Construction Of Defined Mutations Of Rabbit Bordetella Bronchiseptica And Property Research

Bordetella bronchiseptica (Bb) is an important pathogenic bacterium which can cause acute and chronic respiratory system disease. Bb can infect numerous animals, such as swine, horse, cat, canine, rabbit, mouse, cavia cobaya and so on. It always result in rhinitis and bronchial pneumonia of the infect animals. Human beings are also infected by Bb, especially children and immune deficient individuals. The high infection rate in rabbits impact rabbit product industry seriously, and affect the quality of experiment rabbits used for research.Pertactin(PRN) encoding by prn gene is one of outer membrane protein composition of Bb with antigen protection.It is also an important adhesion molecule. PRN is the most important component of adhesion molecules and the antigen of the Bb, and the main component of the commercialization of pertussis vaccine widely used.Much work has been done about Bordetella bronchiseptica disease on swine. But less was known of that on rabbits. As yet, there is no report about the PRN deletion mutant of rabbit Bordetella bronchiseptica.We construct PRN deletion mutant strain of rabbit Bordetella bronchiseptica to study the function and the pathogenesis of Bb, and to provide a theoretical basis for researching Bb live attenuated vaccine.The main experiments and results are as follows:1. Construction of defined mutations of PRN of rabbit Bordetella bronchiseptica.Suicide vectors for deletion of the PRN genes were constructed as follows. The 550-bp fragments of p1 and 440-bp fragment of the p2 were generated by PCR respectively. Both of these two fragments together with GM gene were cloned into pUC18, named pUC18-PRN1GMPRN2. The fragment PRN1GMPRN2 was insterted into suicide vector named pMEG375-PRN1GMPRN2. The suicide vector was transformed into host strain SM-10.SM-10 containing the suicide vector mate with receptor strain Bb on the solid phase membrane, suicide vector transfer into the recipient bacteria. According to homologous recombination, the principle of resistance screening, Mutant of PRN were generated and were named BbΔPRN.2. biological characteristics and immunogenicity of PRN mutant strainsTo study the biological characteristics of the mutant strain BbΔPRN, we compared BbΔPRN with wild type (WT) in genetic stability,growth characteristics, hemolytic activity and cell adhesion properties. The results showed that the mutant strain stabilized geneticly and grew slower than the wild type. There is no significant difference between them in hemolytic activity and adhesion on Hep-2 cells. Median lethal dose (LD50) values were 1.1×107CFU per mouse for mutant strain and 5.36×106CFU per mouse for wild-type strain. Compared with parent strain, mutant stain was attenuated about two fold. The mice were immunized with different doses of mutant, and were attacked by wild type strain 21 days after vaccination, the results showed that, BbΔPRN could produce a strong immune capacity, and indicated that mutant strain with good immunogenicity is expected to a vaccine candidate strain.We construct PRN deletion mutant strian of rabbit Bordetella bronchiseptica by Suicide vectors,and the mutant strain stabilize geneticly. The mutant strain grow slower than the wild type strain, There is no significant difference between them in hemolytic activity and adhesion on Hep-2 cells. Compared with parent strain, mutant stain was attenuated about two fold. Potency tests in mice showed the mutant with good immunogenicity could play a protective role on mice and provide a theoretical basis for researching Bb live attenuated vaccine.