鉤端螺旋體Lig蛋白在倉(cāng)鼠動(dòng)物模型中的免疫效果評(píng)價(jià)
發(fā)布時(shí)間：2018-07-26 13:51
【摘要】：鉤端螺旋體病是一種全球分布重要的急性人獸共患病，由鉤端螺旋體屬致病性問(wèn)號(hào)鉤端螺旋體引起，在世界范圍內(nèi)廣泛流行，對(duì)人類的健康和畜牧業(yè)經(jīng)濟(jì)發(fā)展產(chǎn)生了嚴(yán)重的威脅。從事戶外職業(yè)，洪水暴發(fā)，與動(dòng)物密切接觸等因素，會(huì)增加此病的傳播風(fēng)險(xiǎn)，加大感染機(jī)率。鉤端螺旋體病有多種臨床癥狀，包括亞臨床，自限，無(wú)黃疸，發(fā)熱等，嚴(yán)重程度可到致死癥狀。鉤端螺旋體廣泛分布在熱帶，亞熱帶和溫帶的城市及農(nóng)村地區(qū)。在過(guò)去的幾十年中，流行病學(xué)模式的變化強(qiáng)調(diào)了該病作為一個(gè)公共健康問(wèn)題的重要性。每年報(bào)道的鉤體病例有50萬(wàn)余例，超過(guò)10%的病死率。 我國(guó)受鉤體病疫情困擾已久，令其防治成為一項(xiàng)迫在眉睫的任務(wù)，但已取得的研究進(jìn)展中對(duì)致病機(jī)制和宿主免疫反應(yīng)機(jī)理的研究還未十分清楚，普遍認(rèn)為鉤端螺旋體的感染過(guò)程主要是在各種膜表面分子（毒力因子）的幫助下侵入和粘附特定宿主細(xì)胞，形成持久性定殖和免疫逃避。由于鉤端螺旋體LPS碳水化合物的組成有差異性，使得鉤體菌型復(fù)雜，血清型多樣，超過(guò)280種。LPS作為鉤端螺旋體的一種免疫保護(hù)性抗原，已經(jīng)被用于研制疫苗，但商品化的滅活全菌苗或弱毒苗會(huì)產(chǎn)生一些毒性反應(yīng)，需要加強(qiáng)免疫來(lái)維持免疫時(shí)間，同時(shí)不同血清型的LPS成分不同，對(duì)其他血清型無(wú)法產(chǎn)生交叉保護(hù)作用。 現(xiàn)有研究表明，，致病性鉤體的一些外膜蛋白（OMP）如OmpL1、LipL32、LipL41、LigA、LigB、Lp1454等在各種血清型之間存在抗原保守性，因此免疫后可以對(duì)多種血清型起到交叉保護(hù)作用，作為候選亞單位疫苗表現(xiàn)出良好的發(fā)展前景，有可能替代全細(xì)胞滅活疫苗。 鉤端螺旋體通過(guò)表面粘附素作用結(jié)合到宿主細(xì)胞外基質(zhì)（ECM）引發(fā)感染。在鉤體外表面暴露的多種粘附素中，Lig蛋白含量豐富且較為保守，廣泛分布在各種致病性血清型中。本試驗(yàn)選擇問(wèn)號(hào)鉤端螺旋體秋季型臨4株基因組為模板，目標(biāo)基因?yàn)長(zhǎng)igAcon、LigAvar、LigBcen1，采用高保真PCR擴(kuò)增片段測(cè)序后，分別與原核表達(dá)載體pGEX-4T-1連接構(gòu)建重組表達(dá)載體pGEX-LigAcon，pGEX-LigAvar、pGEX-LigBcen1轉(zhuǎn)入大腸桿菌BL21(DE3)中誘導(dǎo)表達(dá)重組蛋白，將親和層析純化后的可溶性蛋白混合弗氏佐劑，作為免疫抗原皮下注射3~4周齡的倉(cāng)鼠，試驗(yàn)組為rLigAcon、rLigAvar、rLigBcen1、rLigAcon+rLigAvar，3周后加強(qiáng)免疫，對(duì)照組PBS組和GST標(biāo)簽組相同免疫程序。重組蛋白的免疫保護(hù)效果由倉(cāng)鼠模型中存活率和組織病理學(xué)切片觀察來(lái)評(píng)價(jià)。 試驗(yàn)結(jié)果顯示誘導(dǎo)表達(dá)的融合蛋白大小分別約為89kDa(LigAcon)、92kDa(LigAvar)、68kDa(LigBcen1)，試驗(yàn)組中免疫的重組蛋白LigA對(duì)急性感染鉤體的倉(cāng)鼠有明顯保護(hù)作用，與PBS對(duì)照組、GST標(biāo)簽組對(duì)比，差異顯著（P＜0.05）。病理學(xué)切片顯示存活的倉(cāng)鼠肝、腎、肺臟組織內(nèi)的炎癥反應(yīng)和出血狀況與對(duì)照組相比明顯改善。本研究中體現(xiàn)了Lig蛋白作為問(wèn)號(hào)鉤端螺旋體屬的特異性保護(hù)抗原的免疫保護(hù)效果，鉤端螺旋體LigA基因?qū)Φ挚雇豌^體感染能力較好，這些初步試驗(yàn)結(jié)果為鉤端螺旋體亞單位疫苗的進(jìn)一步研究奠定了基礎(chǔ)。
[Abstract]:Leptospirosis, an important acute zoonosis in the world, is caused by the leptospirosis, which is caused by the leptospirosis, which is widely popular in the world, and poses a serious threat to the health of human beings and the development of animal husbandry. The factors such as outdoor occupation, flood outbreaks, close contact with animals, and so on, will increase Leptospirosis has a variety of clinical symptoms, including subclinical, self limiting, non jaundice, fever, and fatal symptoms. Leptospirosis is widely distributed in tropical, subtropical and temperate cities and rural areas. In the past few decades, changes in epidemiological patterns have emphasized this The importance of disease as a public health problem is reported. There are more than 500 thousand cases of leptospirosis reported annually, with a mortality rate of more than 10%.
The prevention and control of leptospirosis has become an urgent task in China for a long time. However, the research progress on the pathogenesis and host immune response mechanism is not very clear. It is generally believed that the infection process of leptospirosis is mainly under the help of various membrane surface molecules (virulence factors) to invade and adhere. Specific host cells, forming persistent colonization and immune escape. Due to the difference in the composition of carbohydrates of the Leptospira LPS, the Leptospira type is complex and the serotypes are diverse, more than 280.LPS as an immuno protective antigen of the Leptospira, which has been used in the study of vaccines, but commercially inactivated total or weakly poisonous seedlings will be inactivated. To produce some toxic reactions, we need to strengthen the immune system to maintain the immune time. At the same time, the LPS components of different serotypes are different, and there is no cross protection to other serotypes.
Existing studies have shown that some of the outer membrane proteins (OMP) of pathogenic Leptospira such as OmpL1, LipL32, LipL41, LigA, LigB, Lp1454, and so on are conserved in various serotypes. Therefore, the immunization can play a cross protective effect on a variety of serotypes. As a candidate subunit vaccine, it has a good prospect of development and may replace whole cell death. Live vaccines.
The leptospirosis is associated with the extracellular matrix (ECM) of the host by adhesion to the host cell matrix (ECM). In a variety of adhesion elements exposed to the surface of the hook, the content of Lig protein is rich and conservative, widely distributed in various pathogenicity serotypes. This test selected the genome of the 4 strains of leptospirosis in the Leptospira as a template and the target gene. For LigAcon, LigAvar, LigBcen1, the recombinant expression vector pGEX-LigAcon, pGEX-LigAvar, pGEX-LigBcen1 was transferred into Escherichia coli BL21 (DE3) to express the recombinant protein after sequencing with high fidelity PCR amplification fragment, and pGEX-LigAvar, pGEX-LigBcen1 was transferred into Escherichia coli BL21 (DE3), and the soluble protein mixed with purified soluble protein after affinity chromatography was used as a free adjuvant. The pestilence antigen was injected subcutaneously for 3~4 weeks old hamster. The test group was rLigAcon, rLigAvar, rLigBcen1, rLigAcon+rLigAvar. After 3 weeks, the immunization was strengthened. The immune protection of the control group was the same as that of the PBS group and the GST label group. The immune protective effect of the recombinant protein was evaluated by the survival rate and histopathology section of the hamster model.
The results showed that the size of the induced fusion protein was about 89kDa (LigAcon), 92kDa (LigAvar), 68kDa (LigBcen1). The recombinant protein LigA in the test group had obvious protective effect on the hamster with acute infection of the leptospira. Compared with the PBS control group, the GST label group was significantly different (P < 0.05). The pathological section showed the surviving hamster liver and kidney. The inflammatory response and bleeding status in the lung tissue were significantly improved compared with those in the control group. In this study, the Lig protein was used as a specific protective antigen of the Leptospira interroponate, and the Leptospira LigA gene was better in resistance to the same type of Leptospira. These preliminary results were the Leptospira subunit. Further research has laid the foundation for the vaccine.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S855
本文編號(hào)：2146221
[Abstract]:Leptospirosis, an important acute zoonosis in the world, is caused by the leptospirosis, which is caused by the leptospirosis, which is widely popular in the world, and poses a serious threat to the health of human beings and the development of animal husbandry. The factors such as outdoor occupation, flood outbreaks, close contact with animals, and so on, will increase Leptospirosis has a variety of clinical symptoms, including subclinical, self limiting, non jaundice, fever, and fatal symptoms. Leptospirosis is widely distributed in tropical, subtropical and temperate cities and rural areas. In the past few decades, changes in epidemiological patterns have emphasized this The importance of disease as a public health problem is reported. There are more than 500 thousand cases of leptospirosis reported annually, with a mortality rate of more than 10%.
The prevention and control of leptospirosis has become an urgent task in China for a long time. However, the research progress on the pathogenesis and host immune response mechanism is not very clear. It is generally believed that the infection process of leptospirosis is mainly under the help of various membrane surface molecules (virulence factors) to invade and adhere. Specific host cells, forming persistent colonization and immune escape. Due to the difference in the composition of carbohydrates of the Leptospira LPS, the Leptospira type is complex and the serotypes are diverse, more than 280.LPS as an immuno protective antigen of the Leptospira, which has been used in the study of vaccines, but commercially inactivated total or weakly poisonous seedlings will be inactivated. To produce some toxic reactions, we need to strengthen the immune system to maintain the immune time. At the same time, the LPS components of different serotypes are different, and there is no cross protection to other serotypes.
Existing studies have shown that some of the outer membrane proteins (OMP) of pathogenic Leptospira such as OmpL1, LipL32, LipL41, LigA, LigB, Lp1454, and so on are conserved in various serotypes. Therefore, the immunization can play a cross protective effect on a variety of serotypes. As a candidate subunit vaccine, it has a good prospect of development and may replace whole cell death. Live vaccines.
The leptospirosis is associated with the extracellular matrix (ECM) of the host by adhesion to the host cell matrix (ECM). In a variety of adhesion elements exposed to the surface of the hook, the content of Lig protein is rich and conservative, widely distributed in various pathogenicity serotypes. This test selected the genome of the 4 strains of leptospirosis in the Leptospira as a template and the target gene. For LigAcon, LigAvar, LigBcen1, the recombinant expression vector pGEX-LigAcon, pGEX-LigAvar, pGEX-LigBcen1 was transferred into Escherichia coli BL21 (DE3) to express the recombinant protein after sequencing with high fidelity PCR amplification fragment, and pGEX-LigAvar, pGEX-LigBcen1 was transferred into Escherichia coli BL21 (DE3), and the soluble protein mixed with purified soluble protein after affinity chromatography was used as a free adjuvant. The pestilence antigen was injected subcutaneously for 3~4 weeks old hamster. The test group was rLigAcon, rLigAvar, rLigBcen1, rLigAcon+rLigAvar. After 3 weeks, the immunization was strengthened. The immune protection of the control group was the same as that of the PBS group and the GST label group. The immune protective effect of the recombinant protein was evaluated by the survival rate and histopathology section of the hamster model.
The results showed that the size of the induced fusion protein was about 89kDa (LigAcon), 92kDa (LigAvar), 68kDa (LigBcen1). The recombinant protein LigA in the test group had obvious protective effect on the hamster with acute infection of the leptospira. Compared with the PBS control group, the GST label group was significantly different (P < 0.05). The pathological section showed the surviving hamster liver and kidney. The inflammatory response and bleeding status in the lung tissue were significantly improved compared with those in the control group. In this study, the Lig protein was used as a specific protective antigen of the Leptospira interroponate, and the Leptospira LigA gene was better in resistance to the same type of Leptospira. These preliminary results were the Leptospira subunit. Further research has laid the foundation for the vaccine.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S855
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