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Objective To investigate the dynamic changes in intestinal alpha-defensin-5 (RD-5),beta-defensin-2 (BD-2) mRNA after acute liver failure(ALF),and to explore their role in ALF.Methods A total of 60 C57BL5 mice were divided into 4 groups by means of random number table method:normal control group,ALF group,E.coli via gavage group and ALF + E.coli via gavage group.Intraperitoneal injection of D-galactosamine (500 mg/kg) and lipopolysaccharide(10 μg/kg) to make the model,in addition,ALF mice were fed with E.coli,and the observation time was 6 hours,12 hours,and 24 hours after modeling,and each time point had 6 specimens.Real-time PCR was used to test the RD-5 mRNA and BD-2 mRNA levels in the ileum tissue.Results The levels of RD-5 and BD-2 showed dynamic change in the experiment of ALF.Compared with the levels of RD-5 and BD-2(11.25 ±0.74,23.86 ±0.39) of the normal control group,the levels of RD-5 and BD-2 in ALF group and E.coli via gavage group increased at 6 hours after modeling(14.19 ±0.39,26.79 ± 0.36 and 12.57 ± 0.68,26.45 ± 0.85),and the differences were significant(all P＜0.05);at 12 hours after modeling,the RD-5 and BD-2 reached to the maximum concentration(15.76 ±0.33,29.10 ± 0.61 and 12.90 ± 0.96,27.42 ± 0.71),and the differences were statistically signi-ficant (all P ＜ 0.05).The degree of elevation of BD-2 was higher than RD-5.Later,they gradually declined.Conclusions RD-5 and BD-2 may play an important role in the pathogenesis of intestinal endotoxemia in experimental ALF.
RESUMO
Objective To construct the prokaryotic expression vector for HD-5 and purify the recombinant HD-5 protein then analyze its antifungal activity. Methods The HD-5 gene was cloned by PCR, then was inserted into prokary-otic expression plasmid pQE-30Xa to construct pQE-30Xa/HD-5. After sequencing, pQE-30Xa/HD-5 was transformed in-to E.coli M15 cells. Its expression was induced by IPTG and confirmed by SDS-PAGE. The recombinant protein was purified through Ni-NTA affinity purification system. The antifungal activity was tested by disk diffusion method. Results HD-5 gene and pQE-30Xa/HD-5 vector were obtained successfully. E.coli M15 strains was used to express HD-5 fusion protein. After purification, the fusion protein was confirmed by Western blot. The disk diffusion test confirmed that the fusion pro-tein can inhibit Candida albicans. Conclusion Expression vector pQE-30Xa/HD-5 was successfully constructed. The HD-5 fusion protein was expressed in E.coli successfully, which showed a certain degree of antifungal activity.