Preliminary Study On The Inflammatory Mechanism Of Brucella Omp25 To Embryonic Trophoblast Cells

Object:Brucellosis, which is a zoonosis chronic infection disease caused by Brucella, has occuerd widly in the world. Brucellosis is a severe threaten the economy construction, public health and the national security to our courtry. Brucella spp. is the pathogen of brucellosis. Brucellae are facultative intracellular. Research on The virulence of Brucella depends upon its ability to survive and replicate in host cells. Animal placenta is one of the most favorite locations of surviving and replicating. Embryonic trophoblast is the target cells of brucella. Once destroyed, it will induce abortion. However, its molecular mechanism of abortion is unclear. The results of current etiologic study show that outer membrane proteins (OMPs) and type IV secretion system (T4SS) proteins are putative virulent factors because these elements play an important role in brucella's invasion, survival and multiplication.Therefore, we will base on Brucella virulent factors and Embryonic trophoblast cells in this study.Methods:(1)The omp25 gene was cloned by PCR from B.melitensis 16M and inserted to the expression prokaryotic vector pET32a, successfully constructed a prokaryotic expression vector pET32a-omp25, and induced by IPTG.The result of SDS-PAGE and western blot indicated that an approximately 43.5KD exogenous protein was observed. And the purified Omp25 fusion protein was obtained. (2) As the eukaryotic expression vector pEGFP, the EGFP gene upstream of omp25 gene to construct the expression of Omp25-EGFP fusion protein;according to the nucleotide sequence of omp25 were constructed siRNA-a/b/c three interference vectors,the pEGFP-omp25 siRNAs were co-transfected HPT-8 cells by lipsome,the optimal real-time quantitative PCR screening interference plasmid. (3)The effect on HPT-8 cells Omp25 protein and its mechanism of toxicity as a starting point, respectively, normal-phase (stimulation of the purified Omp25 protein) and reverse (RNAi) analysis, Omp25 protein induced inflammation Expression of signaling molecules to HPT-8 cells.Results:(1)we constructed pET32a-omp25,and the purified Omp25 fusion protein was obtained. (2)The pEGFP-omp25 expression vector were constructed successfully in eukaryotic cells.(3)Three positive siRNA plasmids were successfully transfected into HPT-8 cells. The optimal siRNA-c was screened and its inhibition rate reached to 98%.(3)ELISA test showed:the release amount of NO and the activity of LDH and inflammatory cytokines of TNF-a w with the Omp25 protein concentration as increased by varying degrees. (4) real-time quantitative PCR results showed that Omp25 protein allows the expression of TLR4 and MyD88 mRNA increased.Conclusion:(1)The purified Omp25 protein protein had immunogenicity. (2)The low dose of Omp25 protein may stimulate cytokine production through TLR4 activation, resulting in protective immunity. (3) The high doses of Omp25 protein results in a cytotoxic effect on HPT-8 cells.