|
Major pathogens of respiratory syncytial virus (Respiratory syncytial virus, RSV) lower respiratory tract infections in infants, immunocompromised persons and the elderly, there is still no safe and effective RSV vaccine market. The experiment will GCTL gene (RSV G protein gene 130-230 fragment, which replaced with CTL epitopes the the CX C motif) and AgB5B gene (Mycobacterium tuberculosis early secretory protein the Ag85B gene) fusion expression supplemented mucosal adjuvant immune Kunming mice, analysis and evaluation of the immunogenic protein. The the PCR obtained GCTL and Ag85B gene, build expression the carrier GCTL-tubercle antigen 85B and-pET30a tubercle antigen 85B and-pET28a, these two carriers already designed carrier GCTL-pET22b were transformed into E. coli BL21 expression. Application His-Tag affinity with Ni ions GCTL-Ag85B, Ag85B protein was purified by dialysis refolding can be obtained directly purified GCTL. Three proteins of purified GCTL-Ag85B, Ag85B, GCTL with non-toxic E. coli heat-labile enterotoxin (Heat-labil enterotoxin, LT) the mixed immune Kunming mice, immune total of five groups, respectively : GCTL-Ag85B LT, Ag85B LT, GCTL LT, LT, PBS, immune 0,2,4 weeks. After the end of the immunization for serum IgG, IgA, IgG2aIgG1 respiratory sIgA by ELISA determination count, blood and lung lavage eosinophils and basophils. -Tapping 106pfu/ml RSV intranasally immune to poison the mice for 3 consecutive days. The mice were killed after the end of the challenge, again for serum IgG, IgA in IgG2aIgG1 respiratory sIgA by ELISA determination in the blood and lung lavage eosinophils and basophils count. GCTL-Ag85B, Ag85B, GCTL the form of inclusion bodies in E. coli expression of induced the 4h expression respectively, accounted for 20% of the total protein at 37 ℃, 40%, 30%, expressed by Western Blotting results prove the target protein GCTL-Ag85B, GCTL tubercle antigen 85B and protein. Tubercle antigen 85B and the after GCTL-affinity purified His-Tag and Ni ions, tubercle antigen 85B and protein purity of 90%, after a purity of 92% of the direct GCTL purified dialysis. Immunoassay results show that: the serum IgG in the second, three times after immunization, the GCTL-of Ag85B LT, the GCTL LT group antibody titer higher than that of Ag85B LT, LT, the PBS group (P lt; 0.05); after the third immunization serum IgA and the immune lung lavage slgA GCTL-tubercle antigen 85B and LT GCTL LT group antibody titers higher than Ag85B LT, LT, PBS group (P lt; 0.05); serum after immunization IgGl/IgG2a, results show GCTL- the Ag85 LT group relative to the immune GCTL LT tubercle antigen 85B and LT group polarization equilibrium; simultaneously balanced Th1/Th2 regulation of the immune polarization LT; addicted to the blood and lung lavage fluid protein group mice after immunization The P13S high acidic granulocytes and basophils ratio (P lt; 0.05). Challenge test results showed that: After the challenge GCTL-Ag85B LT, GCTL LT group IgG titers higher than tubercle antigen 85B and LT, The LT, PBS group (P lt; 0.05); IgG2aIgGl no difference after immunization and reactive toxic; Serum IgA and sIgA after challenge GCTL-Ag85B LT, GCTL LT group antibody titers higher than tubercle antigen 85B and LT, LT, PBS group (P lt; 0.05); challenge GCTL-Ag85B LT group eosinophils quantity and basophilic the number of granulocytes below the Ag85B LT group (P lt; 0.05) while higher than GCTL LT group (P lt; 0.05). success of this experiment was constructed the-tubercle antigen 85B and-pET30a expression vector GCTL, tubercle antigen 85B and-pET28a, and expressed and purified this two proteins and GCTT. protein, and measuring the immunogenicity of the protein. Experimental results show that mixed with LT adjuvant to improve the body's mucosal secretion of sIgA amount the GCTL-of Ag85B relative GCTL and Ag85B polarization equilibrium the GCTL-of Ag85B phase for Ag85B can be reduced of eosinophil after challenge and The number of basophils, thereby reducing the inflammatory response, the challenge experiments also demonstrated GCTL-Ag85B immunogenicity. Immunogenicity of respiratory syncytial virus fusion protein GCTL-Ag85B laid the foundation for RSV vaccine development.
|