鼠疫耶尔森菌小肽基因sORF34缺失株毒力及短期免疫保护效果评价

目的 探索小肽基因34(sORF34)缺失对鼠疫耶尔森菌毒力影响,并评价sORF34缺失株免疫保护活性。方法 基于鼠疫耶尔森菌201株和鼠疫耶尔森菌EV76疫苗株利用λ-Red一步法构建小肽基因缺失株。比较鼠疫小肽基因缺失株和亲本株之间的毒力差别,并采用皮下免疫方式对小鼠进行免疫,与EV76疫苗株比较,评价体液免疫、保护率等方面的差异。结果 PCR扩增结果证实,小肽基因缺失株构建成功。通过皮下LD₅₀测定、生存曲线测定,表明小肽基因缺失株较亲本株毒力下降。鼠疫201ΔsORF34和EV76ΔsORF34皮下免疫后可刺激机体产生抗F1-IgG,其中鼠疫201ΔsORF34免疫组抗体滴度与EV76疫苗株免疫组差异无统计学意义(P>0.05),EV76ΔsORF34免疫组较EV76疫苗株免疫组抗体滴度低;鼠疫EV76ΔsORF34株可刺激机体产生低滴度抗LcrV-IgG,而EV76和201ΔsORF34株不能刺激机体产生抗LcrV-IgG。初免后42 d使用致死剂量鼠疫201株进行皮下攻毒和滴鼻攻毒,3组保护率差异无统计学意义(P>0.05)。结论 小肽基因缺失株经皮下途径感染BALB/c小鼠的毒力下降,鼠疫EV76ΔsORF34株较EV76疫苗株残存毒力进一步下降,但保护率差异无统计学意义(P>0.05),EV76ΔsORF34株有作为鼠疫减毒活疫苗候选株的潜力。

Analysis of virulence and short-term immune protection in mice of sORF34 gene deletion mutants of Yersinia pestis

To explore the effects of sORF34 (small open reading frame, sORF) gene mutation on the virulence of Yersinia pestis and evaluate the immune protection efficacy of sORF34. The sORF34 gene was deleted from Y. pestis biovar Microtus strain 201 (avirulent to human) or the attenuated Y. pestis strain EV76, and their virulence were evaluated in a mouse model via subcutaneous injection (s.c.). The virulence of 201ΔsORF34 or EV76ΔsORF34 was significantly reduced compared to their parental strains. Here, the humoral immune responses and protective efficacy were examined after subcutaneous immunization of mice with the above-mentioned mutants and the attenuated Y. pestis strain EV76. The 201ΔsORF34 and EV76ΔsORF34 induced high levels of anti-F1 IgG in the sera of mice immunized by s.c. route. Among them, there was no significant difference between the antibody titer of the 201ΔORF34 immunized group and the EV76 immunized group, but lower antibody titer in the EV76ΔsORF34 immunized group was observed. EV76ΔORF34 strain can stimulate the body to produce low-titer anti-LcrV IgG, while EV76 and 201ΔsORF34 strains cannot. Moreover, after lethal challenge with Y. pestis biovar Microtus strain 201 by subcutaneous and intranasal (i.n.) routes, the protective efficacy of 201ΔsORF34-immunized group and EV76ΔsORF34-immunized group were comparable to that of EV76-immunized group. Thus, the EV76ΔsORF34 represents a live-attenuated vaccine candidate against plague.