The Research Of The Respiratory Syncytial Virus Fusion Protein G_CTL-Ag85B's Immunogenicity

Respiratory syncytial virus is the major pathogen of respiratory infection for infants and young children, immunodeficient people and old people; there are still no effective vaccine available now. In this study, The gene of GCTL (truncated RSV G protein fragment 130-230, which replaces the CX3C motif by CTL epitope of RSV M protein) and Ag85B (the early secretory protein Ag85B gene of MTB) were fused, expressed then proteins were immunized mice with mucosal adjuvant.Through PCR the gene of Ag85B, GCTL were gotten; the express vector GCTL-Ag85B-pET30a,Ag85B-pET28a were constructed, then proteins expressed in E. coli. GCTL-Ag85B, Ag85B protein were purified by His-Tag, GCTL protein refolded by dialysis and purified directly. The purified GCTL-Ag85B, Ag85B, GCTL and non-toxic E. coli heat-labile enterotoxin (LT) proteins were mixed and immunized mice. The immuned mice were divided into 5 groups as following:GCTL-Ag85B+LT, Ag85B+LT, GCTL+LT, LT, PBS. The mice were immunized in 0,2,4 weeks. A week after the last immunization half of the mice were sacrificed, the lefted mice were callenged with RSV. The immunine indices of the mice were measured, included serum IgG, IgA, IgG2a/IgG1, the lung lavage sIgA, eosinophils and basophils quantity in blood and lung lavage fluid.GCTL-Ag85B, Ag85B, GCTL proteins were expressed in 37℃for 4 hours with great quantity by E. coli which were more than 20%,30%,20% of the total proteins, respectively. At last, purity of these proteins'were more than 90% after been purified, and the target proteins were confirmed to be GCTL-Ag85B, Ag85B, GCTL proteins by Westen Blotting.Immunoassay results showed that the serum IgG titer after the second and third immunization of GCTL-Ag85B+LT, GCTL+LT groups were higher than Ag85B+LT, LT, PBS groups (P<0.05); the serum IgA titer after the third immunization of GCTL-Ag85B+ LT, GCTL+LT groups were higher than Ag85B+LT, LT, PBS groups (P<0.05); lung lavage fluid's sIgA after third immunization of GCTL-Ag85B+LT, GCTL+LT groups were higher than PBS, Ag85B+LT and LT groups (P<0.05). The ratio of the IgG2a/IgG after third immunization showed that GCTL-Ag85B could balance the polarization of GCTL and Ag85B while LT has the effect of balance Thl/Th2.Challege results showed that the IgG titers in serum in the GCTL-Ag85B+LT, GCTL+ LT group was higher than Ag85B+LT, LT, PBS groups (P<0.05); the ratio of IgG1/IgG2a had no difference between the ration that after immunization. Serum IgA and lung lavage fluid's sIgA titer results showed that GCTL-Ag85B+LT group and GCTL+LT group were higher than other groups (P<0.05). The number of GCTL-Ag85B+LT groups'eosinophils and basophils in the blood and lung lavage fluid's were between the GCTL+LT group's and Ag85B+LT group's (P<0.05).In this study, proteins of GCTL-Ag85B, Ag85B and GCTL were expressed and purified, and their immunogenicity were determinationed. The results showed that mixing with LT adjuvant increased the titer of sIgA secreted through mucosal; while GCTL-Ag85B protein balanced Thl/Th2 polarization of GCTL and Ag85B; GCTL-Ag85B protein decreaced the number of eosinophils and basophils in blood and lung lavage afer challedged and the inflammatory was decreased. Challege experiment showed that the fusion protein was immunogenicity. This study lay basis for the further research of RSV vaccine.