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为获得可以区分自然感染和疫苗免疫且毒力较弱的布鲁氏菌候选疫苗株,本实验研究了磷酸葡萄糖变位酶基因(pgm)对布鲁氏菌(Brucella melitensis)M5-90疫苗株毒力的影响,并对其进行了免疫评价。本实验构建了重组质粒pGEM-7zf-Δpgm,电转化布鲁氏菌M5-90感受态细胞,筛选获得布鲁氏菌疫苗株M5-90的pgm基因缺失株(Δpgm),并对获得的M5-90 Δpgm株进行遗传稳定性、免疫原性检测。结果显示,Δpgm能稳定传15代;Δpgm组的抗体含量较亲本株M5-90组差异不显著;Δpgm在诱导机体产生IL-2的能力要强于M5-90,产生INF-γ的能力低于M5-90;该基因缺失株采用虎红平板凝集试验和试管凝集试验不发生凝集反应;Western blot证实,Δpgm不能诱导小鼠产生抗pgm蛋白的抗体。本研究构建的Δpgm具有很好的遗传稳定性和免疫原性,为研制布鲁氏菌基因缺失疫苗提供了实验依据。
In order to obtain a Brucella vaccine candidate that can distinguish natural infection from vaccine and virulence, the present study investigated the effect of phosphoglucomutase (pgm) on Brucella melitensis M5-90 vaccine strain The impact of virulence and immunological evaluation. In this study, a recombinant plasmid pGEM-7zf-Δpgm was constructed and electrotransformated into Brucella M5-90 competent cells. The pgm gene deletion mutant (Δpgm) of Brucella vaccine strain M5-90 was screened and the M5- -90 Δpgm strain for genetic stability, immunogenicity detection. The results showed that Δpgm was stable for 15 generations. The antibody level in Δpgm group was not significantly different from that in the parental strain M5-90. Δpgm had stronger ability to induce IL-2 production than M5-90 and INF-γ production ability M5-90; the gene deletion strain by tiger red plate agglutination test and tube agglutination test does not occur agglutination reaction; Western blot confirmed that Δpgm can not induce mouse anti-pgm protein antibody. The Δpgm constructed in this study has good genetic stability and immunogenicity, providing an experimental basis for the development of Brucella gene-deleted vaccine.