牛环形泰勒虫表面抗原蛋白的串联表达与免疫原性分析

发布时间：2018-06-28 11:13

本文选题：环形泰勒虫 + 抗原表位　；参考：《新疆农业大学》2015年硕士论文

【摘要】：牛环形泰勒虫病(Tropical theileriasis)是由寄生于牛的红细胞、淋巴细胞及巨噬细胞内的环形泰勒虫(Theileria annulata)引发的一种蜱传急性、热性血液原虫病。本病除了会直接引起病畜死亡、使病牛体重和产奶量减少,而且病后残存在家畜体内的虫体,还会导致家畜抵抗力下降,在其他疾病暴发时再次诱发此病,造成严重的经济损失,对畜牧业危害很大。环形泰勒虫裂殖子表面抗原(The merozoite surface antigen,Tams1),裂殖体表面抗原(The schizonts surface antigen,Tasp)及子孢子表面抗原(The sporozoite antigen,Spag1)已被确认具有较好的抗原性,但鉴于寄生虫抗原基因存在多态性及感染免疫的特点,以环形泰勒虫单一抗原的生物制品在免疫时会产生不完全保护,做检测抗原时存在敏感性与特异性不高等问题。本研究对牛环形泰勒虫的三种表面抗原做进一步研究,旨在探索表面抗原基因串联表达后的免疫原性,为今后泰勒焦虫亚单位疫苗的研制奠定基础。本实验应用生物信息学方法分析串联表面抗原基因后所表达出的串联蛋白Tams1-Spag1、Tasp-Spag1的主要特性,预测出串联重组蛋白Tams1-Spag1、Tasp-Spag1具有良好的抗原性。在此基础上,将成功构建的重组融合表达质粒p ET-28a-Tams1-Spag1、pET-28a-Tasp-Spag1转化至E.coil BL21(DE3)感受态细胞中,经IPTG诱导重组蛋白的表达并运用Western blot实验检测此串联蛋白的反应原性。在对重组蛋白进行初步纯化后免疫小鼠,采取小鼠血清后检测该血清中的抗体,从而鉴定此重组蛋白的免疫原性。生物信息学分析发现,串联重组蛋白Tams1-Spag1含有219个氨基酸属于稳定的非分泌型亲水性蛋白。分别含有13个与11个可能的糖基化与磷酸化位点,存在两个低复杂性的结构域,一个NOT的结构域。可能有6个B细胞表位优势区段与10个T细胞表位优势区段,存在两段交叉反应性表位肽。串联重组蛋白Tasp-Spag1具有236个氨基酸,属于不稳定的非分泌型、非跨膜亲水蛋白,分别具有33个与21个可能的糖基化与磷酸化位点,有三段低复杂性的结构域,并且含有Sorb、NL的同源区域,可能有10个B细胞表位优势区段与7个T细胞表位优势区段,具有两段交叉反应性表位肽。原核表达后通过SDS-PAGE与Western blot检测显示,得到大小与预期分子量相当的目的蛋白;对表达条件进行优化,发现此两种蛋白的表达均以包涵体形式存在。串联重组蛋白Tams1-Spag1在OD600=1时加入IPTG至终浓度为1.0 mmol/L,37℃过夜诱导时,其表达量最高。串联重组蛋白Tasp-Spag1在OD600=1.2时加入IPTG至终浓度为0.9 mmol/L,37℃过夜诱导时,其蛋白的表达量最高。采用KCL染色切胶后电洗脱法对蛋白进行纯化,通过Western blot检测,表明此两种串联蛋白的生物活性没有改变,仍具有良好的反应原性。将纯化的两组蛋白与弗氏佐剂乳化后免疫小鼠,经间接ELISA和Western Blot检测此小鼠血清,结果表明两组串联蛋白均能够诱导小鼠产生相应抗体,且该抗体可以与牛环形泰勒焦虫病血源细胞疫苗蛋白特异性反应。本次试验结果可以证明串联重组蛋白Tams1-Spag1、Tasp-Spag1具有良好的反应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.
【学位授予单位】：新疆农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.23

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