RÉSUMÉ
Objective:To develop a recombinant protein vaccine based on KPC-2, a drug resistance target in Klebsiella pneumoniae, and evaluate its immunogenicity, protective efficacy and mechanism in a mouse model of pneumonia. Methods:KPC-2 was expressed in Escherichia coli and purified using GST affinity chromatography. A recombinant protein vaccine was prepared with KPC-2 and used to immunize New Zealand rabbits through subcutaneous injection. Serum samples were isolated from cardiac blood and Protein G chromatography was used to purify polyclonal antibodies against KPC-2. Opsonophagocytic killing assay was used to assess the bactericidal activity of the polyclonal antibodies in vitro. Female BALB/c mice were immunized three times with the recombinant protein vaccine, and the titers of specific IgG antibodies in serum were measured by indirect ELISA. One week after the last vaccination, the mice were infected with Klebsiella pneumoniae strain SRT through tracheal intubation, and received a single intravenous dose of meropenem (0.1 mg) 1 h later. The protective efficacy of the KPC-2 recombinant protein vaccine was evaluated by comparing the survival rates, bacterial colonization and histopathological changes between vaccine group and adjuvant group as well as the survival rates between meropenem group and normal saline group. Moreover, the protective efficacy of polyclonal antibodies against KPC-2 was evaluated through passive immunization. Results:The level of specific IgG antibodies in serum was significantly higher in the vaccine group than in the adjuvant group ( t=4.325, P<0.05). The survival rate in the vaccine group was also higher than that of the adjuvant group [70% (7/10) vs 10% (1/10), P<0.05]. Furthermore, lung inflammation was less severe and bacterial burden was reduced in the vaccine group as compared with those of the control group ( t=3.127, P<0.05). Both active and passive vaccination strategies demonstrated strong protective efficacy against Klebsiella pneumoniae infection, and had a synergistic effect when used in combination with antibiotic therapy. The polyclonal antibodies against KPC-2 had bactericidal activity in vitro ( t=5.427, P<0.05). Conclusions:The prepared KPC-2 vaccine has better immunogenicity and protective efficacy. It can induce strong humoral immune responses. This study suggest that drug resistance target may be used as a candidate antigen for future vaccine development.
RÉSUMÉ
Objective To construct recombinant plasmids containing HPV18E7 gene ,and explore the optimization condition of its expression in Escherichia coli .Methods The genomic DNA extracted from HeLa cell line which served as a template to the HPV18 E7 gene was amplified using PCR method ;and the amplified product of HPV18E7 gene was connected to the pET-32a(+ ) vector ,which composed the pET-32a(+ )-HPV18E7 recombinant plasmid ;the positive recombinant plasmids were transformed into BL21-DE3-pLysS competent cells and the optimized expression condition was explored in order to obtain a large amount of HPV18E7 oncogenic protein .Results The fragment length of PCR products of HeLa cell genomic DNA was consistent with that of HPV18 E7 gene .In LB medium ,the expression level of the target protein was not high under such conditions as different concentra-tion of IPTG and lactose ,different temperatures and different induction starting amount .Therefore the ZYM-5052 auto-induction medium was tried in this experiment ,and the expression amount of the fusion protein was much higher than that induced with IPTG and lactose .Conclusion The amount of HPV18E7 fusion protein in ZYM-5052 automatic induction medium is much higher than that induced with IPTG and lactose .