Brucella 19 Weakened, Construction And Preliminary Study Of Immune Effects Of PGM Gene Deleted Strain

Brucellosis is a zoonotic disease caused by Brucella, which caused abortion and sires orchitis in animals, and caused huge economic losses to livestock industry. Many countries have taken measures to prevent brucellosis, such as using vaccination and killing, but it dose not work; However, after the attenuated vaccine immunitied susceptible herd, serological testing is usually positive, it is not effectively distinguish for naturally infected animals. Meanwhile some vaccines are still virulent, causing abortion in pregnant animals. Brucella 019 was isolated at Xinjiang in 1980 s. Therefore, the 019 has important practical significance for developing new vaccines.Objective: In this study, Brucella 019 mutation was induced by physical and chemical methods. Meanwhile, we constructed Brucella 019 pgm(phosphoglucomutase) deletion mutant, and evaluated its virulence and immunity protection.Methods:(1) Brucella 019 was treated with different concentrations of acridine orange(0.1g/L and 0.05g/L) and phenol(0.1g/L and 0.05g/L), induced by UV irradiation in different times(5min and 2min), serially passaged 50 generation times; Its growth curve and generation change were measured; Spleen index and colony-forming units(CFU) of peritoneally injected mice were measured;(2) The upstream and downstream homologous sequences of pgm gene and SacB gene were amplified by PCR. The pgm gene was fused with the upstream and downstream homologous sequences by overlap PCR. The SacB gene was connected to the vector pGEM-7Zf+ after double digestion, respectively, named pGEM-7zf-Δpgm-SacB. The recombinant vector was transformed into competent strain 019, and the 019Δpgm srains were screened through ampicillin resistance and sugar sensitivity; The positive bacteria were serially passaged 10 times for genetic stability testing.(3) After intraperitoneal injection of 019, 019Δpgm, S2 or M5-90, the level of spleen index and CFU were tested, blood was collected, serum was separated, and the content of IgG and IFN-γ was detected every 10 days.Results:(1)After the treatment of different concentrations of acridine orange-induced phenol, as well as different UV irradiation,the growth trend of the treatment groups was similar to the parental strain 019, but it enters the platform earlier than the time period of the parent strain. After the intraperitoneal injection of different Brucella strains, their spleen index are significantly higher than the PBS group(P<0.05), the UV radiation and phenol(0.1g/L) treatment group were no significant difference with M5-90, but significantly lower than the parent strain(P<0.05); and various concentrations of acridine orange-treated group were significantly higher than the vaccine strain M5-90(P<0.05), there was no significant difference with parental strain. Spleen CFU results showed that the UV radiation(5min) and phenol(0.1g/L) treatment groups were significantly lower than 019(P<0.05),and the various treatment group spleen CFU were significantly higher than M5-90(P<0.05).(2) We successfully amplified 950 bp and 973 bp downstream of pgm gene homology arms target band, by overlap PCR fused the homology arms target, then the fusion gene fragment with SacB gene were connected to the suicide vector pGEM-7zf+. After the electrical conversion of recombinant vector into Brucella 019, we successfully obtained Brucella 019Δpgm. After 10 generations the genetic stability and reverse mutation did not occur.(3) After intraperitoneal injection of various Brucella strains, spleen index and CFU results showed that there was no significant difference(P>0.05) between 019Δpgm and its parent strain 019, but 019Δpgm significantly higher than M5-90 and S2 groups(P<0.05); after subcutaneous injection of inactivated whole bacteria adjuvant formulation, serum levels of IgG and IFN-γ found 30 days 019Δpgm inactivated whole bacteria adjuvant formulations with the parent strain 019 inactivated whole bacteria adjuvant formulation compared to its parent strain 019 was not significantly different(P>0.05), but were significantly lower than the vaccine strain M5-90(P<0.05); IFN-γ test results also showed attenuated M5-90 significantly higher than other Brucella inactivated adjuvant formulation(P<0.05), in the first 30 days, 019Δpgm whole bacteria live IFN-γ induced by adjuvant was the lowest, significantly lower than other experimental groups.Conclusion: After the different concentrations of acridine orange, phenol and UV irradiation treatment at different times,the growth of Brucella 019 was inhibited and the virulence was reduced. The group of UV irradiation treatment(18w, wavelength 253.7nm, 5min) was attenuated more effectively. We successfully constructed Brucella 019Δpgm gene deletion strains. Mice experiments showed that the 019?pgm of virulence did not cause significant changes compared with parental strain 019; the 019Δpgm was made inactivated adjuvant formulations, the levels of IgG and IFN-γ were similar with 019, but significantly lower than M5-90.