Study On The Function Of Apobr In Brucella Intracellular Parasitism

Brucellosis，which caused by Brucella,is a zoonotic infectious disease. Infected patientsshowed undulant fever, animal brucellosis mainly cause abortion, orchitis. Brucella long-termparasitic macrophages and inhibit apoptosis of infected cells, causing human and livestockmultiple organ damage and chronic infections, and pose a serious threat to human health anddevelopment of animal husbandry. Therefore, investigation on the pathogenesis of Brucella and ontherapeutic drugs and vaccines is imminent. The outer membrane protein OMP25, presented inBrucella mambrane, directly interacts with macrophage membrane and inhibits production oftumor necrosis factor (TNF), while it does not affect the generation of IL-6, IL-1β, IL-8.Object：The laboratory has confirmed ApoBR to Brucella infection of macrophagespotential receptors. This article explores ApoBR infected by RNAi technology in the role ofmacrophages in Brucella, to further study the mechanism by which Brucella evades macrophagekilling and inhibit TNF production.Methods:(1) RNAi vector was constructed targeting receptor gene. Nine positiveinterference fragments and one negative interference fragment were design by siDESIGN Centerpages, then The interference fragments were connected into the pSIREN-the RetroQ-ZsGreenvector, and screening out the more obvious inhibitory effect RNAi interference fragment byqRT-PCR.(2) To further screening highest inhibitory effect RNAi interference fragment, weconstructed lentivirus vectors.(3) Murine macrophages was transfected by lentiviral vector wereinfected with Brucella2308, and to analyze CFU and TNF-α of murine macrophages RAW264.7were infected with Brucella2308strain to collection cells and supernate at different time points.Lysed infected lentiviral vector transfected macrophages, extracted RNA, and reverse transcriptionof cDNA, application qRT-PCR detection and suppression of pro-apoptotic factor Bax changes inapoptotic factor Bcl-2, and by flow cytometry detection of Brucella infection of cells transfectedwith lentiviral apoptosis.Results:(1) The sequencing results showed that Four interference vector（ApoBR-253,ApoBR-163，ApoBR-457，ApoBR-2668，ApoBR-2053，ApoBR-648，ApoBR-1138，ApoBR-893,ApoBR-1325and CK）were constructed.By qRT-PCR and bioinformatics analysis screened outfour more efficient interference vectors: ApoBR-253, ApoBR-893, ApoBR-1325,ApoBR-2053.(2)Four lentiviral vectors LentiLox3.7-253,pLentiLox3.7-893, pLentiLox3.7-1325,pLentiLox3.7-2053were constructed and screened two more efficient and interference vectorpLentiLox3.7-253pLentiLox3.7-1325, inhibition rate of over60%(3) Brucella infectiontransfected murine macrophage CFU results compared to the control group and negative controlgroup is reduced. ELISA results show that TNF-α compared with the control group and negativecontrol group is decreased. Apoptosis rate of flow cytometry results showed that, the apoptosisrate increased, compared with the control group and negative control group, RT-PCR of Bax,Bcl-2results showed that the relative expression of pro-apoptotic factor Bax compared with thecontrol group and negative control group is increased, while the result of Bcl-2is the opposite.Conclusion： The experimental results show that ApoBR is one of important to identifyreceptor to brucella infection of macrophages and plays an important role for brucella invasionand persistent infection.Inhibition of the expression of ApoBR results in TNF-a elevated,activation the innate immune response to artificially provide a target point and inhibition ofintracellular survival provides a potential regulatory sites, provide a scientific basis for finding an effective treatment for brucellosis drugs.