本文選題：隱孢子蟲鼠基因型　切入點：核酸疫苗　出處：《中國農(nóng)業(yè)科學院》2010年碩士論文　論文類型：學位論文

【摘要】： 隱孢子蟲病(cryptosporidiosis)是由隱孢子蟲(Cryptosporidium spp.)引起的一種以持續(xù)性腹瀉為主要臨床特征的全球性人獸共患寄生蟲病。隱孢子蟲病具有廣泛的宿主類型,其病原可以寄生于哺乳類、鳥類、爬行類、兩棲類、魚類等包括人類在內(nèi)的170多種動物。隱孢子蟲病給畜牧業(yè)生產(chǎn)造成嚴重的經(jīng)濟損失,威脅免疫功能缺陷者的生命安全。當前,對隱孢子蟲病的研究已經(jīng)成為全球寄生蟲學領域的熱點之一。目前還沒有治療隱孢子蟲病的特效藥物,因此隱孢子蟲病的免疫預防研究就顯得愈發(fā)重要。 本研究選取了隱孢子蟲鼠基因型子孢子表面蛋白P23、CP15/60以及卵囊壁蛋白CP41 3個主要保護性抗原基因,利用DNA重組技術將這3個基因分別克隆到真核表達載體pVAX1上,制成核酸疫苗。用制備的3個隱孢子蟲核酸疫苗免疫BALB/c小鼠,對疫苗誘導的IgG水平、小鼠排卵囊量情況進行了檢測。結果顯示,免疫小鼠血清中特異性抗體IgG滴度水平隨免疫次數(shù)的增加而增加,呈現(xiàn)逐步升高的趨勢,在第3次免疫后達到最高。實驗組IgG滴度水平極顯著高于對照組(P0.01),而實驗組內(nèi)、對照組組內(nèi)差異不顯著(P0.05)。與生理鹽水、pVAX1對照組相比,實驗組小鼠卵囊排出數(shù)量顯著小于對照組(P0.05),減卵率在85% ~100%之間,而實驗組內(nèi)、對照組組內(nèi)差異不顯著(P0.05);試驗組小鼠開始排出卵囊時間延后,排出卵囊的持續(xù)時間縮短。表明本研究制備的3個核酸疫苗可以誘導小鼠產(chǎn)生良好的特異性免疫應答,對BALB/c小鼠隱孢子蟲鼠基因型感染有較好的免疫保護作用。 將重組質(zhì)粒pGEX-4T-1-P23、pGEX-5x-3-CP41、pET-28b(+)-CP15/60在大腸桿菌中成功表達。Western blot分析表明制備的3個重組蛋白具有良好的免疫原性,可被隱孢子蟲鼠基因型感染小鼠血清所識別。將3個重組蛋白與弗氏佐劑充分乳化后制成亞單位疫苗免疫BALB/c小鼠,對疫苗誘導的IgG水平、小鼠排卵囊情況進行了檢測。間接ELISA檢測結果表明重組蛋白rP23、rCP41、rCP15/60可引起小鼠產(chǎn)生良好的體液免疫應答,免疫小鼠血清中特異性抗體IgG滴度水平隨免疫次數(shù)的增加而增加,呈現(xiàn)逐步升高的趨勢,在第3次免疫后達到最高,實驗組IgG滴度水平極顯著高于對照組(P0.01)。與PBS、佐劑、rGST(4T)和rGST(5x)對照組相比,實驗組卵囊排出量顯著低于對照組(P0.05),減卵率在73%~80%之間,而實驗組內(nèi)、對照組組內(nèi)差異不顯著(P0.05);試驗組開始排出卵囊時間延后,排出卵囊持續(xù)時間縮短。表明本實驗制備的3個亞單位疫苗可明顯地誘導小鼠產(chǎn)生特異性免疫應答,對BALB/c小鼠隱孢子蟲鼠基因型感染有較好的免疫保護作用。 本實驗對隱孢子蟲病的免疫預防進行了初步的探索,制備的3個核酸疫苗和3個亞單位疫苗對隱孢子蟲病的疫苗研究進行了有益的補充,為下一步深入研制具有高保護性的隱孢子蟲預防疫苗提供基礎。
[Abstract]:Cryptosporidiosism is a global zoonosis caused by Cryptosporidium spp.which is characterized by persistent diarrhea. Cryptosporidiosis has a wide range of host types and can parasitize mammals and birds. Reptiles, amphibians, fish, etc. More than 170 species of animals, including human beings. Cryptosporidiosis causes serious economic losses to animal husbandry and threatens the lives of people with immune deficiency. The research on Cryptosporidiosis has become one of the hotspots in the field of parasitology. There are no effective drugs to treat Cryptosporidiosis, so the immunological prevention of Cryptosporidiosis becomes more and more important. In this study, three major protective antigen genes of Cryptosporidium genetype P23 CP15 / 60 and oocystin wall protein CP41 were selected and cloned into eukaryotic expression vector pVAX1 by DNA recombination technique. Nucleic acid vaccine was prepared. BALB/c mice were immunized with three Cryptosporidium nucleic acid vaccines. The levels of IgG induced by the vaccine and the number of ovulation sac of the mice were determined. The titer of specific antibody IgG in serum of immunized mice increased gradually with the increase of immunization times, and reached the highest level after the third immunization. The titer of IgG in the experimental group was significantly higher than that in the control group (P 0.01), while in the experimental group, the titer of IgG was significantly higher than that of the control group (P 0.01). Compared with normal saline pVAX1 control group, the number of oocysts in the experimental group was significantly lower than that in the control group (P 0.05), and the oocyte reduction rate was between 85% and 100% in the control group, while in the experimental group, the number of oocysts in the control group was significantly lower than that in the control group. In the control group, the difference was not significant (P0.05), the time of oocyst excretion was delayed and the duration of oocyst excretion was shortened in the experimental group. The results showed that the three nucleic acid vaccines prepared in this study could induce the mice to produce a good specific immune response. It can protect the BALB/c mice against Cryptosporidium genetype infection. The recombinant plasmid pGEX-4T-1-P23 pGEX-5x-3-CP41 pET-28b (pET-28b) was successfully expressed in Escherichia coli. Western blot analysis showed that the three recombinant proteins had good immunogenicity. Three recombinant proteins were emulsified with Freund's adjuvant and subunit vaccine was prepared to immunize BALB/c mice. The results of indirect ELISA test showed that the recombinant protein rP23rCP41 rCP15 / 60 could induce a good humoral immune response in mice, and the titer of specific antibody IgG in serum of immunized mice increased with the increase of immunization times. The level of IgG titer in the experimental group was significantly higher than that in the control group (P 0.01). Compared with the PBSs, adjuvant rGST4T) and rGST5x), the egg sac excretion in the experimental group was significantly lower than that in the control group (P 0.05), and the egg reduction rate was between 7380% and 73.8%. In the experimental group, there was no significant difference between the control group and the control group (P 0.05), but the time of oocyst excretion in the experimental group was delayed and the duration of the oocyst excretion was shortened. The results showed that the three subunit vaccines prepared in this experiment could induce the specific immune response of the mice. It can protect the BALB/c mice against Cryptosporidium genetype infection. Three nucleic acid vaccines and three subunit vaccines were prepared to supplement the research of Cryptosporidiosis vaccine. It provides the foundation for the further development of the highly protective Cryptosporidium prophylaxis vaccine.
【學位授予單位】：中國農(nóng)業(yè)科學院
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392

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