Construction Of Brucella△omp25Strain And It’s Virulence Evaluation

Brucellosis is a zoonotic infectious diseases. The performance of the animals infected with the disease as abortion and sire of the dams of infertility.For humans, the disease can cause the people heat waves, arthritis, muscle pain and so on. Brucella as brucellosis pathogen which survival mechanism within the host has always been research hotspots. Brucella itself does not produce endotoxins,but it evade the host’s immune system killing effect is achieved through a variety of virulence factors. Outer membrane protein is the important Brucella virulence factors. Therefore, In order to do some preparation for further insight into the mechanism of survival of Brucella, this article from the outer membrane protein gene omp25of Brucella as start to begin, We through three experiments to explore autophagy in host cells when the Brucella invasion of host cell1.According to the nucleotide sequences of Brucella outer membrane protein gene published in GenBank, a pair of primers of omp25was designed and synthesized. The omp25fragments was amplified by polymerase chain reaction(PCR). The PCR product was cloned into the pMD18-T vector and then detected by PCR and sequencing. pET28a-GFP and pMD-T-omp25were digested by EcoR Ⅰ and Sal Ⅰ and The prokaryotic expression vector pET28a-GFP-omp25was constructed. The resultant pET28a-GFP-omp25was transformed into E. coli BL21cells and induced by IPTG. pET28a-GFP-omp25was constructed, and SDS-PAGE and Western-blot indicated that exogenous protein was observed. It indicated that fusion gene GFP-omp25was expressed in Bacillus coli., purified the OMP25proteins with green fluorescent and analyzed with western blot, The results showed that the protein having a good Reactogenicity.2. According to the The upper and lower arm nucleotide sequences of Brucella omp25gene published in GenBank, two pair of primers of the upper and lower arm of omp25gene was designed and synthesized. The upper and lower arm of omp25gene fragments was amplified by polymerase chain reaction(PCR).And The upper and lower arm of omp25gene fragments will be recycled, then using the fusion PCR method fusion and amplification. Bacillus subtilis SacB gene specific primers were designed and SacB gene was amplified.Then fusion the fragments and SacB gene sequence is connected to the cloning vector pMD19-T simple. Fusion fragments of the upper and lower arm gene was digested by Xhol Ⅰ and Sac Ⅰ, then connection to the suicide vector pGEM-7zf+. To get suicide plasmid pGEM-7zf-△omp25-SacB, the SacB gene was digested by Sac Ⅰ and connection to suicide vector with the purpose of gene. Suicide plasmids transformed into Brucella2308competent cells were treated with100mg/L ampicillin-resistant screening and8%sucrose sensitivity screening after homologous recombination in bacteria internal. omp25deletion mutant upload15generations in non-resistant Brucella medium and stable genetic.2308A omp25strain infect human embryonic trophoblast cells (HPT-8) to CFU count of intracellular bacteria and evaluate the virulence of the2308△omp25strain at the cellular level; With the2308△omp25immunized mice to CFU count of spleen and evaluate the virulence of the2308△omp25strain at the individual level. We found that the virulence of the deletion mutant between the virulent strain2308and the vaccine strain RB51.3. With gene deletion mutant2308△omp25, Brucella abortus virulent strain2308, as well as the vaccine strain RB51infection human embryonic trophoblast cells HPT-8. Using rabbit anti-human LC3, rabbit anti-human Beclin-1as first antibody and goat anti-rabbit IgG fluorescent secondary antibodies. By immunohistochemistry prepared immunofluorescence slice,through confocal microscopy immunofluorescence observe the immunofluorescence slice and record the average fluorescence intensity of individual slice, analysis the role of various strains in cell autophagy. At the same time, all strains infect human embryonic trophoblast cells HPT-8, extraction of total cellular RNA, reverse transcription of cDNA as a template, using real-time quantitative PCR method to detect cell autophagy-related gene expression of LC3and Beclinl amount, analysis influence of2308△omp25strain in autophagy. Comprehensive analysis found that2308△omp25strain have a greater impact in autophagy.