Study On The Purification And Stability Of PCI-chIL-4-chIL-2-EGFP Recombinant Plasmid

The order to understand the stability of pCI-chIL-4-chIL-2-EGFP recombinant plasmid,optimize the purification scheme of plasmid,reduce the residual amount of foreign protein,make it meet the standard of Food and drug administration(FDA)and improve the freeze-dried bacterial survival rate of pCI-chIL-4-chIL-2-EGFP recombinant strain.In this study,the improved alkali lysis method of plasmid extraction,the diatomite method by orthogonal test to purify recombinant gene adjuvant plasmid,and the preservation stability and use stability test of plasmid were optimized,which could provide dates to establish the extraction,purification process and stability of plasmid.Orthogonal test was used to screen the best formula of freeze-dried protective agent for pCI-chIL-4-chIL-2-EGFP recombinant Escherichia coli,which provided the basis for the quantitative production of the recombinant gene adjuvant.The research results showed that:(1)The effects of skimmed milk(range R 10.00),trehalose(range R 12.31)and glycerol(range R 29.74)on freeze-drying protection of pCI-chIL-4-chIL-2-EGFP recombinant bacteria increased in turn.The results showed that Ij value of glycerol:Ij3>Ij2>Ik1;Ij value of trehalose:Ij3>Ij2>Ij1;Ij value of skimmed milk Ij2>Ij1>Ij3,therefore the best formula of freeze-dried protective agent was A3B3C2,which was composed of 15%trehalose,3%glycerol and 10%skim milk.The survival rate of pCI-chIL-4-chIL-2-EGFP recombinant strain reached 96.15%.(2)The plasmid yield of modified alkali lysis method was significantly higher than that of CTAB method and LiCL method(P<0.01).In the improved alkali lysis method,the effects of the improved alkaline lysis method on the extraction quantity of supercoiled plasmid were final concentration of NaOH(range R 112.65),OD value of bacterial solution(range R 63.90)and lysis time(range R 41.05).The results showed that Ij value of lysis time:Ij3>Ij2>Ij4>Ij1;Ij value of final concentration of NaOH:Ij3>Ij4>Ij2>Ij1;Ij value of OD value of bacterial suspension:Ij1>Ij4>Ij3>Ij2,so A3B3C1 was the best combination in the extraction process of supercoiled plasmid.The modified alkaline lysis method with the final concentration of NaOH of 0.18 moL/L and the lysis time of 5 min was used to extract plasmid from the bacterial suspension with OD600 value of 0.6.The results showed that high-yield supercoiled plasmid samples could be obtained by this method.(3)In the orthogonal test,the effects of guanidine hydrochloride,NaCl and diatomite on plasmid yield and protein residue decreased in turn.The Ij value of guanidine hydrochloride:Ij3>Ij2>Ij1;the Ij value of NaCl:Ij3>Ij2>Ij1;the Ij value of diatomite:Ij3>Ij2>Ij1,so the optimal level of plasmid extraction was A3B3C3.The Ij value of guanidine hydrochloride:Ij1>Ij2>Ij3;the Ij value of NaCl:Ij3>Ij2>Ij1;the Ij value of diatomite:Ij3>Ij2>Ij1,so the optimal level of protein residue was A1B3C1.In the validation test,there was no significant difference in the protein content among A3B3C1,A2B3C1 and A1B3C1 groups(P>0.05),and they were all less than the FDA standard of 0.01 μg/μg plasmid.The production of plasmid in A3B3C3,A3B3C1,A2B3C1 and A1B3C1 groups decreased in turn.There was no significant difference between A3B3C3 and A3B3C1 groups(P>0.05),and there was no significant difference between A2B3C1 and A1B3C1 groups(P>0.05),but the former two groups were significantly higher than the latter two groups(P<0.01).Therefore,A3B3C1 was the combination for removing proteins from plasmid purified by diatomite method.When the concentrations of diatomite,guanidine hydrochloride and NaCl were 0.1 g/mL,5 mol/L and 0 mol/L,the purity,protein residue and endotoxin content of purified plasmid samples were 1.82,0.008 μg/μg plasmid and 24.54 EU/mg plasmid,which meet FDA standards.(4)There was no significant difference in the purity,concentration and pH of plasmids between TE solution and PBS solution stored at-80℃ and-20℃(P>0.05)for 12 months.At 12 months,the supercoiled ratio of TE plasmid solution at-80℃ was not significantly different from that before preservation(P>0.05).The supercoiled ratio of PBS plasmid solution stored at-80℃ for 9～12 months was significantly lower than that before storage(P<0.01),and that of PBS plasmid solution stored at-20℃ for 6～12 months was significantly lower than that before storage(P<0.01).At 12 months,the supercoiled ratio of all preservation groups was more than 80%,and there were about 900 bp and about 4.7 kb bands in gel electrophoresis of plasmid double enzyme digestion products,which indicated that the recombinant plasmid could be stored stably in TE solution and PBS solution within 12 months,the storage stability of TE solution was better than PBS solution,and the stability of plasimd at-80℃ was better than-20℃.(5)The purity,concentration,pH and supercoiled ratio of plasmid samples which placed at 25℃ for 8 h were not significantly different from those before 0 h(P>0.05),and there were about 900 bp and about 4.7 kb bands in gel electrophoresis of plasmid double enzyme digestion products,which indicated that the recombinant plasmid could be stably placed at 25℃ for at least 8 h.