| Objective: Recombinant lactococcus lactis(r L.lactis)expressing CTB-Cap fusion protein was constructed and its immunological activity was assessed.Methods:1.The gene sequence of CTB and PCV2 Cap protein was obtained from the NCBI gene database,joined in tandem to generate the chimeric gene(CTB-Cap).Firstly,r L.lactis was constructed to express CTB-Cap in the expression vector p NZ8148 electricity into L.lactis NZ9000 after connection.After nisin induced expression of r L.lactis,SDS-PAGE and Western blot were used to identify the induced expression products while the concentration of CTB-Cap fusion protein was determined by ELISA.2.r L.lactis was placed in a simulatedgastric fluid,intestinal fluid and high osmotic pressure solution,colony counts were performed at different time points to detect the tolerance of r L.lactis.CFSE-labeled r L.lactis was given to mice by gavage,and the retention of the r L.lactis in the intestinal tract was detected by flow cytometry.3.r L.lactis was administered orally to immunized mice with doses of 1010 CFU,109 CFU,108 CFU and 107 CFU for 3 times,with an interval of 7 days and continuous immunization for 3 days each time.ELISA was used to detect the levels of Cap-specific Ig G in serum and Cap-specific s Ig A in feces and intestinal mucosa.4.r L.lactis was injected subcutaneously to immunized mice with doses of 2.5×1010 CFU,5×109 CFU,and 5×108 CFU doses for 2 times,with an interval of 14 d.ELISA was used to detect the levels of Cap-specific Ig G in serum.Results:1.The results of SDS-PAGE and Western blot showed that CTB-Cap fusion protein was expressed in r L.lactis,and 50.5 mg of fusion protein was obtained in 100 m L medium.2.The analysis results of the tolerance showed that the survival rates of r L.lactis were about 100%,85.7%,1.4% and 0 in the simulated gastric juice environment at p H 3.5,3.0,2.5 and 2.0 for 5 hours,respectively.The survival rate of r L.lactis was 89.4% in simulated intestinal fluid culture for 12 hours.The survival rate of recombinant bacteria was about 99.2%,81.7%,70.2% and 64.9% in 1%,3%,5% and7% solution of salt concentration for 5h,and about 9.5%,10.5%,5.8% and 1.4% in 12 h,respectively.The results showed that r L.Lactis has poor tolerance to simulated gastric fluid and good tolerance to simulated intestinal fluid and high osmotic pressure solution.Retention of r L.lactis in the intestinal tract showed that no fluorescence signal was detected in the intestinal tract of the mice after single gavage or continuous gavage,it showed that r L.lactis could not survive in the intestines of the mice.3.The analysis results of oral immunization showed that there was no significant change in Cap-specific Ig G and s Ig A levels(p>0.05)in r L.lactis mice compared with the blank bacteria group and the PBS group.The results showed that r L.lactis could not induce mucosal and humoral immune responses after oral immunization.4.Compared with the PBS group,the results of subcutaneous immunization showed that the level of Cap-specific Ig G was significantly increased in 2.5×1010 CFU group(0.05),while there was no significant change in the 5×109 CFU and 5×108 CFU groups(p>0.05).The results showed that r r L.lactis could induce humoral immune response after 2.5×1010 CFU immunization.Conclusion: r L.lactis,which can stably express CTB-Cap fusion protein,was constructed.After oral immunization,r L.lactis could not induce specific immune response,but could induce specific immune response after subcutaneous immunization. |
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