Establishment Of Porcine Intestinal Mucosa Protection Factor Of Brevibacillus Expression System

Piglet diarrhea was mainly caused by which the intestinal mucosa barrier function was impaired.Antisecretory factor(AF),Heme oxygenase(HO-1)and Trefoil factor family 3(TFF3)can repair the damaged intestinal epithelial cells,which plays an important role in maintaining intestinal mucosal barrier function.Brevibacillus expression system enables highly efficient production of heterologous protein in the secreted form,and does not produce endotoxin,and regulates the intestinal micro ecological balance.However,No only the cost of natural separation mucosa protection factor is expensive,but also the production and activity of which are low,it is difficult for the production of industry or being used as feed additive.The article aims to establish porcine intestinal mucosa protection factor(AF,HO-1,TFF3)of Brevibacillus expression system in order to provide new ideas and new methods to reduce the occurrence of piglet diarrhea and protect the intestinal mucosal barrier.To generate pNCMO2-AF,pNCMO2-HO-1 and p NCMO2-TFF3 expression vectors,the DNAs for AF,HO-1 and TFF3 were cloned from porcine tissues by PCR,and connected respectively to p NCMO2 vector.pNCMO2-AF,pNCMO2-HO-1 and pNCMO2-TFF3 expression vectors were transformed into Brevibacillus,and analyzed the porcine AF,HO-1 and TFF3 genes fusion expression in Brevibacillus with SDS-PAGE and Western Blot methods.The results showed that the cloning of porcine AF,HO-1 and TFF3 genes were 1164 bp,897bp and 273 bp,encoding 387,298 and 87 amino acids,the results of electrophoresis and sequence were consistent with the expected size of the target gene;and successfully constructed recombinant expression vector pNCMO2-AF,pNCMO2-HO-1 and pNCMO2-TFF3,identification of bacteria PCR and digestion with double restriction enzymes were correct;after electroporation identification results of digestion showed that pNCMO2 vector was 5200 bp and the target gene AF,HO-1 and TFF3 were 1164 bp,897bp and 273bp;SDS-PAGE and Western Blot analysis showed that the size of inducible expression protein AF,HO-1 and TFF3 were approximately 46.3KD,36.5KD and 13.6KD,which was in line with expectation,and was extracellular secretion.In conclusion the results showed that porcine intestinal mucosa protection factor of Brevibacillus expression system was successfully constructed,which provided a new idea for the prophylaxis and treatment of piglet diarrhea.