Study On Lactose Induction Of Hybrid Antimicrobial Peptide CC34 And Its Fermentation Technology

In order to reduce the production costs and simplify downstream purification, the work attempts to use lactose instead of IPTG to induct the hybrid antimicrobial peptide CC34 ecpression, and optimized the lactose induction condition and compositon of the nutrient medium. We also combined the lactose induction and fermentor technology together in expanding culture.The exploration results were as follows:1. In our work, we used lactose induct engineering bacteria pGEX-6P-1/CC34/BL21 (DE3) express CC34 fusion protein, which was induced by IPTG before, both were very good induction. During the optimization of lactose induction, we found that 0.5mmol/L lactose could effectively induce the expression of CC34 fusion protein, but the price of lactose was inexpensive, so the success of lactose induction significantly reduced the production cost?2. In order to improve expression level, the lactose induce conditions were optimized. The optimized conditions were as follows. (1) Rotation speed of 180r/min, (2) induction temperature of 33?, (3) induction time of 10h.3. Based on the above work of condition optimization, we using single factor experiment design, orthogonal design and uniform design to optimize the composition of the medium, the conclusions were as follows. (1) High concentrations of glucose inhibit the induction of lactose, in the single factor experiment we choose the best glucose density was lg/L, this was confirmed in the uniform design again. (2) Improved gene engineering bacteria pGEX-6P-1/CC34/BL21 (DE3) lactose induction of expression of the antimicrobial peptide fusion protein CC34 heterozygous efficiency, so that the overall expression level was generally more than 25%, the maximum expression level was 39.6%. (3) By regression analysis of the uniform design, we got the model between factors and the results as follows.Y= 183.501+22.376X,+0.710X3-44.483X4-872.168X5+4.416X42+4673.818X52-59.618X62, R=0.9940, the equation was significant. Using the model we got the forecast score of 129.33. In the model confirmatory test, its expression level was 40.02%, the score was 140.67. Overall, the model predicted a reliable result.4. Finally, the work applied 20L fermenter to expand the gene engineering bacteria pGEX-6P-1/CC34/BL21 (DE3) cultivation to 10L, the fusion protein expression level at 30h fermentation was 41.60%, bacteria density was OD600=11.76, fusion protein purification was 545.25mg/ml, it means that 1L fermentation broth could get 545.25mg active CC34 fusion protein. So we achieved the combination between lactose induction and fermentation, and established the basis for the fermentative production of hybrid peptide CC34.