Expression And Functional Analysis Of Recombinant Anti-C.difficile Toxins Nanobodies

Clostridium difficile,a Gram-positive anaerobic bacillus,is one of the leading factors of infectious diarrhea caused by antibiotics.When the balance of intestinal microflora is disturbed,C.difficile multiplies and overgrows,becoming the main flora of the intestine,which causes Clostridium difficile infection(CDI),leading to mucosal necrosis and intestinal mucosa of intestinal epithelial cells.Epithelial cell apoptosis,increased mucosal permeability,eventually leading to severe diarrhea and intestinal inflammation.In recent years,due to the serious abuse of antibiotics,the incidence and mortality of C.difficile infection have increased year by year in the world,and it will become one of the epidemic diseases threatening public safety in the future.Studies have shown that two exotoxins(Tcd A and Tcd B)secreted by clostridium difficile are the main causes of CDI.The project is based on the nanobody of AA6 and E3,they were fused to build the nano-body synthesis through genetic engineering method,the synthesis can highly improve its stability and affinity;The expression of the target protein was optimized by Lactococcus lactis,and improve the protein expression technique;The inhibitory effect of recombinant Nanobody E3-AA6 on Tcd A and Tcd B was examined by in vitro cell assay.This study will provide a new way to prevent,control and treat CDI.The specific research is as follows: 1.Express the Tcd A and Tcd B in B.megaterium.Transform the p His1525-Tcd A?p His1525-Tcd B plasmids into B.megaterium competent cells,screen out the positive recombinants and induce the expression by xylose.Then the ni-affinity chromatography could be used to separate and purify Tcd A and T-cd B from the recombinant proteins,expression purity is 86% and 82% respectively.It was found that Tcd A of 0.5 ?g/m L or Tcd B of 0.4 ng/m L was both able to cause 100%of HCT-8 cells to turn round by the cell rounding experiment,and the flow cytometry experiments indentified that Tcd A and Tcd B could induce significant apoptosis of HCT-8 cells through 6h.2.Construct the nano-antibody lactococcus lactis.The expression vector p NZ8148-E3-AA6 could be constructed through the fractional PCR.The vector is electrically transformed into the L.lactis NZ9000 competent cells,named p NZ8148-E3-AA6/NZ9000.Induce the expression by nisin,isolate and pure E3-AA6 protein by ni-affinity chromatography with a purity of 95.2%.MTT also tested its good biological activity.3.Analysis of neutralization effect of nano-antibody E3-AA6 on toxins Tcd A and Tcd B.The Woundhealing method was used to analyze the migration of HCT-8 cells,to investigate the inhibitory effect of E3-AA6 on the pathological effects of Tcd A and Tcd B.The activity of HCT-8 cells was analyzed by CCK8 method to investigate the toxicity of Tcd A and Tcd B.The results showed that the relative inhibition rates of pathological effects of Tcd A and Tcd B by E3-AA6 at 50 ?g/m L were 23% and 41.7%,respectively.The relative inhibition rates of toxicity of Tcd A and Tcd B were 21.1% and 39%,respectively.It provides a new therapeutic approach to the treatment of infection caused by clostridium difficile and an experimental basis for the development and application of oral drugs.