EgM9、EgM123蛋白免疫犬持續(xù)期抗體檢測與免疫組織化學研究
發(fā)布時間：2018-09-10 16:38
【摘要】：細粒棘球蚴病是細粒棘球?qū)俚募毩＜蚪{蟲幼蟲所引起的人畜共患寄生蟲病,俗稱包蟲病(Hydatid disease)。據(jù)統(tǒng)計,由于動物感染包蟲病,每年使我國畜產(chǎn)品經(jīng)濟損失達5億元以上,給當?shù)鼐用竦纳眢w健康和畜牧業(yè)的生產(chǎn)與發(fā)展帶來巨大的危害。研究顯示,用EgM家族(EgM9、EgM123)蛋白免疫過的犬可獲得80%以上的免疫保護效果。但是有關(guān)EgM家族(EgM9、EgM123)蛋白免疫效果的持續(xù)期研究尚且不全。因此,本研究對EgM9、EgM123兩種蛋白免疫犬后0~27周相關(guān)血清抗體效價、淋巴因子、小腸組織及腸系膜淋巴結(jié)抗體靶位點分布情況進行了檢測,旨在了解免疫后兩種抗體的持續(xù)變化情況,為終末宿主免疫預(yù)防研究提供試驗依據(jù)。1.采用SDS堿裂解法提取pET41b-EgM123質(zhì)粒,酶切獲得EgM123基因,對EgM123基因進行亞克隆并測序,克隆后基因與原核表達載體pET28a連接、轉(zhuǎn)入大腸桿菌BL21。測序分析和SDS-PAGE分析表明,獲得的EgM123基因片段大小為657 bp,與GenBank中登錄號為AF482718的基因序列同源性為100%,誘導(dǎo)表達的pET28a-EgM123分子量30 kD。Western blott檢測顯示,陽性血清與pET28a-EgM123重組蛋白在30 kD處有清晰的特異性結(jié)合條帶,顯示出良好的反應(yīng)活性。2.將純化的pET28a-EgM123重組蛋白免疫健康新西蘭大白兔,并通過ELISA檢測免疫過的新西蘭大白兔的血清中特異性抗體水平。結(jié)果顯示,獲得了高效價的特異性抗血清,其效價達1:320000以上。3.實驗犬分為三組,分別用EgM9蛋白、EgM123蛋白及PBS進行免疫,免疫三次,每次免疫間隔2周,第5周經(jīng)口服感染源頭蚴,0~27周之間定期采血,用ELISA檢測法檢測血清抗體效價及血清中(IFN-γ、IL10)和第27周血漿中(IFN-γ、IL4)兩種淋巴因子含量。結(jié)果顯示,三免后一周抗體水平達到最高,攻擊原頭蚴后,抗體水平隨之下降,免疫時間達到16周,免疫組抗體與對照組的差異不顯著(P0.05)。第5周時各組血清中IFN-γ的含量均達到最高,之后呈現(xiàn)下降趨勢;第5周后PBS組血清中IL10含量顯著高于EgM9蛋白免疫組及EgM123蛋白免疫組(P0.05)。第27周PBS組血漿中IFN-γ含量低于EgM9蛋白免疫組及EgM123蛋白免疫組(P0.05);各組血漿中IL4含量差異不顯著(P0.05)。4.采用免疫組織化學技術(shù)分別對三組犬腸系膜淋巴結(jié)及小腸組織進行IgG抗體靶位點檢測,EgM9蛋白免疫犬7周的腸系膜淋巴結(jié)及小腸組織作為標準陽性I組。結(jié)果顯示四個實驗組都存在IgG抗體靶位點,進一步進行三步法,I組(標準陽性樣)呈現(xiàn)棕色,反應(yīng)強陽性,27周的腸系膜淋巴結(jié)及小腸組織樣品II組(EgM9)和III組(EgM123)弱陽性,PBS組免疫組化反應(yīng)陰性;三步法可以定位檢測免疫犬腸系膜淋巴結(jié)和小腸上的IgG靶位點抗體富集程度。免疫組織化學及抗體檢測結(jié)果顯示隨著免疫持續(xù)期延長,EgM家族蛋白免疫犬血清抗體水平呈現(xiàn)下降趨勢,腸系膜淋巴結(jié)和小腸組織特異性抗體也隨之減少。
[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- 緯 nil 10) and 27 week plasma (IFN- 緯 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- 緯 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- 緯 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.
【學位授予單位】：新疆農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.4
本文編號：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- 緯 nil 10) and 27 week plasma (IFN- 緯 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- 緯 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- 緯 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.
【學位授予單位】：新疆農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.4
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