Study On The Culture Conditions And Stability Of PCI-chIL-4-chIL-2-EGFP Recombinant Escherichia Coli

In order to improve the immune response of live coccidiosis vaccine,we developed a eukaryotic expression vector p CI-ch IL-4-ch IL-2-EGFP of chicken interleukin 4(ch IL-4)and chicken interleukin 2(ch IL-2)fusion gene.The ch IL-4-ch IL-2 fusion protein expressed and secreted by this vector can enhance the immune protection of the live vaccine of chicken coccidiosis and reduce the side effects of live vaccine.In order to establish a large-scale plasmid p CI-ch IL-4-ch IL-2-EGFP production,extraction and purification process,and to establish the quality control system of the product,this study screened the fermentation culture conditions of recombinant plasmid-carrying bacteria,and the extraction and purification process of the plasmid was improved.The preservation stability and genetic stability of the recombinant bacteria carrying the plasmid p CI-ch IL-4-ch IL-2-EGFP were studied,and a tertiary seed bank was established.The results showed that:(1)The highest plasmid yield obtained by the recombinant strain in four kinds of medium of 2�YT,LB,SOB,TB decreased sequentially under the conditions of 37?,42? and logarithmic growth phase from 37?to 42?,and the difference was extremely significant(P<0.01),but the cost of producing 1g plasmid,LB,SOB,2�YT,TB medium increased sequentially.Indicating that LB medium is more suitable.The plasmids were produced by large-scale fermentation;the yields of the four cultures were decreased at37?,42?,and 42?,and the difference was extremely significant(P<0.01).Indicating that the growth phase was the induction of the recombinant bacteria at 42? can significantly increase the plasmid yield.(2)The recombinant bacteria was passaged by a continuous passage method,and the recombinant bacteria which carried the plasmid p CI-ch IL-4-ch IL-2-EGFP in 0 to 30 generations were all red corynebacteria,can be used for fermenting the glucose and the mannitol without fermenting the lactose,and the homology with the escherichia coli 16 s r RNA fragments were more than 95%.The supercoiled ratio of each generation plasmid was greater than 90%,the loss rate of the plasmid was 0,and the double-enzyme digestion of the plasmid appeared to be about 882 bp and about 4.7 kb.Indicating that the30-generation plasmid was split and the structure was stable,and the transfection rate of each of the cellswas increased.The expression of the fusion protein was not significant(P>0.05),and the secretion protein could effectively promote the proliferation of the spleen lymphocytes of the chicken,but the difference of the stimulation index was not significant(P>0.05).Indicating that the function of the recombinant plasmid was stable.The results showed that the recombinant bacteria carrying the plasmid p CI-ch IL-4-ch IL-2-EGFP can be stably passaged to 30 generations.At the same time,the three-stage seed bank was established according to the 2015 edition.(3)Within three months,there was no significant difference in the number of living bacteria between the freeze-dried group and the pre-preservation group(P>0.05),while the number of living bacteria stored at 4? 8% glycerol for two months for three months was significantly(P<0.05)lower than that before preservation,and the decreasing rate increased in turn.When preserved for three months,the decrease rate of 8% glycerol preservation bacteria(10.56%)was significantly higher than that of-80 ? gelatin sucrose preservation bacteria(-2.78%)(P <0.05).There was no significant difference(P >0.05),no significant difference in plasmid content(P > 0.05),and there were about 882 bp and about 4.7 kb bands in gel electrophoresis of plasmid double enzyme digestion products in the other condition preservation bacteria(P>0.05),and there was no significant difference in plasmid content(P>0.05).The results showed that the recombinant Escherichia coli could be preserved stably in three months by sucrose-skim milk freeze-drying,gelatin-sucrose freeze-drying and 8% glycerol preservation,except 4? 8% glycerol.(4)The minimum of CTAB concentration of the completely precipitated plasmid DNA was0.006%.The RNA and the g DNA were not detected in the plasmid DNA extracted with the 0.006% CTAB of the final concentration and the 0.4 mol/L Mg Cl2,the yield of the plasmid was 590 ?g/m L,and the purity was 1.84.The contents of endotoxin and protein were 0.014 EU/?g and 0.036 ?g/?g of plasmid DNA.