| Brucellosis, which is caused by Brucella, is one of the most important bacterial zoonosis endemic in many countries. Data from WHO showed that the economic loss caused by Brucellosis is over 3 billions. The economic loss in Xinjiang and Qinhai of our country is about one hundred million each year. And until now, the Malta Fever and chronic infection, abortion and sterility in ruminant can not be cured. Moreover, this pathogen has been used to develop biological weapons by terrorists. Therefore, Brucellosis is a severe threaten to the economy construction, public health and the national security to our courtry. In the middle of 1980s, a reemergence of brucellosis risen, and appeared in 40 to 50 countries and areas .The main epidemic regions include Africa,Asia,south America and parts of Europe. For our country, it is reported that the morbidity of brucellosis has beyond the peak in the histry, provoking great attentions from the government.Brucella spp. is the pathogen of brucellosis. Brucellae are facultative intracellular, gram-negative coccobacilli without spore, flagella and capsule. They are oxidase, catalase and urease positive. Brucella has breath metabolism and special needs for oxygen. Its most suitable growth temperature is 37℃and pH value 6.6-7.4. Six species are recognized within the genus Brucella: Brucella abortus, Brucella melitensis, Brucella suis, Brucella ovis, Brucella canis, and Brucella neotomae . This classification is mainly based on the differences in host preference and pathogenicity. Among these, B. melitensis, B. abortus and B. suis are virulent to humans. The broad spectrum of Brucella isolates has recently been extended to marine mammals. This indicates that there may be some different types of Brucella and some do not infect human. B. melitensis, B. abortus and B. suis are prevalent in our country. B. melitensis and B. abortus mainly cause abortion in female and sterility in male. In some countries, B.ovis is the main for abortion and sterility of sheep. Human is infected by Brucella during interaction with infected animals. Pathogenic Brucella can invade host through skin abrasions. It has quite intense invasiing, multiplying and diffusing capacity in the host, which related to the production of alidase and its capsule antiphagocytosis. Once invaded into the host, pathogenic Brucella is internalized by phagocyte. Because of the caspsule, brucella can survive the phagocytosis and replicate in host cells. The bacteria enter regional lymph node and multiply there. When the cell number reached a threshold, they enter into blood vessel and interact with host defence system, which results in bacteremia. At this stage, patients manifect fever, malaise and other clinical symptoms. Then the bacteria invade into spleen, liver and marrow cells through blood. As the decrease of the bacteria, the body temperature descends accordingly. The human disease recurs when the bacteria invade into blood again, and at this time body temperature increase, this phenomena is called Malta Fever. Being mainly located in host cells, Brucella spp. can escape the host immune response and drug treatment. And therefore, it is hard to cure completely and easily turn into chronic infection, which may exist for 1-20 years.One important characteristic of Brucellosis is that once infected, it is hard to cure completely. Therefore, prevention is the most important means to control it. Vaccination is most efficient means for brucella prevention. For the present, there are mainly five live attenuated vaccines for animal: S19,104M,S2,M5 and RB51, and killed two vaccine: 45/20 and H38. Two attenuated live strains 19-B and 104M are applied for human. Among the vaccines, live attenuated strains are the most efficient ones.The vaccines that widely used in our courty are live attenuated vaccine strains, such as B.melitensis M5, B.abortus S19 and B.suis S2. M5 developed in our country is an live attenuated vaccine strain. It is extensively used in our country and very effective for prevention of animal brucellosis. It is also found that these vaccines also have cross immunity. For example, goat and pig immuned with M5 also have protective immune response against brucellosis. And also, S2 can also induce protective immunse response in goat and pig. The application of live attenuated vaccine strain plays very important roles in controlling brucellosis. However, these vaccines also have some limitations or defects. For example, although these vaccines have good efficencies, but they are still virulent and on occasions, cause infections. At some regions, the mobility increased with the application of vaccines. Another defect of these vaccines is that, they have very similar antigenicity to virulent strains, and therefore, it is very difficult to differentiate the immunization and infection. Therefore, how to resolve these problems of these vaccines is important for brucellosis prevention.BP26 is an important diagnostic antigen for Brucella. It is extensively used in Brucella diagnosis. BP26 is a surface antigen, and play some roles in virulence. However, it is not a protective antigen. These characteristics make it a good candidate antigen for gene tagging vaccine development. Deletion of this antigen can decrease the virulence, but also provide a means for differentiation. So, in the present study, wo planed to modify the live attenuated strain by delete the BP26 gene. And then, the survival capability of the tagged strains in host cell and mice were tested to making sure that it can replicate both in host cells and mice. Based on the sequence differences between the wild type strain and tagged strain, a diagnostic method was developed to differentiate immunation and infection. By these means, we can improve immunization protection efficency for one hand, and also develop differiating methods for infection monitor. With these aims, two studies were carried out:Part one: Construction and primary analysis of BP26 tagging strains. Live attenuated vaccine M5 was selected for modification and BP26 was chosen as the target gene. By using homologous recombination, BP26 gene was replaced by resistance gene to construct tagged strain. Firstly, N and C terminal arms of BP26 were amplified and cloned into pUC19K sequentially. The N terminal arm was firstly cloned into the MCS upstream of kanamycin gene of pUC19K to generate pUC19K-N. And then the C terminal arm was cloned into MCS downstream of kanamycin gene of pUC19K-N, generating pUC19K-NC, which was transformed into M5 competent cells. The transformant was spread on TSA plastes containing kanamycin and cultured at 37℃for 3-5d to select for resistant clones. By using primers located in kanamycin gene and that located outside of the homologous arms, the resistant clones were PCR verified. The results showed that the BP26 was corrected replaced by kanamycin gene, and the tagged strain was correctly constructed. The wild type strain and tagged strain were used to infect macrophage for survival capability comparison. The tagged strain could replicate in macrophage but showed reduced survival capability when compared with the wild type strain. Then, the two strains were used to infect BLAB/c mice. At 1 and 2 weeks post the infection, bacteria was isolated from spleen and liver and enumerated. The results showed that the tagged strain could survive in mice, but the survival capability decreased. These results indicated that, the tagged strain could be used as live attenuated strain, and when compared with the wild type strain, the virulence was reduced, implying that the vice effects might be decreased. The construction of tagged live attenuated strain provides a new vaccine candidate for further research.Part II, development of differentiating PCR for tagged live attenuated strain. According to the differences of host and pathogenisis, Brucella is divided into six species, of which B.melitensis, B.abortus, and B.suis are pathogenic for human. Therefore, the detection and differentiation of the three strains is very important for brucellosis diagnosis. In this part, the genus specific signature gene was chosen to develop genus specific real time PCR. Then, a multiplex PCR was developed to differentiate the most popular species B.melitensis, B.abortus and B.suis. At last, based on the sequence difference, a duplex PCR was developed to differentiate tagged strain. BP26 is conserved in brucella genus, and therefore, it is a very important signature gene for brucella genus. A pair of primers and a Taqman probe were designed and synthesized. A fragment of BP26 was PCR amplified and cloned into pET-32a to generate pET-32a-BP26, which was then used as positive plasmid. By omptimizing the concentration of Mg2+ and probe, a real time PCR method for brucella genus detection was developed.According to the differences in location of IS711 in genomes, 4 primers were designed, of which 3 were located in B. melitensis, B.abortus and B.suis specific locuses, and the fourth primer was located in IS711. The four primers was used to fabricate a multiplex PCR. By omptimization of primer concentration, a triplex PCR for differentiating the 3 species was developed. At last, a pair of primers targeting the delected region of the tagged strain and one another pair targeting DnaK were designed. The two pairs of primers were used to set up a duplex PCR. This duplex PCR could differentiate wild type strain and tagged strains, where for the former, two product bands were generated, but for the latter one, only one band was generated. By this means, the wild type strain and tagged strains can be differentiated.
|
PDF Full Text Request