PAN Yue;CAO Qi;REN Honglin;HU Pan;LI Yansong;ZHOU Yu;LIU Zengshan;LU Shiying;Key Laboratory of Zoonosis Research,Ministry of Education,Institute of Zoonosis,Jilin University;

CD40L;;adjuvant;;tetraodotoxin;;immune effect

The aim of this study was to express recombinant protein CD40 L from mice by E.coli expression system,and investigate its immunopotentiating effect on tetrodotoxin(TTX) artificial complete antigen in BALB/c mice.Total RNA from spleen of BALB/c mice was extracted by Trizol reagent and retranscribed into cDNA.Primers were designed according to CD40 L CDS region,and the target gene was amplified by PCR.The recombinant vector pGEX4 T-1 was constructed for prokaryotic expression,and the recombinant CD40 L protein was purified.TTX-BSA and TTX-OVA were prepared by formaldehyde method according to Mannich reaction principle.The adjuvant of artificial recombinant protein CD40 L was used as the experimental group and the adjuvant of Freund's was used as the control group.BALB/c mice were immunized.The immune effects of each group was analyzed using ELISA method combined with SPSS 19.0 software to explore the effect of recombinant CD40 L protein on the immune effect during TTX-BSA immunization.This experiment successfully amplified 783 bp CD40L gene,prokaryotic expression and purification of GST-tagged 55 ku mice CD40 L recombinant protein.Compared with TTX-BSA synergistic immunization in mice,CD40 L had extremely significant immune enhancement effect at the initial stage of immunization(P<0.01) than Freund's adjuvant.In conclusion,the recombinant protein CD40 L and TTX-BSA complete antigen synergistically immunized mice could enhance the body's response to hapten at the initial stage of immunization,which laid a foundation for further development of immunopotentiation adjuvants suitable for the efficient preparation of small hapten antibodies.