CAJ | 학술논문

目的构建布鲁杆菌M5-90Δbp26基因缺失株,对比观察其免疫原性与M5-90亲本株的区别,研制毒力弱、安全性能好并能进行鉴别诊断的布鲁杆菌弱毒候选苗.方法利用常规分子生物学技术构建布鲁杆菌M5-90Δbp26基因缺失株并进行遗传稳定性检测和常规细菌学性质鉴定；用1.0×109 CFU/2 ml剂量的M5-90亲本株和M5-90Δbp26基因缺失株分别免疫绵羊,所得血清与纯化的BP26蛋白进行Western blot免疫印迹实验,比较M5-90Δbp26基因缺失株与M5-90亲本株在体液免疫中对BP26蛋白识别上的差异；用试管凝集试验(SAT)检测受试绵羊不同时期(0、7、14、21、30、45 d)的血清效价；安全性试验用1.0×106、6.0×106、2.0×107 CFU/0.2 ml的M5-90亲本株和M5-90Δbp26基因缺失株分别免疫接种小鼠,观察、记录小鼠接种疫苗后的临床症状和死亡情况,比较二者的毒力.结果遗传稳定性检测M5-90亲本株PCR扩增出长度约1279 bp的片段,而2～15代的M5-90Δbp26基因缺失株扩增出长度约629 bp的片段,后者测序结果表明出现bp26基因650 bp的缺失；常规细菌学性质鉴定结果,M5-90Δbp26由M5-90亲本株的单因子血清试验M型转变为R型,BK2噬菌体裂解实验由阳性转变为阴性,鉴定为深度变异菌株；Western blot免疫印迹实验表明M5-90Δbp26基因缺失株免疫绵羊所获得的血清与纯化的BP26蛋白不发生反应,而M5-90亲本株可发生反应；STA试验表明M5-90Δbp26基因缺失株诱导绵羊产生抗体水平(1∶50)明显低于M5-90亲本株(＞1:800)；安全性试验表明M5-90Δbp26基因缺失株毒力比M5-90亲本株明显减弱.结论成功构建M5-90Δbp26基因缺失株.与M5-90亲本株比较,M5-90Δbp26基因缺失株具有毒力弱、能区分自然感染与疫苗免疫的特点,可作为标记疫苗的候选株.
목적 구건포로간균M5-90Δbp26기인결실주,대비관찰기면역원성여M5-90친본주적구별,연제독력약、안전성능호병능진행감별진단적포로간균약독후선묘.방법 이용상규분자생물학기술구건포로간균M5-90Δbp26기인결실주병진행유전은정성검측화상규세균학성질감정；용1.0×109 CFU/2 ml제량적M5-90친본주화M5-90Δbp26기인결실주분별면역면양,소득혈청여순화적BP26단백진행Western blot면역인적실험,비교M5-90Δbp26기인결실주여M5-90친본주재체액면역중대BP26단백식별상적차이；용시관응집시험(SAT)검측수시면양불동시기(0、7、14、21、30、45 d)적혈청효개；안전성시험용1.0×106、6.0×106、2.0×107 CFU/0.2 ml적M5-90친본주화M5-90Δbp26기인결실주분별면역접충소서,관찰、기록소서접충역묘후적림상증상화사망정황,비교이자적독력.결과 유전은정성검측M5-90친본주PCR확증출장도약1279 bp적편단,이2～15대적M5-90Δbp26기인결실주확증출장도약629 bp적편단,후자측서결과표명출현bp26기인650 bp적결실；상규세균학성질감정결과,M5-90Δbp26유M5-90친본주적단인자혈청시험M형전변위R형,BK2서균체렬해실험유양성전변위음성,감정위심도변이균주；Western blot면역인적실험표명M5-90Δbp26기인결실주면역면양소획득적혈청여순화적BP26단백불발생반응,이M5-90친본주가발생반응；STA시험표명M5-90Δbp26기인결실주유도면양산생항체수평(1∶50)명현저우M5-90친본주(＞1:800)；안전성시험표명M5-90Δbp26기인결실주독력비M5-90친본주명현감약.결론 성공구건M5-90Δbp26기인결실주.여M5-90친본주비교,M5-90Δbp26기인결실주구유독력약、능구분자연감염여역묘면역적특점,가작위표기역묘적후선주.
Objective To construct the bp26 deletion mutant of Brucella vaccine strain M5-90 (M5-90Δbp26),to compare its immunogenicity with parental strain(M5-90),and to develope a new candidate vaccine strain of Brucella melitensis with reduced virulence that can be used to distinguish vaccinated livestock from infected animals.Methods Mutant vaccine strain of Brucella melitensis was constructed by conventional molecular biology techniques then the genetic stability of mutant M5-90Δbp26 was tested and its conventional bacteriological nature was identified; 1.0 × 109 CFU/2 ml doses of M5-90Δbp26 strain and the parental strain were used to vaccinate 3 sheep; sera were analyzed for reactivity against BP26 by Western blotting and for agglutination activity; to analyze the virulence of mutant and parental strain,mice were injected with 1.0 × 106,6.0 × 106 and 2.0 × 107 CFU/0.2 ml doses of M5-90 and M5-90Δbp26,respectively,and clinical symptoms were monitored and the death of mice was recorded.Results The M5-90Δbp26 was successfully generated and reversion was not observed in 15 generations.The size of PCR products was 629 bp while the parental strain was 1279 bp.The sequence analysis showed a 650 bp missing in M5-90Δbp26.The conventional bacteria identification tests confirmed that the mutant was depth variant strain,including mono-specific antiserum M type transformed into R,and the BK2 phage based splitting assay converted from the positive to the negative.Western blotting showed the purified BP26 protein was recognized by the serum against the parental strain while not by the serum against M5-90Δbp26 strain.Agglutination test showed the level of the serum antibody induced by M5-90Δbp26 strain(1:50) was significantly lower than that of serum induced by parental strain(＞ 1:800).Virulence test showed that M5-90Δbp26 strain was less virulent than parental strain.Conclusions M5-90Δbp26 has been successfully constructed.M5-90Δbp26 of Brucella melitensis has the characteristic of reduced virulence and has a potential as brucellosis candidate vaccine strain permitting serological discrimination between diseased and vaccinated livestock.