Fusion expression of antigen dominant epitopes from outer membrane protein A and adhesin protein in Klebsiella pneumoniae and its immune protection in mice
Objective To evaluate the immunoprotective effect of the dominant epitope fusion protein of Klebsiella pneumoniae(KP)outer membrane protein A(OmpA)and adhesin MrkD on KP-infec-ted mice.Methods According to the KP ompA and mrkD sequences published by the National Center for Biotechnology Information(NCBI),the dominant epitopes of these two proteins were selected through bioin-formatics analysis,and the epitopes were amplified by overlap polymerase chain reaction(PCR).The fu-sion sequence was cloned into the pColdI vector,and transformed into Escherichia coli BL21(DE3)stored in our department,induced by isopropyl β-D-thiogalactoside(IPTG),and the recombinant protein rAD(recombinant OmpA and MrkD) was obtained after Ni+chromatography gel purification.A total of 22 BALB/c mice were subcutaneously immunized with rAD,and the adjuvant control group was set at the same time.After the immunization procedure,the serum anti-rAD antibody level in the mice and the con-tents of cytokines after rAD stimulation in the spleen supernant in vitro were measured by enzyme-linked immunosorbent assay(ELISA),and BALB/c mice were challenged intranasally with KP.The survival rate of mice and the CFUs of KP in the lungs were measured.The t test was used for comparison between groups.Results The epitope sequences of ompA and mrkD were respectively amplified and successfully fused.The length of the fusion fragment was 1 056 bp,which was consistent with the predicted size.After being cloned into the pColdI vector and sequenced,the recombinant expression strain BL21(DE3)-pColdI-rAD was induced by IPTG,and the supernatant was collected after ultrasonic cracking then purified to obtain soluble rAD with a size of 38.4 × 103,which was consistent with the predicted size.After the BALB/c mice were boosted twice,the anti-rAD antibody titer in the immunized group was significantly higher than that in the control group(170 667.00±59 121.00 vs.0.00±0.00,t=5.00,P＜0.01).After mice were chal-lenged with KP,the survival rate of the mice in the immunization group was significantly higher than that of the control group[(65.00±5.00)％vs.(0.00±0.00)％,t=22.52,P＜0.01],and the CFUs of KP in the lungs of the mice in the immunization group was significantly lower than those of the control group[(9 500.00±1 384.00)vs.(87 833.00±25 365.00),t=7.55,P＜0.01].After splenocytes were cul-tured and stimulated by rAD in vitro,the counts of tumor necrosis factor-α(TNF-α),interleukin-4(IL-4)and interferon-γ(IFN-γ)in the immune group were significantly higher than those in the control group[(115.70±17.21)pg/ml vs.(24.00±9.17)pg/ml,t=8.14,P＜0.01;(200.30±8.51)vs.(9.00±3.00)pg/ml,t=36.75,P＜0.01;(92.33±6.11)vs.(9.67±1.53)pg/ml,t=22.73,P＜0.01].Conclusion Immunization of BALB/c mice with KP OmpA and MrkD dominant antigen epitope fusion protein rAD contributes to its resistance to KP infection.
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