| Brucellosis is a worldwide zoonotic infectious disease, which leads to a great harm to human and animals. The Ministry of health of the people’s Republic of China has listed it as class B infectious diseases. Brucella is a pathogen of Brucellosis, there are more than 60 kinds of susceptible animals, including sheep,pigs, cattle and so on. Brucella are able to cause infertility and abortion in mammals and intermittent fever and joint pain. Brucella can survive and replicate in host cells and transmit organism through skin, digestive tract and respiratory mucosa. Brucella are mainly resident in macrophage cells, then survive and rely on its immune evasion mechanism, after that, pathogens partly transferred to lymph node form the primary lesion. When Brucella reach a certain degree, macrophages will be broken down and the pathogens will spread to other host cells. During this period, the LPS and capsular from Brucella will lead to fever. After Brucella reach a new host cell,intracellular parasite arise again. Based on this cycle mode, there is the undulant fever, which is the typical symptom caused by Brucella. Currently, the mechanisms of Brucella being survival in macrophage have not been identified.Therefore study on the proteins related with Brucella intracellular parasite would be significant.Peroxiredoxin 6(Prdx6) is a member of the non-selenium-dependent peroxidase family, differing from other members of the family, primarily in that the protein is a 1-Cys enzyme. Prdx6 is a bifunctional protein, showing both GSH peroxidase activities snd phospholipase A2 activities. So, Prdx6 plays an important role against oxidative damage, and in regulating apoptosis.Previous studies have reported that Prdx6 showed obvious differencial expression after infecting sheep with Brucella, But, it was not clear about the relation between Brucella and Prdx6. In order to study the relationship between prdx6 and Brucella intracellular parasitic, in this study we cloned the mouse(Mus musculus) prdx6(Mu Prdx6) coding sequence, then the prokaryotic expression vector of Prdx6 was constructed, and the recombinant mouse Prdx6(r Mm Prdx6) protein was expressed and purified, and the purified Mu Prdx6 was applied to prepare polyclonal antibody and monoclonal antibody for subsequent detection. With Brucella suis S2 strain infecting a murine macrophage cell line RAW264.7, analyzed Prdx6 the differential expression of Mu Prdx6 were analyzed. The regulated expressions of Mu Prdx6 by overexpression vectors and sh RNA interference vectors,and the Brucella infection and intracellular parasitic bacteria amount were detected for analyzing the relationship between intracellular prdx6 expression and Brucella infection.The results showed that mouse Prdx6 coding sequence was correct, and prokaryotic expression vector of Mu Prdx6 was constructed successfully, the recombinant mouse Prdx6 protein was expressed by inducing and purified using affinity chromatography, and its antioxidant activity was validated. A specific polyclonal antibody against mouse Prdx6 was obtained by immunizing a rabbit with the purified Prdx6, and a specific monoclonal antibody against mouse Prdx6 was obtained by immunizing BALB/c mice. With Brucella suis S2 strain infecting macrophages, the intracellular prdx6 expression level displayed a trend in first decrease and then increase. The increased expression level of Prdx6 in macrophage could improve the infection and survival ability of Brucella. The decreased expression level of Prdx6 in macrophage could reduce its infection and survival ability.This study showed that the differential expression of Prdx6 was able to affect the infection and intracellular survival activities of Brucella. The increased expression level of Prdx6 in macrophage cells could improve Brucella to infect andsurvival in cells. Our study provided the basis for further studying how prdx6 affects Brucella intracellular survival, and whether prdx6 or not could be regarded as a target protein for effective prevention and treatment of brucellosis. |
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