表达结核分支杆菌Ag85B抗原重组牛布鲁氏菌S19疫苗株的构建
发布时间：2019-06-18 18:17
【摘要】： 布鲁氏菌病(brucellosis)是由布鲁氏菌(brucella)引起的一种全球性流行的人畜共患病。布鲁氏菌感染宿主广泛,目前已知宿主包括60多种家畜、禽及野生动物。B.abortus感染牛后主要引起母畜的流产、死胎、弱仔。牛结核病(Bovine Tuberculosis)是由牛分枝杆菌(Mycobacterium bovis)和结核分支杆菌(Mycobacterium tuberculosis)引起的另一种人兽共患的慢性消耗性传染病,可引起乳房炎,结核性胸膜炎,产乳率和乳质下降等症状。奶牛最为易感。结核病病牛是本病的主要传染源。牛布鲁氏菌病和牛结核不仅造成巨大经济损失,而且可以成为人类的感染源,构成严重公共卫生危害。 免疫接种是预防牛布鲁氏菌病的重要手段。B.abortus S19疫苗株长期应用于牛布鲁氏菌的免疫防制,其安全性和有效性得到充分证明。牛结核一直缺乏有效防制疫苗,其防控主要依赖被动的扑杀淘汰。近年来的研究进展表明,结核分支杆菌胞壁蛋白Ag85B抗原免疫动物可诱导有效保护性免疫反应。布鲁氏菌与结核分支杆菌均为间性胞内寄生的病原菌,二者致病与免疫反应机制相似;两种传染病经常共同持续感染牛群,特别是在发展中国家和边远落后地区。 bp26基因是布鲁氏菌基因缺失标记疫苗的候选靶标。bp26基因的缺失突变不影响疫苗的免疫保护效率,但其诱导的免疫反应可以通过血清学方法与自然感染相区别。为此,本研究以bp26基因作为重组靶位点,将牛结核分支杆菌ag85b基因的开放阅读框和卡那抗性基因(筛选基因)同源重组到S19疫苗株的基因组中,取代bp26基因,筛选出双交叉插入外源目的基因的重组疫苗株S19-ag85b。Western blot结果显示Ag85B抗原蛋白在重组疫苗株获得正确表达。该重组疫苗株在连续含有卡那霉素的TSA-YE平板上传20代,仍保持ag85b基因稳定遗传和表达。 重组疫苗株S19-ag85b与S19疫苗株分别以108CFU菌量腹腔途径菌株接种4-6周龄BALB/c小鼠,分别于接种后第1,3,6,9,12,15周扑杀小鼠,称量脾脏重量并检测脾脏荷菌情况。结果显示,随着时间推移,S19-ag85b和S19在小鼠脾脏内的数量持续减少,二者的荷菌量及消减趋势基本吻合,分别于15周左右基本清除。结果表明,S19-ag85b保持S19的低致病性生物安全特性。 进一步对重组疫苗株与野生疫苗株进行免疫保护效力的比较试验。S19-ag85b和S19分别以105CFU剂量接种4-6周龄BALB/c小鼠。免疫后6周,分别以马耳他种M28强毒株104CFU菌量和牛种544强毒株5×104CFU菌量,通过腹腔接种途径对免疫小鼠进行攻击试验。分别于攻毒后15扑杀小鼠,称量脾脏重量并检测脾脏荷菌情况。结果显示,无论M28还是544攻击后,S19-ag85b和S19免疫鼠之间脾脏重量及荷菌量均无显著差异,但都显著低于PBS接种对照小鼠。结果表明,重组疫苗株S19-ag85b保持S19疫苗的免疫保护效力。 本研究为牛布鲁氏菌病和结核的重组二联新型疫苗的探索研究奠定了初步的基础。
[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.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.1
本文编号：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.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.1
【引证文献】
相关硕士学位论文 前1条
1 高宇哲;virB12基因缺失马耳他型布氏杆菌M5-90疫苗株的研究[D];中国农业科学院;2011年
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