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目的观察由卡介苗(BCG)与结核分枝杆菌国际标准弱毒株H37Ra(H37Ra菌株)构建的融合菌株(B/R菌株)生长特性及其在免疫小鼠体内的生长情况。方法分别测定用7H9培养基培养3、6、9、12、15、18、21、24、27和30d的B/R菌株、BCG和H37Ra菌液在波长600nm处的A值,以时间为横坐标,A600值为纵坐标,绘制细菌生长曲线。将C57BL/6小鼠随机分为B/R菌株、BCG和H37Ra菌株3组,每组30只,分别皮下接种B/R、BCG和H37Ra菌液0.1ml(含活菌数为1.0×106个),免疫15、30、45、60和75d后无菌条件下取小鼠肝脏、脾脏和肺脏,制成脏器组织匀浆,接种罗氏固体培养基斜面,培养20d后计数各脏器菌落数(Log10cfu)。结果细菌生长曲线显示,B/R菌株的适应调整期较长,6d后进入生长对数期,27d后进入稳定期,生长周期约30d左右。免疫15、30、45、60和75d小鼠肝脏、脾脏、肺脏定植细菌数(Log10cfu)B/R菌株组高于BCG和H37Ra菌株组。小鼠肝脏定植细菌数(Log10cfu)B/R菌株组与BCG和H37Ra菌株组比较差异无统计学意义(P>0.05);免疫15、60及75d小鼠脾脏定植细菌数(Log 10cfu)B/R菌株组与BCG和H37Ra菌株组比较差异有统计学意义(P<0.05),免疫30和45d小鼠脾脏定植细菌数(Log10cfu)B/R菌株组与BCG组比较差异有统计学意义(P<0.05),与H37Ra菌株组比较差异无统计学意义(P>0.05);免疫15和45d小鼠肺脏定植的细菌数(Log10cfu)B/R菌株组与BCG和H37Ra菌株组比较差异有统计学意义(P<0.05),免疫30、60和75d小鼠肺脏定植细菌数(Log10cfu)B/R菌株组与BCG组比较差异有统计学意义(P<0.05),与H37Ra菌株组比较差异无统计学意义(P>0.05)。结论 B/R菌株的生长周期为30d,在小鼠体内至少存活2个月,定植能力较强。
Objective To observe the growth characteristics and the growth of the fusion strain (B / R strain) of BCG and Mycobacterium tuberculosis H37Ra (H37Ra strain). Methods The B / R strains at 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30 days after culture with 7H9 medium were measured respectively. The values of A in BCG and H37Ra were 600 nm, Coordinates, A600 values for the vertical axis, draw the bacterial growth curve. C57BL / 6 mice were randomly divided into three groups: B / R, BCG and H37Ra, with 30 mice in each group. The mice were inoculated subcutaneously with 0.1 ml B / R, BCG and H37Ra ), Immunized 15, 30, 45, 60 and 75d after aseptic conditions, mouse liver, spleen and lungs, made of tissue homogenate, inoculated Roche solid medium slant, cultured 20d after counting the number of colon organs (Log10 cfu). Results The bacterial growth curve showed that the adaptation period of B / R strain was longer and reached the logarithmic phase after 6 days, and reached the stationary phase after 27 days. The growth period was about 30 days. The numbers of colonized bacteria (Log10cfu) in B / R group were higher than those in BCG and H37Ra groups on 15, 30, 45, 60 and 75 days after immunization. The number of colonized bacteria in the liver of mice (Log10cfu) was no significant difference between the B / R strain and BCG and H37Ra strains (P> 0.05) (P <0.05). The numbers of colonized bacteria (Log10cfu) in spleens of mice on the 30th and 45th days after immunization were significantly different from those of the BCG and H37Ra strains (P <0.05). There was no significant difference between H37Ra and H37Ra strains (P> 0.05). The number of colonized Bacillus subgroups (Log10cfu) at 15 and 45 days after immunization was significantly lower than that of BCG and H37Ra strains (P <0.05). The numbers of colonized bacteria (Log10cfu) in the lungs of mice immunized at 30, 60, and 75 days after immunization were significantly lower than those in the BCG group (P <0.05) Significance (P> 0.05). Conclusion The growth cycle of B / R strain was 30 days, and it survived for at least 2 months in mice and the colonization ability was strong.