EgM9、EgM123蛋白免疫犬持续期抗体检测与免疫组织化学研究
发布时间：2018-09-10 16:38
【摘要】：细粒棘球蚴病是细粒棘球属的细粒棘球绦虫幼虫所引起的人畜共患寄生虫病,俗称包虫病(Hydatid disease)。据统计,由于动物感染包虫病,每年使我国畜产品经济损失达5亿元以上,给当地居民的身体健康和畜牧业的生产与发展带来巨大的危害。研究显示,用EgM家族(EgM9、EgM123)蛋白免疫过的犬可获得80%以上的免疫保护效果。但是有关EgM家族(EgM9、EgM123)蛋白免疫效果的持续期研究尚且不全。因此,本研究对EgM9、EgM123两种蛋白免疫犬后0~27周相关血清抗体效价、淋巴因子、小肠组织及肠系膜淋巴结抗体靶位点分布情况进行了检测,旨在了解免疫后两种抗体的持续变化情况,为终末宿主免疫预防研究提供试验依据。1.采用SDS碱裂解法提取pET41b-EgM123质粒,酶切获得EgM123基因,对EgM123基因进行亚克隆并测序,克隆后基因与原核表达载体pET28a连接、转入大肠杆菌BL21。测序分析和SDS-PAGE分析表明,获得的EgM123基因片段大小为657 bp,与GenBank中登录号为AF482718的基因序列同源性为100%,诱导表达的pET28a-EgM123分子量30 kD。Western blott检测显示,阳性血清与pET28a-EgM123重组蛋白在30 kD处有清晰的特异性结合条带,显示出良好的反应活性。2.将纯化的pET28a-EgM123重组蛋白免疫健康新西兰大白兔,并通过ELISA检测免疫过的新西兰大白兔的血清中特异性抗体水平。结果显示,获得了高效价的特异性抗血清,其效价达1:320000以上。3.实验犬分为三组,分别用EgM9蛋白、EgM123蛋白及PBS进行免疫,免疫三次,每次免疫间隔2周,第5周经口服感染源头蚴,0~27周之间定期采血,用ELISA检测法检测血清抗体效价及血清中(IFN-γ、IL10)和第27周血浆中(IFN-γ、IL4)两种淋巴因子含量。结果显示,三免后一周抗体水平达到最高,攻击原头蚴后,抗体水平随之下降,免疫时间达到16周,免疫组抗体与对照组的差异不显著(P0.05)。第5周时各组血清中IFN-γ的含量均达到最高,之后呈现下降趋势;第5周后PBS组血清中IL10含量显著高于EgM9蛋白免疫组及EgM123蛋白免疫组(P0.05)。第27周PBS组血浆中IFN-γ含量低于EgM9蛋白免疫组及EgM123蛋白免疫组(P0.05);各组血浆中IL4含量差异不显著(P0.05)。4.采用免疫组织化学技术分别对三组犬肠系膜淋巴结及小肠组织进行IgG抗体靶位点检测,EgM9蛋白免疫犬7周的肠系膜淋巴结及小肠组织作为标准阳性I组。结果显示四个实验组都存在IgG抗体靶位点,进一步进行三步法,I组(标准阳性样)呈现棕色,反应强阳性,27周的肠系膜淋巴结及小肠组织样品II组(EgM9)和III组(EgM123)弱阳性,PBS组免疫组化反应阴性;三步法可以定位检测免疫犬肠系膜淋巴结和小肠上的IgG靶位点抗体富集程度。免疫组织化学及抗体检测结果显示随着免疫持续期延长,EgM家族蛋白免疫犬血清抗体水平呈现下降趋势,肠系膜淋巴结和小肠组织特异性抗体也随之减少。
[Abstract]:Echinococcus granulosus is a zoonotic parasitic disease caused by Echinococcus granulosus larvae, commonly known as hydatidosis (Hydatid disease). According to statistics, the annual economic loss of livestock products in China is more than 500 million yuan due to the infection of hydatid disease in animals, which brings great harm to the health of local residents and the production and development of animal husbandry. More than 80% of the dogs immunized with EgM family (EgM9,EgM123) proteins were immunized. But the duration of immune response of EgM family (EgM9,EgM123) proteins is not complete. Therefore, the antibody titers, lymphoid factors, small intestinal tissues and mesenteric lymph nodes were detected in dogs immunized with EgM9,EgM123 proteins at 27 weeks after immunization, in order to understand the continuous changes of the two antibodies after immunization. To provide experimental basis for the study of terminal host immunity prevention. 1. The pET41b-EgM123 plasmid was extracted by SDS base lysis method, the EgM123 gene was digested by enzyme, the EgM123 gene was subcloned and sequenced. After cloning, the EgM123 gene was ligated with the prokaryotic expression vector pET28a and transferred into E. coli BL21.. Sequencing and SDS-PAGE analysis showed that the size of the EgM123 gene fragment was 657 bp, and the homology of the AF482718 gene sequence in GenBank was 100. The molecular weight of induced expression of pET28a-EgM123 was detected by 30 kD.Western blott assay. The positive serum and the pET28a-EgM123 recombinant protein have clear and specific binding bands at 30 kD, showing good reaction activity. 2. The purified pET28a-EgM123 recombinant protein was immunized with healthy New Zealand white rabbits and the specific antibody levels in the sera of the immunized New Zealand rabbits were detected by ELISA. The results showed that the specific antiserum with high titer was obtained and its titer was over 1: 320 000. The experimental dogs were divided into three groups. They were immunized with EgM9 protein EgM123 protein and PBS respectively, three times, with the interval of 2 weeks each time. The blood samples were collected regularly during the fifth week after oral infection. The titer of serum antibody and the contents of two kinds of lymphoid factors in serum (IFN- 纬 nil 10) and 27 week plasma (IFN- 纬 L 4) were detected by ELISA assay. The results showed that the level of antibody reached the highest one week after three immunizations, and the antibody level decreased after attacking protocaria, and the immunization time reached 16 weeks. There was no significant difference between the immunized group and the control group (P0.05). At the 5th week, the content of IFN- 纬 in serum of each group reached the highest level, and then showed a downward trend. After 5 weeks, the content of IL10 in serum of PBS group was significantly higher than that of EgM9 protein immunized group and EgM123 protein immunized group (P0.05). The plasma IFN- 纬 level in PBS group was lower than that in EgM9 protein immunized group and EgM123 protein immunized group (P0.05), but there was no significant difference in IL4 content in each group (P0.05). 4. The IgG antibody target sites in mesenteric lymph nodes and small intestine tissues of three groups of dogs immunized with EgM9 protein for 7 weeks were detected by immunohistochemical technique as standard positive group I. The results showed that all the four experimental groups had IgG antibody targets. The strongly positive mesenteric lymph nodes and small intestine tissues of II group (EgM9) and III group (EgM123) were weakly positive for 27 weeks, and three-step method could be used to detect the concentration of IgG target antibody in mesenteric lymph nodes and small intestine of immunized dogs. The results of immunocytochemistry and antibody detection showed that the serum antibody level of immunized dogs with EgM family proteins decreased with the prolongation of the immune duration, and the specific antibodies in mesenteric lymph nodes and small intestine also decreased.
【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】：S852.4
本文编号：2235009
[Abstract]:Echinococcus granulosus is a zoonotic parasitic disease caused by Echinococcus granulosus larvae, commonly known as hydatidosis (Hydatid disease). According to statistics, the annual economic loss of livestock products in China is more than 500 million yuan due to the infection of hydatid disease in animals, which brings great harm to the health of local residents and the production and development of animal husbandry. More than 80% of the dogs immunized with EgM family (EgM9,EgM123) proteins were immunized. But the duration of immune response of EgM family (EgM9,EgM123) proteins is not complete. Therefore, the antibody titers, lymphoid factors, small intestinal tissues and mesenteric lymph nodes were detected in dogs immunized with EgM9,EgM123 proteins at 27 weeks after immunization, in order to understand the continuous changes of the two antibodies after immunization. To provide experimental basis for the study of terminal host immunity prevention. 1. The pET41b-EgM123 plasmid was extracted by SDS base lysis method, the EgM123 gene was digested by enzyme, the EgM123 gene was subcloned and sequenced. After cloning, the EgM123 gene was ligated with the prokaryotic expression vector pET28a and transferred into E. coli BL21.. Sequencing and SDS-PAGE analysis showed that the size of the EgM123 gene fragment was 657 bp, and the homology of the AF482718 gene sequence in GenBank was 100. The molecular weight of induced expression of pET28a-EgM123 was detected by 30 kD.Western blott assay. The positive serum and the pET28a-EgM123 recombinant protein have clear and specific binding bands at 30 kD, showing good reaction activity. 2. The purified pET28a-EgM123 recombinant protein was immunized with healthy New Zealand white rabbits and the specific antibody levels in the sera of the immunized New Zealand rabbits were detected by ELISA. The results showed that the specific antiserum with high titer was obtained and its titer was over 1: 320 000. The experimental dogs were divided into three groups. They were immunized with EgM9 protein EgM123 protein and PBS respectively, three times, with the interval of 2 weeks each time. The blood samples were collected regularly during the fifth week after oral infection. The titer of serum antibody and the contents of two kinds of lymphoid factors in serum (IFN- 纬 nil 10) and 27 week plasma (IFN- 纬 L 4) were detected by ELISA assay. The results showed that the level of antibody reached the highest one week after three immunizations, and the antibody level decreased after attacking protocaria, and the immunization time reached 16 weeks. There was no significant difference between the immunized group and the control group (P0.05). At the 5th week, the content of IFN- 纬 in serum of each group reached the highest level, and then showed a downward trend. After 5 weeks, the content of IL10 in serum of PBS group was significantly higher than that of EgM9 protein immunized group and EgM123 protein immunized group (P0.05). The plasma IFN- 纬 level in PBS group was lower than that in EgM9 protein immunized group and EgM123 protein immunized group (P0.05), but there was no significant difference in IL4 content in each group (P0.05). 4. The IgG antibody target sites in mesenteric lymph nodes and small intestine tissues of three groups of dogs immunized with EgM9 protein for 7 weeks were detected by immunohistochemical technique as standard positive group I. The results showed that all the four experimental groups had IgG antibody targets. The strongly positive mesenteric lymph nodes and small intestine tissues of II group (EgM9) and III group (EgM123) were weakly positive for 27 weeks, and three-step method could be used to detect the concentration of IgG target antibody in mesenteric lymph nodes and small intestine of immunized dogs. The results of immunocytochemistry and antibody detection showed that the serum antibody level of immunized dogs with EgM family proteins decreased with the prolongation of the immune duration, and the specific antibodies in mesenteric lymph nodes and small intestine also decreased.
【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】：S852.4
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