Prokaryotic Expression Of Murine CD40L And Its Immunopotentiating Effect On Two Toxic Haptens

The aim of this study is to express recombinant murine CD40 L protein by E.coli expression system,and to investigate its immunopotentiating effect on Tetrodotoxin(TTX)artificial complete antigen in BALB/c,for further to development a suitable immune enhancement adjuvants for hapten efficient preparation of monoclonal antibody provides new train of thought and experimental basis.Total RNA from BALB/c’s spleen is extracted by Trizol reagent and reverse transcription into cDNA.Primers are designed according to CD40L’s CDs region and soluble CD40L’s(sCD40L)genes sequence.The target genes sequence are amplified by PCR.The recombinant vector pGEX4T-1-CD40 L and pET-28a-sCD40 L is constructed for prokaryotic expression,and the recombinant CD40L/sCD40 L protein is purified.According to Mannich reaction principle,TTX-BSA and TTX-OVA were prepared by formaldehyde method.BALB/c mouse are immunized with the adjuvant of artificial recombinant CD40 L as the experimental group,while immunized with the adjuvant of Freund’s as the control group.Antibody levels of each group in serum are analyzed by ELISA and SPSS to investigate the effect of recombinant CD40 L protein on the immune effect during TTX-BSA immunization.MC-LR is coupled with BSA and OVA by Carbon Diimide method to prepare immunogen MC-LR-BSA and detection MC-LR-OVA.BALB/c mouse inject with the artificial recombinant sCD40 L adjuvant as the experimental group,and the Freund’s adjuvant as the control group.The immune effects of the recombinant sCD40 L protein in MC-LR and MC-LR-BSA is analyzed by ELISA and SPSS software.With reference to the conserved sequences of β-actin,IL-2,IFN-γ,IL-4,IL-5,CD19,CD21,CD80,CD86,CD138 in GenBank,the mRNA’s forward and reverse primers are designed respectively by referring to the GeneBank and Primer Premier 5.0.The transcription levels of Cytokines and Cluster of differention are detected by SYBR Green I Quantitative Real-time PCR,and the reverse transcription product cDNA of the mouse spleen total RNA is used as the template.Using mouse interleukin ELISA to expression the levels of IL-2,IL-4,IL-5 and IFN-γ,the serum of mouse is detected by one-step double antibody Sandwich ELISA.The results showed that the 783 bp CD40 L and the 505 bp target gene containing sCD40 L regions are successfully amplified,and expression and purification of the 55 kD CD40 L recombinant protein fused with GST-tag and 18 kDa sCD40 L recombinant protein fused with His-tag by prokaryotic.The results of mouse immunized with recombinant CD40 L protein and TTX-BSA showse that CD40 L has a better immune-enhancing effect compared with ordinary Freund’s adjuvant at the initial stage of immunization(P < 0.01).The experimental results of mouse immunized by sCD40 L recombinant protein with MC-LR and MC-LR-BSA showed that sCD40 L not only make the body produce an immune response to MC-LR hapten,but also achieved an immune enhancement similar to that of Freund’s adjuvant,and the effect is faster than Freund’s adjuvant.Further studies confirme that sCD40 L can bind to CD40,facilitate the mature of DCs,stimulate the transcriptional expression of CD80 and CD86,promote the proliferation and transformation of B cells into plasma cells,stimulate the transcription and expression of Th1 and Th2 cytokines,and enhance the immune response of Th2.In conclusion,whether CD40 L or sCD40 L can enhance the immune response to small hapten,than Freund’s adjuvant which not only need to emulsion,but also need to make small hapten coupling with a carrier protein to adjuvant,CD40L/sCD40 L adjuvants is simple to use,quick effect,which lead a foundation for further development of immunopotentiation adjuvants suitable for the efficient preparation of small hapten antibodies.