression of these two proteins all existed in the form of inclusion body. The expression of series recombinant protein Tams1-Spag1 was 1 mmol/L at IPTG to the final concentration at OD600=1, and the expression amount was the highest when 37 degrees centigrade was over night. Series recombinant protein Tasp- Spag1 added IPTG to the final concentration of 0.9 mmol/L at OD600=1.2, and the protein expression was the highest when 37 centigrade was induced overnight. The protein was purified by electroelution by KCL staining and Western blot, indicating that the biological activity of the two series of series proteins was not changed, and it still had a good reactivity. The purified two groups of proteins were still being purified. The mice were immunized with the emulsification of Freund's adjuvant and the serum was detected by indirect ELISA and Western Blot. The results showed that the two groups of series proteins could induce the corresponding antibody in mice, and the antibody could react with the specific protein of the bovine ring-shaped Taylor char cell vaccine protein. The results of this test could prove that the series recombinant protein Tams1-Sp could be proved. AG1 and Tasp-Spag1 have good reactivity and immunogenicity.
【學(xué)位授予單位】：新疆農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.23

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