Polycistronic Expression Of CGA-N46 Gene In Bacillus Subtilis And Its Fermentation Optimization

It is significant to research new effective antifungal agents with low toxicity because the death rate of patients with impaired immune functions such as AIDS is increasing due to fungal infections caused by Candida albican and other pathogenic fungi, and the existing antifungal drugs have potential toxicity to host cells and the rise of drug-resistant pathogents are on. Chromogranin A is a precursor of many peptides with various biological activities, one of the N-terminal derived peptides CGA-N46 is a short peptide with highly efficient antifungal activity to Candida albican and is expected to become a novel antifungal agent with low molecular mass. In this study, a pair of restriction endonucleases with same stick end Nheâ… and Spe I were used to construct the recombinant T vectors pT-N46-Kpnâ… pT-2N46 and pT-3N46 through recombining single, double and three copies of the heterogenous DNA fragment which contained SD sequence, start codon, sacB signal peptide sequence, CGA-N46 encoding sequence and stop codon with the cloning vector pMD18-T respectively. The monocistronic, bicistronic and tricistronic expression plasmid p-N46 p-2N46 and p-3N46 were constructed by subcloning the DNA fragment of different copies in recombinant T vector to the expression plasmid pSBPTQ and the result recombinant expression plasmids were transformed to the protease deficient Bcillus subtilis strain DB1342 to form engineered strains DB1342(p-N46),DB1342(p-2N46) and DB1342(p-3N46). The expression of engineered Bacillus subtilis was studied by SDS-PAGE. The results demostrated that the monocistronic and bicistronic expression system expressed low yield of CGA-N46, while tricistronic expression system enhanced the production of CGA-N46, and the peptide was secreted into the the culture medium as soluble monomer after induced by sucrose. In order to enhance the production of the engineered strain DB1342(p-3N46), the expresion conditions were optimized. The effect of 1% carbon sources such as soluble starch, dextrin maltoe, lactose, glycero and glucose on the yield of recombinant CGA-N46 was studied, and the results showed that 1% dextrin could improve the expression of CGA-N46; The effect of 2% nitrogen soure such as peptone, casein, (NH4)2SO4, and 2%peptone+2%casein, 2%casein+2%(NH4)2SO4, 2%peptone+2%(NH4)2SO4, and 2%peptone+2%casein+2%(NH4)2SO4 on theproduction of recombinant CGA-N46 was studied, and the results showed that 2%peptone+2%casein was the optimal nitrogen soure; The effect of initial pH of culture medium and the inducing time on the expression of recombinant CGA-N46 were investigated, and the results showed that pH 7.5 was the optimal initial pH and that the production of CGA-N46 reached the highest level after induced by surcrose for 56h. The inhibition of CGA-N46 to the growth of Candida albican was studied by moisture-retaining culture method and agar diffusion method, the results showed that recombinant CGA-N46 could inhibit the growth of Candida albican and the diameter of inhibition zone reached 20mm. The results of this thesis will provide basis for development of CGA-N46 to be a antifungal agent.