The Expression Of Porcine GM-CSF In Lactobacillus Casei And Evaluation Of Its Bioactivity

Granulocyte and macrophage colony stimulating factor (gm-csf) is composed of T cells, Bcells, macrophages, endothelial cells and fibroblasts secrete, it is an important hematopoieticgrowth factors and immune adjustment factor which can contribute to the survival, proliferationand differentiation of precursor cells and promote differentiation and granulocyte and monocytemacrophage colony formation, gm-csf play a key role in promoting dendritic cells’s differentiation,and antigen presenting cells gathered the the antigen at the injection site. Therefore, GM-CSF isconsidered a new generation of highly effective molecular adjuvant which has huge applicationpotential and development prospect. In this study, we designed primer to amplify pGM-CSF genewithout the signal peptide and got the protein in the form of secretion in L.casei ATCC393. Thenthe pGM-CSF protein was used with Pseudorabies Vaccine in order to research the effect about theimmune response which pGM-CSF played on.In this study, We designed special primers to the pUC57-pGM-CSF template and acquiredpGM-CSF gene without signal peptide area. The pGM-CSF gene was cloned into pET30b vectorand expressed in E.coli. SDS-PAGE and Western blot analysis showed that the pGM-CSF proteinwas about23ku and mainly in the form of inclusion body. Then in the help of molecularchaperone, we successfully acquired the soluble protein and its purified production through theNi-NTA. The results showed that the purified protein had better biological activity after testingPBMC’s proliferation, monocytes proliferation and colonies formation in soft agar.In addition, the pGM-CSF gene fragment was directly cloned into Lactobacillus expressionvector pPG-2, and constructed secretary expression system pPG-2-pGM-CSF/L.casei393. Therecombinant strains were induced by1%lactose, and the secreted expression protein in theexpression system was detected by Western blot. Furthermore, we detected the biological activityof the protein expressed in L.casei393in the same way of the protein expressed in E.coli, and itshowed better bioactivity on the proliferation of PBMC and monocytes. In order to explore theeffects of this immunoadjuvant, after the recombinant strains was induced, we concentrated thecultured supernatant, and100μg、50μg、25μg、12μg was used to combine with PseudorabiesVaccine. pGM-CSF group and pPG-2/L.casei393group were injected3days before or after theinjection of vaccine, and the anti-PRV immunoglobulin G (IgG) antibody was detected by indirect ELISA in the serum of immunized mice at0、4、7、10and14days after immunization. The resultsof ELISA showed that the groups of vaccine combining with pGM-CSF had higher antibody levelthan the group of vaccine. And in the4groups with different concentration of pGM-CSF, theantibody level of50μg group was higher than other3groups, and had significant difference(p<0.01) compared with the vaccine group. The25μg groups indicated significantly difference(p<0.05) than vaccine group. Meanwhile, we detected the antibodies’ neutralization titer by cellneutralization test, the neutralization titer of vaccine group was1:44.36; vaccine combining withpPG-2/L.casei.393group was1:46.3; vaccine combining with pGM-CSF group was1:62.5. Thespleen lymphocyte proliferation activity of immunized mice was tested by MTT assay. The resultshowed that lymphocytes proliferation index of vaccine combining with pGM-CSF group wassignificantly difference (p<0.01) compared with vaccine group and the group of vaccine combiningwith pPG-2/L.casei393. It indicated that vaccine combining with pGM-CSF group induced thebody producing a higher level of specificity immune response.14days after immunity,, allexperimental mice were challenged with PRV then observed the morbidity and deaths of eachgroup. The results showed that the protective rate of vaccine group and vaccine combining withpPG-2/L.casei393was33%and the group of vaccine combining with pGM-CSF was50%. All theresults showed that GM-CSF had good immunological effect as adjuvant, it could enhance thespecificity immune response levels, and to a certain extent, it could improve the protective efficacyof virus infection.In summary, the results obtained in this study showed that pGM-CSF protein expressed inlactobacillus casei had good immunogenicity and biological activity, and when used it withvaccine, it showed good adjuvant features, this provides experimental data and theoretical basis forthe application of the pGM-CSF, and laid the foundation for the further study of other vaccineadjuvants.