| CD40L is a type II transmembrane glucoprotein on the surface of activated T cells. CD40L belongs to tumor necrosis factor superfamily. The interaction of CD40L and CD40 plays animportant role in the interaction between antigen-specific T lymphocytes and antigen-presenting cells. More and more studys show that CD40L can enhance the immunized effect of vaccine significantly.In this study, CD40L gene was amplified by RT-PCR, and then the souble gene fragment (sCD40L) was cloned. sCD40L gene was contceted with Leucine zipper motif LZ and melittin. The recombinant CD40L was expressed with Bac-To-Bac expression system and immunoenhancement effect for PRRSV and FMDV vaccine was investigated in this paper.1. Cloning and sequence analysis for porcine CD40LIn this chapter, the full length of porcine CD40L gene was amplified from swine perpheral blood lymphocytes via RT-PCR, and the slouble fragment of porcine CD40L was amplified. Nucleotide sequence analysis shows that CD40L gene is relatively conservative between different species of pigs. However, the homology with other animals is relatively lower.2. Expression procine CD40L gene in E.coli and preparation of specific polyclonal antibodyIn this chapter, Porcine CD40L gene was cloned into prokaryotic expression vector pET-32a(+) to obtain prokaryotic recombinant expression plasmid pET-CD40L. CD40L gene was expressed in inclusion body when recombinant expression plasmid pET-CD40L was transformed into E. coli BL21 and induced with IPTG. The specific polyclonal antiserum was obtained from rabbit immunized with the purified recombinant fusion protein CD40L. Serum was separated and detected with indirect ELISA and Western-blot. Results showed that ELISA titer of the serum was obove 1:10000; Western-blot indicates that the antiserum can react with porcine CD40L specifically. 3. The expression and identification of the procine CD40L gene in insect cellsThree recominant baculovirus vectors pFastDual-CD40L, pFastDual-LZsCD40L and pFastDual-MLZsCD40L were sucessfully constructed in this chapter. Sf-9 cell was transfected with the positve rBacmids, following three recombinant plasmids were transformed into DH10Bac competent cells and identified with blue-white screening and PCR analysis. When the cytopathic effect (CPE) was obvious, the transfected sf-9 cell was harvested from the cell plate, and then positive recombinant virus was named as rBac-CD40L, rBac-LZsCD40L and rBac-MLZsCD40L. Insertion of target gene into Baculovirus genome was proved with PCR analysis; expression for target protein was identified with SDS-PAGE, Western-blot and fluorescence microscope observation.4. The study of the CD40L enhancement of the immune for PRRSV and FMDVIn order to test the immunoenhancement for recombinant protein CD40L and MLZsCD40L obtained in former chapter,40 piglets were randomly divided into eight groups with five of them in each group. Pigs were vaccined with inactivated PRRSV and FMDV antigen mixed with CD40L, MLZsCD40L or blank Baculovirus, and then emulsionized with mineral oil as adjuant. At the same time PRRSV and FMDV antigen only were used as control group, respectively. The immunized piglets were two weeks intervals after primary vaccination, and PRRSV and FMDV antibodies was detected with an commercial ELISA kit; Neutralizing antibody titer for each group was detected with virus neutralization test assay to evaluate the humoral immune response; The cellular immunity response for the immunized chicken was estimated according to lympocyte subtype ananlysis with cell flow cytomatry and virus specific cytokine detection. |
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