肺炎克雷伯菌外膜蛋白A和黏附素蛋白抗原优势表位融合表达与小鼠免疫保护效果

Fusion expression of antigen dominant epitopes from outer membrane protein A and adhesin protein in Klebsiella pneumoniae and its immune protection in mice

目的探讨肺炎克雷伯菌(KP)外膜蛋白A(OmpA)和黏附素MrkD优势抗原表位融合蛋白对KP感染小鼠的免疫保护作用。方法根据(美国)国家生物技术信息中心(NCBI)公布的KP ompA和mrkD序列,通过生信分析选取这两个蛋白的优势抗原表位,经融合聚合酶链反应(PCR)扩增表位序列后克隆至pColdI载体,并转入本科室保存的大肠杆菌BL21(DE3),经异丙基硫代半乳糖苷(IPTG)诱导,Ni+层析凝胶纯化后获得重组蛋白rAD。rAD经皮下免疫22只BALB/c小鼠,同时设置22只佐剂对照组。完成免疫程序后通过酶联免疫吸附试验(ELISA)对小鼠血清中的抗体水平和脾脏体外抗原刺激后细胞因子分泌水平进行测定,并对小鼠鼻腔注射KP,观察免疫后的小鼠存活率和肺脏细菌数量。组间比较采用t检验。结果分别扩增出ompA和mrkD抗原表位序列,并成功融合,融合片段大小为1056 bp,与预测大小一致,克隆至pColdI载体后测序,测序正确。重组表达菌BL21(DE3)-pColdI-rAD经IPTG诱导后破碎收集上清纯化,获得可溶性rAD,大小为38.4×10^(3),与预测大小一致。rAD 3次免疫BALB/c小鼠后,免疫组抗体滴度显著高于对照组(170667.00±59121.00比0.00±0.00,t=5.00,P<0.01)。KP感染小鼠后,免疫组小鼠存活率显著高于对照组[(65.00±5.00)%比(0.00±0.00)%,t=22.52,P<0.01],免疫组小鼠肺脏细菌数显著低于对照组[(9500.00±1384.00)CFU比(87833.00±25365.00)CFU,t=7.55,P<0.01]。脾细胞分离后经rAD刺激,免疫组肿瘤坏死因子-α(TNF-α)、白细胞介素-4(IL-4)、干扰素-γ(IFN-γ)分泌量均显著高于对照组[(115.70±17.21)pg/ml比(24.00±9.17)pg/ml,t=8.14,P<0.01;(200.30±8.51)pg/ml比(9.00±3.00)pg/ml,t=36.75,P<0.01;(92.33±6.11)pg/ml比(9.67±1.53)pg/ml,t=22.73,P<0.01]。结论KP OmpA和MrkD优势抗原表位融合蛋白rAD,免疫BALB/c小鼠有助于对KP感染的抵抗。

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-infected 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 bioinformatics analysis,and the epitopes were amplified by overlap polymerase chain reaction(PCR).The fusion sequence was cloned into the pColdI vector,and transformed into Escherichia coli BL21(DE3)stored in our department,induced by isopropylβ-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 contents 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 1056 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 38.4×10^(3),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(170667.00±59121.00 vs.0.00±0.00,t=5.00,P<0.01).After mice were challenged 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[(9500.00±1384.00)vs.(87833.00±25365.00),t=7.55,P<0.01].After splenocytes were cultured 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.