重組全長(zhǎng)副肌球蛋白在小鼠與水牛中杭日本血吸蟲(chóng)感染的保護(hù)力研究
發(fā)布時(shí)間：2019-06-22 15:59
【摘要】：日本血吸蟲(chóng)病是一種嚴(yán)重危害我國(guó)人民身體健康的寄生蟲(chóng)病。根據(jù)2006年的調(diào)查資料,我國(guó)現(xiàn)有血吸蟲(chóng)病人51萬(wàn)多,有6000多萬(wàn)人口受到血吸蟲(chóng)病的威脅。近年,我國(guó)血吸蟲(chóng)病防治以人畜同步化療為主,消滅易感地帶釘螺和加強(qiáng)健康教育為輔的綜合防治措施。多年的防治實(shí)踐證明,這一綜合措施雖然有效,但效果難以鞏固,不僅受到生態(tài)環(huán)境和防治力度變化的挑戰(zhàn),而且面臨著人群化療后再感染的困擾。單純依賴化療難以達(dá)到防治目標(biāo),鑒于疫苗在許多傳染病控制中無(wú)可比擬的作用,研發(fā)血吸蟲(chóng)病疫苗,獲取化療短期效應(yīng)與疫苗接種誘導(dǎo)的長(zhǎng)期免疫預(yù)防作用相結(jié)合的雙重效果,將是未來(lái)有效防治血吸蟲(chóng)病的主要奮斗目標(biāo)。在我國(guó),耕牛作為主要的保蟲(chóng)宿主和傳染源在血吸蟲(chóng)病的流行過(guò)程中起著重要的傳播作用。發(fā)展適合耕牛等主要保蟲(chóng)宿主的動(dòng)物疫苗對(duì)血吸蟲(chóng)病的控制具有重要意義,此舉不僅可通過(guò)控制傳播降低人群血吸蟲(chóng)病流行率,而且為人用血吸蟲(chóng)病疫苗的研制提供一定的理論依據(jù)。 血吸蟲(chóng)病疫苗的研究歷時(shí)70余年,大體上經(jīng)歷了死疫苗、減毒活疫苗、基因工程疫苗、核酸疫苗研究階段。作為血吸蟲(chóng)病疫苗候選分子之一的副肌球蛋白,在曼氏血吸蟲(chóng)或日本血吸蟲(chóng)的動(dòng)物模型中均獲得了不錯(cuò)的保護(hù)效果。相比其他血吸蟲(chóng)病疫苗候選分子,副肌球蛋白誘生的保護(hù)力較為突出,蟲(chóng)源性或重組副肌球蛋白分子片段,以及全長(zhǎng)的DNA疫苗在小鼠、水牛等動(dòng)物實(shí)驗(yàn)中的減蟲(chóng)率可以達(dá)到60%以上。然而,許多保護(hù)力實(shí)驗(yàn)中所用的副肌球蛋白為成蟲(chóng)的生化純化產(chǎn)物,或大腸桿菌中的重組表達(dá)產(chǎn)物。生化純化需要大量的蟲(chóng)源,而重組表達(dá)一直未能解決全長(zhǎng)片段純化困難、得率偏低的問(wèn)題,長(zhǎng)期以來(lái),成為阻止其作為潛在疫苗快速推進(jìn)到大規(guī)模研究,尤其是應(yīng)用于大動(dòng)物實(shí)驗(yàn)的障礙。 免疫佐劑在調(diào)節(jié)免疫和提高疫苗效價(jià)方面具有重要作用,是疫苗獲得理想效果的一條重要途徑。良好的免疫佐劑不僅應(yīng)該能夠加強(qiáng)抗原的免疫原性和免疫保護(hù)效果,同時(shí)也應(yīng)該不引起過(guò)敏反應(yīng)或激發(fā)自身免疫反應(yīng)等副作用。本研究使用的重組副肌球蛋白分子表達(dá)于大腸桿菌,采用改進(jìn)的純化方法后純度能夠達(dá)到95%以上,適于大規(guī)模制備與研究用途,重組蛋白結(jié)構(gòu)和生物學(xué)特性與蟲(chóng)源性副肌球蛋白一致;先后使用多種佐劑與重組副肌球蛋白聯(lián)合免疫小鼠,尋求最佳的疫苗組合;探索了ISA206佐劑與重組副肌球蛋白聯(lián)合免疫水牛后對(duì)日本血吸蟲(chóng)感染的抵抗能力,以此來(lái)尋求適合于家畜(主要為耕牛)等保蟲(chóng)宿主的副肌球蛋白疫苗組合。 本研究獲得如下結(jié)果: 1.大規(guī)模重組表達(dá)的全長(zhǎng)副肌球蛋白分子的純度達(dá)到95%以上,其中LPS含量為0.07EU/ml,與蟲(chóng)源性副肌球蛋白分子具有相同的二級(jí)結(jié)構(gòu)和生物學(xué)功能,后者包括與免疫球蛋白的結(jié)合特性以及與膠原物質(zhì)的結(jié)合特性。 2.在昆明鼠與C57BL/6小鼠模型中,rSj97-Alhydrogel未能誘導(dǎo)理想的保護(hù)力。 3.在C57BL/6小鼠模型中,就減蟲(chóng)率與減卵率而言,與rSj97-FA組和rSj97-CpG-ODN組相比,rSj97-ISA206組對(duì)C57BL/6小鼠具有顯著的保護(hù)效果,分別為45.3%(P0.01)與35.4%(P0.05),該結(jié)果提示,rSj97-ISA206具良好的抗日本血吸蟲(chóng)感染的保護(hù)效果。 4.在水牛模型中,rSj97-ISA206組相比ISA206佐劑組,所獲減蟲(chóng)率為28.01%(P=0.443)、糞便蟲(chóng)卵減少率為28.03%(P=0.584);相比感染對(duì)照組,減蟲(chóng)率為16.08%(P=0.742)、肝臟減卵率為3.64%(P=0.963)、糞便減卵率為38.70%(P=0.462)。 5. rSj97在C57BL/6小鼠體內(nèi)誘生出高水平特異性IgG2a與IgG1抗體,提示rSj97能誘導(dǎo)特異性Th1和Th2特征抗體亞類的產(chǎn)生,可能與誘導(dǎo)C57BL/6小鼠的保護(hù)力存在一定相關(guān)性。 6. rSj97免疫水牛后能誘生出高水平特異性IgG2與IgG1抗體,并誘生出Th1型優(yōu)勢(shì)應(yīng)答,同時(shí)引起的體液免疫與細(xì)胞免疫應(yīng)答可能是水牛產(chǎn)生保護(hù)力的因素之一。 本研究進(jìn)一步豐富了副肌球蛋白的保護(hù)效果研究,尤其是在大動(dòng)物體內(nèi)的研究,為構(gòu)建適合于水牛的副肌球蛋白疫苗組合提供了重要的基礎(chǔ)資料。
[Abstract]:Schistosomiasis japonica is a parasitic disease that is seriously harmful to the health of our people. According to the survey data of 2006, more than 550,000 of the present schistosomiasis has been made in China, and more than 60 million people have been threatened by the schistosomiasis. In recent years, the prevention and control of schistosomiasis in China is the main control measures for the prevention and control of the synchronous chemotherapy of the livestock and the livestock and the elimination of the Oncomelania hupensis and the strengthening of the health education. The years of prevention and control have proved that this comprehensive measure, though effective, is difficult to consolidate, not only is the challenge of the change of the ecological environment and the prevention and control, but also the problem of re-infection after the chemotherapy of the population. It is difficult to achieve the goal of prevention and cure by relying on chemotherapy alone, and in view of the incomparable role of the vaccine in the control of many infectious diseases, the double effects of the combination of the short-term effect of chemotherapy and the long-term immune prevention induced by the vaccine inoculation are obtained, It will be the main goal of the future effective control of the schistosomiasis. In our country, the cultivation of cattle as the main insect-protecting host and the source of infection plays an important role in the epidemic of schistosomiasis. The development of the animal vaccine suitable for the host of the main insect-protecting host such as the cultivated cattle is of great significance to the control of the schistosomiasis, which not only can reduce the prevalence rate of the schistosomiasis in the population by controlling the transmission, but also provides a certain theoretical basis for the development of the schistosomiasis vaccine for human use. The study of schistosomiasis vaccine has lasted for more than 70 years, and has generally undergone the study of dead vaccine, attenuated live vaccine, genetic engineering vaccine and nucleic acid vaccine. para. as the paramyosin of one of the candidate molecules of the schistosomiasis vaccine, a good protective effect is obtained in the animal models of the Schistosoma mansoni or the Schistosoma japonicum Compared with other schistosomiasis vaccine candidate molecules, the protection force of the paramyosin induced by the paramyosin is more prominent, the insect-derived or recombinant paramyosin molecule fragment, and the full-length DNA vaccine can reach 60% in animal experiments such as mice and buffalo. However, the secondary myosin used in many of the protection experiments is the biochemical purification product of the adult, or the recombinant expression in E. coli The biochemical purification requires a large number of entomogenous sources, and the recombinant expression has been unable to solve the problems of difficult purification and low yield of the full-length fragment, and has long been a barrier to the rapid advance of the full-length fragment to a large-scale study, in particular to a large animal experiment The immune adjuvant plays an important role in regulating immunity and improving the titer of the vaccine, and is a weight of the ideal effect of the vaccine. The good immune adjuvant not only can enhance the immunogenicity and the immune protective effect of the antigen, but also does not cause an allergic reaction or stimulate the self-immune reaction, and the like. The recombinant paramyosin molecule used in the study is expressed in E. coli, and the purity can reach more than 95% after the improved purification method, and is suitable for large-scale preparation and research purposes, the structure and biological characteristics of the recombinant protein and the worm-derived submuscle ball A combination of adjuvant and recombinant paramyosin was used to immunize mice, to find the best combination of the vaccine, and to explore the effect of the adjuvant of ISA206 and the recombinant paramyosin on the infection of Schistosoma japonicum. The ability to search for paramyosin phytophthora, which is suitable for the host of insect-protecting, such as cattle, and the like The combination of the seedlings. The present study was The results are as follows:1. The purity of the full-length paramyosin molecule expressed by the large-scale recombinant expression is over 95%, wherein the content of the LPS is 0.07 EU/ ml and the same secondary junction with the insect-derived paramyosin molecule Construction and biological functions, which include binding properties to immunoglobulins and with collagen the binding properties of the substance.2. In the Kunming mice and the C57BL/6 mouse model, rSj97-Hydrogel did not 3. Compared with rSj97-FA group and rSj97-CpG-ODN group, the rSj97-ISA206 group had significant protective effect on C57BL/6 mice compared with rSj97-FA group and rSj97-CpG-ODN group in the C57BL/6 mouse model. 4. Compared with the control group, the deinsectization rate was 16.08% (P = 0.742), the egg reduction rate of the liver was 3.64% (P = 0.963), and the egg reduction rate was 38. ..70% (P = 0.462). rSj97 induced a high level of specific IgG2a and IgG1 antibody in C57BL/6 mice, suggesting that rSj97 can induce the generation of specific Th1 and Th2-specific antibody subclasses, possibly related to the induction of C57BL/6 mice. 6. A high level of specific IgG2 and IgG1 antibodies can be induced by the rSj97 immune buffalo, and a Th1-type dominant response is induced, and humoral immunity and cellular immunity are induced. The response may be one of the factors that the buffalo produces a protective force. The study further enriched the study of the protective effect of paramyosin, especially in large animals, in order to build a suitable water buffalo
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392
本文編號(hào)：2504738
[Abstract]:Schistosomiasis japonica is a parasitic disease that is seriously harmful to the health of our people. According to the survey data of 2006, more than 550,000 of the present schistosomiasis has been made in China, and more than 60 million people have been threatened by the schistosomiasis. In recent years, the prevention and control of schistosomiasis in China is the main control measures for the prevention and control of the synchronous chemotherapy of the livestock and the livestock and the elimination of the Oncomelania hupensis and the strengthening of the health education. The years of prevention and control have proved that this comprehensive measure, though effective, is difficult to consolidate, not only is the challenge of the change of the ecological environment and the prevention and control, but also the problem of re-infection after the chemotherapy of the population. It is difficult to achieve the goal of prevention and cure by relying on chemotherapy alone, and in view of the incomparable role of the vaccine in the control of many infectious diseases, the double effects of the combination of the short-term effect of chemotherapy and the long-term immune prevention induced by the vaccine inoculation are obtained, It will be the main goal of the future effective control of the schistosomiasis. In our country, the cultivation of cattle as the main insect-protecting host and the source of infection plays an important role in the epidemic of schistosomiasis. The development of the animal vaccine suitable for the host of the main insect-protecting host such as the cultivated cattle is of great significance to the control of the schistosomiasis, which not only can reduce the prevalence rate of the schistosomiasis in the population by controlling the transmission, but also provides a certain theoretical basis for the development of the schistosomiasis vaccine for human use. The study of schistosomiasis vaccine has lasted for more than 70 years, and has generally undergone the study of dead vaccine, attenuated live vaccine, genetic engineering vaccine and nucleic acid vaccine. para. as the paramyosin of one of the candidate molecules of the schistosomiasis vaccine, a good protective effect is obtained in the animal models of the Schistosoma mansoni or the Schistosoma japonicum Compared with other schistosomiasis vaccine candidate molecules, the protection force of the paramyosin induced by the paramyosin is more prominent, the insect-derived or recombinant paramyosin molecule fragment, and the full-length DNA vaccine can reach 60% in animal experiments such as mice and buffalo. However, the secondary myosin used in many of the protection experiments is the biochemical purification product of the adult, or the recombinant expression in E. coli The biochemical purification requires a large number of entomogenous sources, and the recombinant expression has been unable to solve the problems of difficult purification and low yield of the full-length fragment, and has long been a barrier to the rapid advance of the full-length fragment to a large-scale study, in particular to a large animal experiment The immune adjuvant plays an important role in regulating immunity and improving the titer of the vaccine, and is a weight of the ideal effect of the vaccine. The good immune adjuvant not only can enhance the immunogenicity and the immune protective effect of the antigen, but also does not cause an allergic reaction or stimulate the self-immune reaction, and the like. The recombinant paramyosin molecule used in the study is expressed in E. coli, and the purity can reach more than 95% after the improved purification method, and is suitable for large-scale preparation and research purposes, the structure and biological characteristics of the recombinant protein and the worm-derived submuscle ball A combination of adjuvant and recombinant paramyosin was used to immunize mice, to find the best combination of the vaccine, and to explore the effect of the adjuvant of ISA206 and the recombinant paramyosin on the infection of Schistosoma japonicum. The ability to search for paramyosin phytophthora, which is suitable for the host of insect-protecting, such as cattle, and the like The combination of the seedlings. The present study was The results are as follows:1. The purity of the full-length paramyosin molecule expressed by the large-scale recombinant expression is over 95%, wherein the content of the LPS is 0.07 EU/ ml and the same secondary junction with the insect-derived paramyosin molecule Construction and biological functions, which include binding properties to immunoglobulins and with collagen the binding properties of the substance.2. In the Kunming mice and the C57BL/6 mouse model, rSj97-Hydrogel did not 3. Compared with rSj97-FA group and rSj97-CpG-ODN group, the rSj97-ISA206 group had significant protective effect on C57BL/6 mice compared with rSj97-FA group and rSj97-CpG-ODN group in the C57BL/6 mouse model. 4. Compared with the control group, the deinsectization rate was 16.08% (P = 0.742), the egg reduction rate of the liver was 3.64% (P = 0.963), and the egg reduction rate was 38. ..70% (P = 0.462). rSj97 induced a high level of specific IgG2a and IgG1 antibody in C57BL/6 mice, suggesting that rSj97 can induce the generation of specific Th1 and Th2-specific antibody subclasses, possibly related to the induction of C57BL/6 mice. 6. A high level of specific IgG2 and IgG1 antibodies can be induced by the rSj97 immune buffalo, and a Th1-type dominant response is induced, and humoral immunity and cellular immunity are induced. The response may be one of the factors that the buffalo produces a protective force. The study further enriched the study of the protective effect of paramyosin, especially in large animals, in order to build a suitable water buffalo
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392
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