Generation Of Bordetella Bronchiseptica Ghost By Coexpression Of Lysis E Gene And Staphylococcal Nuclease A Gene

Traditional inactivated vaccine is one of important means of preventing animal disease. However, because antigenic determinants in traditional vaccine may be destroyed or damaged partly by physical or chemical treatment (formalin) during preparing inactivated vaccine, and the traditional inactivated vaccine is low in protective efficiency or can play a protective role for the vaccinated animals, which could become carriers of this pathogen. Moreover, there are the gene of resistance to antibacterial agents and the gene of virulence-encoding in the inactivated bacterial cells for traditonal vaccines that might be injected into the animal and would be possible risk to the animals vaccinated. Therefore, it is important theoretical and practical significance to research the reservation of natural antigenic structure of bacteria and the elimination of possibe risk of antibiotic resistance and virulence-encoding genes. We constructed recombinant plasmids by inserting the phage PhiX174 lysis gene E and Staphylococcus aureus nuclease A gene (SN) to the same vector. Being transformed into bacteria, the recombinant plasmids were induced to express the Staphylococcus aureus nuclease A by IPTG and lysis protein E at 42℃. A field strain of Bordetella bronchiseptica from pig is used as target bacterium to produce the bacterial ghosts. After the recombinant plasmid carrying lysis gene E and SN gene was transformed into strain DH5 a of E. coli and successfully expressed for producing the ghosts of E. coli, two recombination plasmids, pET29a-S1-E and pET29a-S2-E, with E gene cassette and SN gene were reconstructed for tranforming into field strain of Bordetella bronchiseptica to attempt to eliminate cytoplasma, plasmid-mediated resistance to antibiotics and virulence-encoding genes, hydrolyte DNA and reserve natural antigenic constructures of Bordetella bronchiseptica. The results follow as below:1. In order to ensure the safety of bacterial ghost preparation, a second kill gene, Staphylococcus nuclease A gene (SN) and E gene cassette from PhiX174 phage were commonly inserted to a plasmid, and pGEX-4T-1-S-E containing ampicillin resistance gene, expression plasmid pET29a-S1-E and non-fusion expression plasmid pET29a-S2-E containing kanamycin resistance genes were constructed. The results showed that plasmid pGEX-4T-1-S-E, pET29a-S1-E and pET29a-S2-E was 6822bp, 7211bp and 7106bp, respectively, by a single enzyme cleaving and agarose gel electrophoresis. Molecular weights of three plasmids were consistent with the theoretical sizes. E gene cassette was the same as theoretical sequence by sequencing identification. Sequence results indicated that the sequence of SN gene was the same as the SN gene sequence of Staphylococcus aureus MRSA252 in GenBank, and E cassette was consistent with theoretical sequence.2. The recombinant plasmid pGEX-4T-1-S-E was transformed into strain DH 5a of E. coli and coexpressed by inducing factors. The detection tests of DNAs in Bacterial cells and supernatant of medium showed that the host DNA was degraded into small fragments (=100 base pairs) without interfering the ghost formation process. The co-expression of gene E and staphylococcal nuclease A gene results in the total inactivation of bacterial ghost. The rate of genetic inactivation was up to 99.999%, determined by CFU test.3. The results of antibiotic susceptibility tests suggested that the field strain of Bordetella bronchiseptica was resistant to ampicillin, gentamycin, and sensitive to chloromycetin, tetracycline and kanamycin. Therefore, plasmids containing kanamycin-resistance gene were chosen as expression plasmids in Bordetella bronchiseptica.4. The expression plasmid, pET29a-S1-E containing kanamycin-resistance gene and non-fusion expression plasmid, pET29a-S2-E were respectively transformed into the field strain of Bordetella bronchiseptica and induced to coexpression. DNAs in bacterial cells and supernatant of medium were detected, the result of which showed that the host DNA was degraded into small fragments (=100 base pairs) without interfering the ghost formation process. The co-expression of gene E and staphylococcal nuclease A gene resulted in the total inactivation of bacterial ghost. The rate of genetic inactivation by the expression plasmid pET29a-S1-E was up to 99.994% determined by CFU test, and the rate of genetic inactivation by the non-fusion expression plasmid pET29a-S2-E was also up to 99.993% determined by CFU test. The genetic inactivation of Bordetella bronchiseptica was fully realized. The difference of genetic inactivation rates was non-significant of the expression plasmid pET29a-S1-E and non-fusion expression plasmid pET29a-S2-E.