表達(dá)結(jié)核分支桿菌Ag85B抗原重組牛布魯氏菌S19疫苗株的構(gòu)建
發(fā)布時(shí)間：2019-06-18 18:17
【摘要】： 布魯氏菌病(brucellosis)是由布魯氏菌(brucella)引起的一種全球性流行的人畜共患病。布魯氏菌感染宿主廣泛,目前已知宿主包括60多種家畜、禽及野生動(dòng)物。B.abortus感染牛后主要引起母畜的流產(chǎn)、死胎、弱仔。牛結(jié)核病(Bovine Tuberculosis)是由牛分枝桿菌(Mycobacterium bovis)和結(jié)核分支桿菌(Mycobacterium tuberculosis)引起的另一種人獸共患的慢性消耗性傳染病,可引起乳房炎,結(jié)核性胸膜炎,產(chǎn)乳率和乳質(zhì)下降等癥狀。奶牛最為易感。結(jié)核病病牛是本病的主要傳染源。牛布魯氏菌病和牛結(jié)核不僅造成巨大經(jīng)濟(jì)損失,而且可以成為人類的感染源,構(gòu)成嚴(yán)重公共衛(wèi)生危害。 免疫接種是預(yù)防牛布魯氏菌病的重要手段。B.abortus S19疫苗株長(zhǎng)期應(yīng)用于牛布魯氏菌的免疫防制,其安全性和有效性得到充分證明。牛結(jié)核一直缺乏有效防制疫苗,其防控主要依賴被動(dòng)的撲殺淘汰。近年來(lái)的研究進(jìn)展表明,結(jié)核分支桿菌胞壁蛋白Ag85B抗原免疫動(dòng)物可誘導(dǎo)有效保護(hù)性免疫反應(yīng)。布魯氏菌與結(jié)核分支桿菌均為間性胞內(nèi)寄生的病原菌,二者致病與免疫反應(yīng)機(jī)制相似;兩種傳染病經(jīng)常共同持續(xù)感染牛群,特別是在發(fā)展中國(guó)家和邊遠(yuǎn)落后地區(qū)。 bp26基因是布魯氏菌基因缺失標(biāo)記疫苗的候選靶標(biāo)。bp26基因的缺失突變不影響疫苗的免疫保護(hù)效率,但其誘導(dǎo)的免疫反應(yīng)可以通過(guò)血清學(xué)方法與自然感染相區(qū)別。為此,本研究以bp26基因作為重組靶位點(diǎn),將牛結(jié)核分支桿菌ag85b基因的開放閱讀框和卡那抗性基因(篩選基因)同源重組到S19疫苗株的基因組中,取代bp26基因,篩選出雙交叉插入外源目的基因的重組疫苗株S19-ag85b。Western blot結(jié)果顯示Ag85B抗原蛋白在重組疫苗株獲得正確表達(dá)。該重組疫苗株在連續(xù)含有卡那霉素的TSA-YE平板上傳20代,仍保持ag85b基因穩(wěn)定遺傳和表達(dá)。 重組疫苗株S19-ag85b與S19疫苗株分別以108CFU菌量腹腔途徑菌株接種4-6周齡BALB/c小鼠,分別于接種后第1,3,6,9,12,15周撲殺小鼠,稱量脾臟重量并檢測(cè)脾臟荷菌情況。結(jié)果顯示,隨著時(shí)間推移,S19-ag85b和S19在小鼠脾臟內(nèi)的數(shù)量持續(xù)減少,二者的荷菌量及消減趨勢(shì)基本吻合,分別于15周左右基本清除。結(jié)果表明,S19-ag85b保持S19的低致病性生物安全特性。 進(jìn)一步對(duì)重組疫苗株與野生疫苗株進(jìn)行免疫保護(hù)效力的比較試驗(yàn)。S19-ag85b和S19分別以105CFU劑量接種4-6周齡BALB/c小鼠。免疫后6周,分別以馬耳他種M28強(qiáng)毒株104CFU菌量和牛種544強(qiáng)毒株5×104CFU菌量,通過(guò)腹腔接種途徑對(duì)免疫小鼠進(jìn)行攻擊試驗(yàn)。分別于攻毒后15撲殺小鼠,稱量脾臟重量并檢測(cè)脾臟荷菌情況。結(jié)果顯示,無(wú)論M28還是544攻擊后,S19-ag85b和S19免疫鼠之間脾臟重量及荷菌量均無(wú)顯著差異,但都顯著低于PBS接種對(duì)照小鼠。結(jié)果表明,重組疫苗株S19-ag85b保持S19疫苗的免疫保護(hù)效力。 本研究為牛布魯氏菌病和結(jié)核的重組二聯(lián)新型疫苗的探索研究奠定了初步的基礎(chǔ)。
[Abstract]:Brucellosis (brucellosis) is a worldwide zoonosis caused by brucellosis (brucella). Brucellosis infection has a wide range of hosts, including more than 60 kinds of livestock, poultry and wild animals. B. abortus infection mainly causes abortion, stillbirth and weak offspring of female animals. Bovine tuberculosis (Bovine Tuberculosis) is another chronic human and animal infectious disease caused by Mycobacterium bovis (Mycobacterium bovis) and Mycobacterium tuberculosis (Mycobacterium tuberculosis). It can cause mastitis, tuberculosis pleurisy, milk production rate and milk quality decline. Cows are the most susceptible. Tuberculosis cattle are the main source of infection of the disease. Bovine brucellosis and bovine tuberculosis not only cause huge economic losses, but also become the source of human infection and constitute serious public health hazards. Immunization is an important means to prevent bovine brucellosis. B.abortus S19 vaccine strain has been used in the immune control of brucellosis for a long time, and its safety and effectiveness have been fully proved. Bovine tuberculosis has been lack of effective prevention and control vaccine, its prevention and control mainly rely on passive culling elimination. Recent research progress has shown that Mycobacterium tuberculosis cell wall protein Ag85B antigen can induce effective protective immune response in animals. Brucellosis and Mycobacterium tuberculosis are both intracellular parasitic pathogens, and the pathogenesis and immune response mechanism of the two infectious diseases are similar. The two infectious diseases often continue to infect cattle, especially in developing countries and remote and backward areas. Bp26 gene is a candidate target for Brucella gene deletion marker vaccine. The deletion mutation of bp26 gene does not affect the immune protection efficiency of the vaccine, but its induced immune response can be distinguished from natural infection by serological method. In this study, using bp26 gene as recombinant target site, the open reading frame and kana resistance gene (screening gene) of ag85b gene of Mycobacterium tuberculosis were homologous recombined into the genome of S19 vaccine strain to replace bp26 gene. The recombinant vaccine strain S19-ag85b.Western blot with double cross insertion of foreign target gene showed that Ag85B antigen protein was correctly expressed in the recombinant vaccine strain. The recombinant vaccine strain was uploaded to TSA-YE plate containing kanamycin for 20 generations, and the ag85b gene was still inherited and expressed stably. The recombinant vaccine strains S19-ag85b and S19 vaccine strains were inoculated with 108CFU strain at the age of 4 and 6 weeks, respectively. The mice were culled at the 1st, 3rd, 9th, 12th and 15th week after inoculation. the weight of spleen was weighed and the load of spleen was detected. The results showed that the number of S19-ag85b and S19 in the spleen of mice continued to decrease with the passage of time, and the amount of bacteria and the decreasing trend of S19 and S19 were basically consistent with each other, and were basically cleared at about 15 weeks. The results showed that S19-ag85b maintained the low pathogenicity biosafety of S19. The immune protective efficacy of the recombinant vaccine strain was compared with that of the wild vaccine strain. S19-ag85b and S19 were inoculated with 105CFU for 4 weeks and 6 weeks old BALB/c mice, respectively. Six weeks after immunization, the immunized mice were challenged by intraabdominal vaccination with the amount of 104CFU strain of Maltese M28 virulent strain and 5 脳 104CFU strain of bovine strain 544, respectively. The mice were killed at 15 days after challenge, the weight of spleen was weighed and the load of spleen was detected. The results showed that there was no significant difference in spleen weight and bacterial load between S19-ag85b and S19 immunized mice, but both of them were significantly lower than those of control mice inoculated with PBS. The results showed that the recombinant vaccine strain S19-ag85b maintained the immune protective effect of S19 vaccine. This study laid a preliminary foundation for the exploration of recombinant double vaccine for brucellosis and tuberculosis.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392.1
本文編號(hào)：2501689
[Abstract]:Brucellosis (brucellosis) is a worldwide zoonosis caused by brucellosis (brucella). Brucellosis infection has a wide range of hosts, including more than 60 kinds of livestock, poultry and wild animals. B. abortus infection mainly causes abortion, stillbirth and weak offspring of female animals. Bovine tuberculosis (Bovine Tuberculosis) is another chronic human and animal infectious disease caused by Mycobacterium bovis (Mycobacterium bovis) and Mycobacterium tuberculosis (Mycobacterium tuberculosis). It can cause mastitis, tuberculosis pleurisy, milk production rate and milk quality decline. Cows are the most susceptible. Tuberculosis cattle are the main source of infection of the disease. Bovine brucellosis and bovine tuberculosis not only cause huge economic losses, but also become the source of human infection and constitute serious public health hazards. Immunization is an important means to prevent bovine brucellosis. B.abortus S19 vaccine strain has been used in the immune control of brucellosis for a long time, and its safety and effectiveness have been fully proved. Bovine tuberculosis has been lack of effective prevention and control vaccine, its prevention and control mainly rely on passive culling elimination. Recent research progress has shown that Mycobacterium tuberculosis cell wall protein Ag85B antigen can induce effective protective immune response in animals. Brucellosis and Mycobacterium tuberculosis are both intracellular parasitic pathogens, and the pathogenesis and immune response mechanism of the two infectious diseases are similar. The two infectious diseases often continue to infect cattle, especially in developing countries and remote and backward areas. Bp26 gene is a candidate target for Brucella gene deletion marker vaccine. The deletion mutation of bp26 gene does not affect the immune protection efficiency of the vaccine, but its induced immune response can be distinguished from natural infection by serological method. In this study, using bp26 gene as recombinant target site, the open reading frame and kana resistance gene (screening gene) of ag85b gene of Mycobacterium tuberculosis were homologous recombined into the genome of S19 vaccine strain to replace bp26 gene. The recombinant vaccine strain S19-ag85b.Western blot with double cross insertion of foreign target gene showed that Ag85B antigen protein was correctly expressed in the recombinant vaccine strain. The recombinant vaccine strain was uploaded to TSA-YE plate containing kanamycin for 20 generations, and the ag85b gene was still inherited and expressed stably. The recombinant vaccine strains S19-ag85b and S19 vaccine strains were inoculated with 108CFU strain at the age of 4 and 6 weeks, respectively. The mice were culled at the 1st, 3rd, 9th, 12th and 15th week after inoculation. the weight of spleen was weighed and the load of spleen was detected. The results showed that the number of S19-ag85b and S19 in the spleen of mice continued to decrease with the passage of time, and the amount of bacteria and the decreasing trend of S19 and S19 were basically consistent with each other, and were basically cleared at about 15 weeks. The results showed that S19-ag85b maintained the low pathogenicity biosafety of S19. The immune protective efficacy of the recombinant vaccine strain was compared with that of the wild vaccine strain. S19-ag85b and S19 were inoculated with 105CFU for 4 weeks and 6 weeks old BALB/c mice, respectively. Six weeks after immunization, the immunized mice were challenged by intraabdominal vaccination with the amount of 104CFU strain of Maltese M28 virulent strain and 5 脳 104CFU strain of bovine strain 544, respectively. The mice were killed at 15 days after challenge, the weight of spleen was weighed and the load of spleen was detected. The results showed that there was no significant difference in spleen weight and bacterial load between S19-ag85b and S19 immunized mice, but both of them were significantly lower than those of control mice inoculated with PBS. The results showed that the recombinant vaccine strain S19-ag85b maintained the immune protective effect of S19 vaccine. This study laid a preliminary foundation for the exploration of recombinant double vaccine for brucellosis and tuberculosis.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392.1
【引證文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 高宇哲;virB12基因缺失馬耳他型布氏桿菌M5-90疫苗株的研究[D];中國(guó)農(nóng)業(yè)科學(xué)院;2011年
，本文編號(hào)：2501689
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