Recombinant protein Schistosoma japonicum-derived molecule attenuates dextran sulfate sodium-induced colitis by inhibiting miRNA-217-5p to alleviate apoptosis

BACKGROUND Inflammatory bowel disease(IBD)affects millions of people worldwide and has emerged as a growing problem in industrialized nations.The lack of therapeutic targets has limited the treatment of IBD.Studies found that parasitic nematode infections can ameliorate clinical and experimental colitis.Our previous study found that rSj16,a 16-kDa secreted protein of Schistosoma japonicum produced by Escherichia coli,has protective effects on dextran sulfate sodium(DSS)-induced colitis in mice.Apoptosis is an important factor in the pathogenesis of colitis.However,it is not clear whether the effect of rSj16 on colitis is related to apoptosis.AIM To investigate whether the protective effects of rSj16 on colitis is related to apoptosis and its mechanism.METHODS In-vivo,colitis was induced by DSS.The severity of colitis was assessed.WB was used to detect the changes of apoptosis-related genes in colon tissues.Q-PCR was used to detect the changes of miRNA-217-5p and HNF1B.In-vitro,WB was used to detect the changes of apoptosis-related genes in intestinal epithelial cells.TUNNEL staining and flow cytometry were used to detect cell apoptosis.RESULTS rSj16 attenuates clinical activity in DSS-induced colitis mice.TUNNEL staining and WB results showed that apoptosis was increased in colon tissue after treatment with DSS,and the apoptosis of colon tissue was significantly reduced after treatment with rSj16.Compared with normal mice,the expression of miR-217-5p was increased in colon tissue of DSS-induced colitis mice.In addition,the miR-217-5p target gene hnf1b was decreased after administration of DSS.After treatment with rSj16,the expression of miR-217-5p was decreased and the expression of HNF1B was increased compared with the DSS-treated group.When Etoposide was used in combination with miR-217-5p mimic on MODE-K cells,the expression of cleaved-Caspase-3 and Bax was increased,and Bcl-2 was decreased compared with only Etoposide treatment,the expression of HNF1B was significantly reduced,suggesting that miR-217-5p acts as a pro-apoptotic in colon epithelial cells and down-regulates the target gene hnf1b.After rSj16 administration in MODE-K cells,miR-217-5p expression was significantly decreased,HNF1B expression was increased,and apoptosis was reduced.CONCLUSION The protective effects of rSj16 on colitis is related to apoptosis and miRNA-217-5p may be a further target for therapeutic intervention against IBD.

日本血吸虫中国大陆株62kDa重组抗原的表达、纯化及鉴定

目的 获得并鉴定日本血吸虫 6 2 k Da重组抗原。方法 将 Sj6 2 / p GEX- 1λT/ TG2 克隆菌用 IPTG诱导表达 ,经超声粉碎 ,离心后取上清过 GS4B柱 ,用还原型谷胱甘肽洗脱液洗脱后获得纯化的 r Sj6 2 / 2 6 GST重组融合蛋白 ,用 Throm bin酶切后获得纯化的 r Sj6 2重组蛋白 ,并对重组蛋白进行 SDS- PAGE和 Western blotting鉴定。结果 重组蛋白纯度较高 ,且可被血吸虫感染的鼠血清识别。

重组日本血吸虫14-3-3蛋白的纯化和免疫特性分析

目的 :纯化日本血吸虫信号蛋白质 14 3 3编码基因的原核表达产物 ,分析纯化产物的免疫学特性。方法 :SDS PAGE鉴定重组日本血吸虫 14 3 3(rSj14 3 3)蛋白包涵体 ,超声破碎细胞收集包涵体 ,尿素溶解包涵体 ,NiSO4平衡层析柱 ,亲和层析纯化rSj14 3 3,Western blot鉴定纯化产物的免疫学特性。结果 :rSj14 3 3是以包涵体形式在大肠埃希菌中表达 ,通过亲和层析在 32 .5kDa附近得到单一蛋白条带 ,Western blot证实纯化产物为rSj 14 3 3,且具有与天然Sj 14 3 3相同的抗原表位。结论 :成功纯化了rSj14 3 3蛋白 ,为研究 14 3 3在血吸虫信号转导中的作用奠定了基础。