| Brucellosis caused by brucella in the world led to huge economic losses.Brucella,a member of the bacterial genus Brucella,is pathogen of the disease,which are small,gram-negative,facultative intracellular bacteria.Brucella invad into the body mainly through the digestive tract,skin and respiratory mucosa and led to infection and morbidity.Most of Brucella parasitize mainly in lymphocyte and lymphatic tissue,resulting serious lesion of the immune system,followed by bacteriemia and toxaemia,which impair to nervous system,reproductive system and skeleton system.Livestock especially bovine and ovine is a major source of infection.Vaccination is an important measure for prevention of brucellosis,but the existence of the shortcomings of vaccines made it impossible to effectively distinguish between vaccine immunization and natural infection because of the persistence of antibody after immunization when using the classic serological methods to diagnosis,which is adverse to prevention and clean of this disease.Marker vaccine is a promising means to solve the issue in order to not only prevent and cut off the spread of brucellosis,but also distinguish between natural infection and immunization.B.melitensis vaccine strain M5-90 originated from M5,compared with the M5,it is safer for pregnant ewes.Its safety and effectiveness meet the world advanced level of.M5-90 has been widely used in China as vaccine,which achived a great success in brucella control for human and animal for its high efficacy.Bp26 protein,a periplasmic protein in the Brucella,has a good immune activity,which is often used as a serological target protein,bp26 gene deletion in mutant vaccine strain does not change the biological characteristics and the immune protection in mice model.When using recombinant BP26 protein as antigen in i-ELISA,it possesses the same sensitivity and specificity as i-ELISA which using brucella extract s-LPS as antigen.Bp26 gene is considered one of the most appropriate marker genes for maker vaccine.In this study,double-crossover recombinant strain is screened by kanamycin resistance gene (Kana?) which recombinated homologously into brucella M5-90 genome using bp26 gene as the target site and M5-90 as the parents.ORF of bp26 gene is entirely replaced by kanamycin resistance gene.Through SDS-PAGE and Western blot,protein of migration rate about 29kd is expressed in M5-90 strain and is recognized by mouse anti-recombinant protein serum,but no recognization occurred in M5-90-26.In addition,recombinant BP26 can not react with serum from mice immunized with bp26 gene deletion strain,but can reacted with serum from mice immunized with M5-90.These results show that the BP26 protein in the bp26 gene deletion strains are not the expressed,deletion strains is named M5-90-26.Biological and biochemistry characteristics of deletion mutant strain did not changed according tests' results.M5-90-26 maintained the same smooth type,proliferation ability,biochemistry characteristics and normal morphology as M5-90.Mice model of safety experiment revealed that M5-90-26 has the same residual virulence as M5-90.Recover time is about 13 week.Residual virulence differed in various time points.Curve of CFU isolation displayed M5-90 and M5-90-26 reduced gradually from body.The virulent strain M28 of B.melitensis maintained higher level at 3Log10 than M5-90 and M5-90-26 even after 15weeks post injection,which showed virulence and feasibility as the challenge strain.Mice model of protection experiment proved that M5-90-26 and M5-90 possess same protection efficacy against B.melitensis virulent strain M28 and B.abortus virulent strain 544 after immunized. CFU number isolated from spleens 45 days post vaccination in groups immunized with M5-90-26 and M5-90 was significant different lower than that of PBS control at 2.48～2.97Log10/spleen, spleens weight was lower 2.86～3.48 times than PBS control,difference between M5-90-26 and M5-90 group is not considered significant.CFU number isolated from spleens 150 days post vaccination in groups immunized with M5-90-26 and M5-90 was significant different lower than that of PBS control at 2.6～4.6Log10/spleen,spleens weight was lower 2 times than PBS control, difference between M5-90-26 and M5-90 group is not considered significant.M5-90-26 maintained the same immune protection in the short and term as M5-90.Immune duration compared with the effectiveness of short-term immunity,M5-90-26 and M5-90 stand at same high level.Serological test indicated that,same high titer of humoral antibody was primed by M5-90-26 and M5-90,i-ELISA coated with s-LPS detected anti-sLPS antibody appeared post immunized at lweek, to summit at 12 week,then declined which was not considered significant different.Memory immune response are induced after challenge by virulent strain with OD values raise sharply,group of M5-90-26 and M5-90 are not considered significant different which shows that M5-90-26 can induce same high lever humoral immune response.I-ELSIA using rBP26 as antigen detecting the sera from mice injected with M5-90,M5-90-26 and M28 indicated that anti-BP26 antibody was induced in after the first week by M5-90 and M28, then increased greatly,and P/N value was more than 5 at 4th week.No antibody was primed by M5-90-26.Conclusion can be draw no anti-BP26 antibody has been induced.So i-ELISA could be used to discriminate sera from M5-90-26 immunized mice from M28 infected,which provided a theory and practical foundation to use the combined diagnose method and proved a feasibility of M5-90-26 as vaccine.Ovine immunized with M5-90 and M5-90-26 did not transfer the Brucella to the unvaccinated in horizontal transmission assay.No serum of unvaccinated ovine changed into positive after 1 and 3 month through RBT and i-ELISA.No M5-90 and M5-90-26 bacteria was isolated from eyes,nose, mouth and tractus genitalis after immunized at 0,2,4,8,14,21 and 42 days,which indicated injection at articulation of elbow subcutaneously can not spread brucella to environment.Isolation from tissues showed that M5-90 and M5-90-26 existed in ovine for about 2moths,no bacteria was isolated from lymph nodes,liver,spleen and kidney after 2.5 and 3 month.Three generations of propagate in ovine did not induced reversion to virulence,bacteria before propagated are not considered significant different,which showed virulence of M5-90-26 was stable.Results indicate that safety and reproduction ability of deletion mutant M5-90-26 was not changed.M5-90-26 and M5-90 can induce same humoral immunize respose in ovine.By RBT,98%ovine transfer to positive after 10 days,then start to decline at 70～80d,20%～30%positive after 6 months, not considered different significantly between both.Cell mediated immunized between M5-90-26 and M5-90 was almost same.Altogether these results indicate that B.melitensis M5-90-26 might be a promising vaccine strain in preventing,monitoring and cleaning brucellosis.
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