霍亂弧菌O139的LPS-CTB結(jié)合疫苗的研究
發(fā)布時(shí)間：2018-11-16 13:52
【摘要】： 霍亂是一種烈性腸道傳染病,是由革蘭氏陰性霍亂弧菌引起的嚴(yán)重水樣腹瀉疾病。人類是霍亂弧菌的天然宿主,通過(guò)食入被霍亂弧菌污染的水或食物而感染。其感染的特點(diǎn)是嚴(yán)重腹瀉和嘔吐,由于水和電解質(zhì)的流失導(dǎo)致在數(shù)小時(shí)內(nèi)造成血容量減少性休克,代謝性酸中毒和鉀的缺乏。并發(fā)癥包括腎衰竭、動(dòng)脈閉塞、肺水腫、孕婦流產(chǎn)和兒童癲癇。在感染人群中的病死率可達(dá)20%以上,而全球每年約有12萬(wàn)人死于霍亂�；魜y的流行特點(diǎn)是爆發(fā)式,歷史上曾經(jīng)爆發(fā)過(guò)7次世界大流行,開始在數(shù)個(gè)不同地點(diǎn)同時(shí)發(fā)生,然后迅速傳播流行可累及許多國(guó)家并持續(xù)多年,尤其在非洲、亞洲和拉丁美洲等一些發(fā)展中國(guó)家較為嚴(yán)重。而控制它的有效途徑之一就是免疫預(yù)防接種,因此尋求研制一種安全有效適用于各種人群的霍亂疫苗極其重要。 已證實(shí)霍亂弧菌細(xì)胞壁上的脂多糖(LPS)既是毒力因子又是重要的保護(hù)性抗原,能誘導(dǎo)機(jī)體產(chǎn)生殺弧菌抗體。但是LPS分子量較小,免疫原性弱,且再次免疫后不能產(chǎn)生免疫加強(qiáng)效應(yīng)。并且多糖類抗原不能誘導(dǎo)嬰幼兒體內(nèi)產(chǎn)生具保護(hù)水平的抗體,但此年齡組又是細(xì)菌性傳染病發(fā)病的高危人群。霍亂毒素B亞單位(CTB)是霍亂毒素?zé)o毒的部分,具有良好的免疫原性,能產(chǎn)生重要的抗毒抗體。并且已經(jīng)證實(shí)CTB是一種很好的粘膜佐劑,能夠誘導(dǎo)機(jī)體產(chǎn)生粘膜免疫反應(yīng)。 本研究將CTB作為載體蛋白與LPS共價(jià)結(jié)合制備成結(jié)合疫苗,給多糖成分賦予“載體效應(yīng)”,既能增強(qiáng)LPS的免疫原性,使其由T細(xì)胞非依賴抗原成為T細(xì)胞依賴抗原,又使誘導(dǎo)的免疫反應(yīng)具有免疫記憶和加強(qiáng)效應(yīng),產(chǎn)生大量的殺菌抗體;而CTB本身又能產(chǎn)生高效價(jià)的抗毒抗體,這正是抗霍亂疫苗應(yīng)該包括的兩個(gè)方面。目的是制備一種安全有效的能針對(duì)各種人群的口服霍亂疫苗。 本研究首先采用熱酚水法從霍亂弧菌O139中提取純化LPS,最終獲得純度良好的LPS干粉。然后用1-氰-4-二甲基氨基吡啶四氟硼酸(CDAP)將提取的LPS活化后與載體蛋白CTB共價(jià)偶聯(lián),產(chǎn)物經(jīng)過(guò)Superdex 200凝膠過(guò)濾層析柱進(jìn)行純化,最終制備成高純度的LPS-CTB共價(jià)結(jié)合物。用制備的LPS-CTB結(jié)合物免疫小鼠,取小鼠血清和糞便進(jìn)行ELISA檢測(cè)。采用給以鹽水的小鼠做陰性對(duì)照,同時(shí)進(jìn)行單獨(dú)給LPS、單獨(dú)給CTB以及給rBS-WC疫苗的對(duì)照實(shí)驗(yàn)。實(shí)驗(yàn)結(jié)果證明,本研究制備的LPS-CTB結(jié)合物不僅能有效誘導(dǎo)小鼠機(jī)體產(chǎn)生血清抗LPS和抗CTB的IgG和IgA抗體,其中,抗LPS的IgG和IgA的抗體滴度分別達(dá)到1:970和1:1690,抗CTB的IgG和IgA抗體滴度更是高達(dá)1:163840。并且該結(jié)合物能很好的誘導(dǎo)小鼠的腸粘膜免疫系統(tǒng)產(chǎn)生對(duì)霍亂免疫極為重要的分泌型IgA抗體。另外在多次免疫后都能產(chǎn)生免疫加強(qiáng)效應(yīng)。說(shuō)明該結(jié)合物具有很好的免疫原性。 本研究初步制備的這種霍亂結(jié)合疫苗為霍亂及其他細(xì)菌性疾病的免疫預(yù)防提供了良好的基礎(chǔ)和思路。
[Abstract]:Cholera is a severe intestinal infectious disease caused by Gram-negative Vibrio cholerae. Humans are natural hosts of Vibrio cholerae, infected by ingestion of water or food contaminated by Vibrio cholerae. Its infection is characterized by severe diarrhea and vomiting, resulting in reduced blood volume shock, metabolic acidosis and potassium deficiency within hours due to the loss of water and electrolyte. Complications include renal failure, artery occlusion, pulmonary edema, miscarriage and childhood epilepsy. The fatality rate among infected populations can be more than 20%, and cholera kills about 120000 people worldwide every year. Cholera is characterized by explosive outbreaks, which have occurred seven times in the history of the world, began at the same time in several different locations, and then spread rapidly in many countries and for many years, especially in Africa, Some developing countries, such as Asia and Latin America, are more serious. One of the effective ways to control it is immunization, so it is very important to develop a safe and effective cholera vaccine for all kinds of people. It has been proved that lipopolysaccharide (LPS) on the cell wall of Vibrio cholerae is not only a virulence factor but also an important protective antigen, which can induce the body to produce vibrio antibody. However, the molecular weight of LPS is small and the immunogenicity is weak. And the polycarbohydrate antigen can not induce the infant to produce the protective level antibody, but this age group is the high risk population of bacterial infectious disease. Cholera toxin B subunit (CTB) is a nontoxic part of cholera toxin. It has good immunogenicity and can produce important anti-virus antibodies. And it has been proved that CTB is a good mucosal adjuvant, which can induce mucosal immune response. In this study, CTB was covalently combined with LPS as a carrier protein to prepare a conjugated vaccine. The conjugated vaccine was endowed with "carrier effect" to the polysaccharide component, which could enhance the immunogenicity of LPS and make it become T-cell dependent antigen from T-cell independent antigen. The induced immune response has immune memory and enhancement effect and produces a large number of bactericidal antibodies. CTB itself can produce high-titer anti-virus antibodies, which are two aspects of anti-cholera vaccine. The aim is to prepare a safe and effective oral cholera vaccine for various populations. In this study, LPS, was extracted and purified from Vibrio cholerae O139 by thermophenol water method. Finally, LPS dry powder with good purity was obtained. The extracted LPS was covalently coupled with the carrier protein CTB after activation with 1-cyano-4-dimethylpyridine tetrafluoroboric acid (CDAP). The product was purified by Superdex 200 gel filtration chromatography. Finally, high purity LPS-CTB covalent conjugate was prepared. Mice were immunized with the prepared LPS-CTB conjugate. Serum and feces were collected for ELISA detection. Mice treated with saline were used as negative control, and CTB and rBS-WC vaccine were given to LPS, alone. The results showed that the LPS-CTB conjugate could not only induce the production of IgG and IgA antibodies against LPS and CTB, but also the titers of IgG and IgA against LPS were 1: 970 and 1: 1690, respectively, in which the titers of IgG and IgA against LPS were 1: 970 and 1: 1690, respectively. The titers of IgG and IgA antibodies against CTB were as high as 1: 163840. The conjugate can well induce the intestinal mucosal immune system of mice to produce secretory IgA antibodies which are very important for cholera immunity. In addition, the immune enhancement effect can be produced after multiple immunization. It shows that the conjugate has good immunogenicity. The cholera conjugate vaccine which was prepared in this study provides a good basis and thought for the immune prevention of cholera and other bacterial diseases.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
[Abstract]:Cholera is a severe intestinal infectious disease caused by Gram-negative Vibrio cholerae. Humans are natural hosts of Vibrio cholerae, infected by ingestion of water or food contaminated by Vibrio cholerae. Its infection is characterized by severe diarrhea and vomiting, resulting in reduced blood volume shock, metabolic acidosis and potassium deficiency within hours due to the loss of water and electrolyte. Complications include renal failure, artery occlusion, pulmonary edema, miscarriage and childhood epilepsy. The fatality rate among infected populations can be more than 20%, and cholera kills about 120000 people worldwide every year. Cholera is characterized by explosive outbreaks, which have occurred seven times in the history of the world, began at the same time in several different locations, and then spread rapidly in many countries and for many years, especially in Africa, Some developing countries, such as Asia and Latin America, are more serious. One of the effective ways to control it is immunization, so it is very important to develop a safe and effective cholera vaccine for all kinds of people. It has been proved that lipopolysaccharide (LPS) on the cell wall of Vibrio cholerae is not only a virulence factor but also an important protective antigen, which can induce the body to produce vibrio antibody. However, the molecular weight of LPS is small and the immunogenicity is weak. And the polycarbohydrate antigen can not induce the infant to produce the protective level antibody, but this age group is the high risk population of bacterial infectious disease. Cholera toxin B subunit (CTB) is a nontoxic part of cholera toxin. It has good immunogenicity and can produce important anti-virus antibodies. And it has been proved that CTB is a good mucosal adjuvant, which can induce mucosal immune response. In this study, CTB was covalently combined with LPS as a carrier protein to prepare a conjugated vaccine. The conjugated vaccine was endowed with "carrier effect" to the polysaccharide component, which could enhance the immunogenicity of LPS and make it become T-cell dependent antigen from T-cell independent antigen. The induced immune response has immune memory and enhancement effect and produces a large number of bactericidal antibodies. CTB itself can produce high-titer anti-virus antibodies, which are two aspects of anti-cholera vaccine. The aim is to prepare a safe and effective oral cholera vaccine for various populations. In this study, LPS, was extracted and purified from Vibrio cholerae O139 by thermophenol water method. Finally, LPS dry powder with good purity was obtained. The extracted LPS was covalently coupled with the carrier protein CTB after activation with 1-cyano-4-dimethylpyridine tetrafluoroboric acid (CDAP). The product was purified by Superdex 200 gel filtration chromatography. Finally, high purity LPS-CTB covalent conjugate was prepared. Mice were immunized with the prepared LPS-CTB conjugate. Serum and feces were collected for ELISA detection. Mice treated with saline were used as negative control, and CTB and rBS-WC vaccine were given to LPS, alone. The results showed that the LPS-CTB conjugate could not only induce the production of IgG and IgA antibodies against LPS and CTB, but also the titers of IgG and IgA against LPS were 1: 970 and 1: 1690, respectively, in which the titers of IgG and IgA against LPS were 1: 970 and 1: 1690, respectively. The titers of IgG and IgA antibodies against CTB were as high as 1: 163840. The conjugate can well induce the intestinal mucosal immune system of mice to produce secretory IgA antibodies which are very important for cholera immunity. In addition, the immune enhancement effect can be produced after multiple immunization. It shows that the conjugate has good immunogenicity. The cholera conjugate vaccine which was prepared in this study provides a good basis and thought for the immune prevention of cholera and other bacterial diseases.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
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