| Brucellosis is a worldwide and important bacterial zoonosis, which hits a serious threat to the healthy development of animal husbandry and human health. It outbreaks and spreads again in many countries and regions around the world, especially in developing countries in recent years.Classical serodiagnosis method, live attenuated vaccine and some corresponding measures usually taken have played important roles on preventing and controlling brucellosis. Unfortunately, they also brought about some disadvantages. For example, antibodies produced in animals vaccinated with live attenuated vaccines against Brucella spp. are indistinguishable from those produced in infected animals using current conventional serological tests. Attenuated vaccines may cause bacterial persistence within the target host, potential transmission to unintended recipients, and the possibility of reversion to virulence.A novel approach to control Brucellosis is to develop a marker vaccine through deletion the bp26 gene from these parental vaccine strains with good immunogenicity and vaccine efficacy, and to develop a novel iELISA based on BP26 protein as antigen to differentiate animals vaccinated with bp26 mutants of Brucella spp. from those infected naturally and those vaccinated with the parental vaccine strains. However, serological test based on the entire sequence of recombinant bp26 protein (BP26) often lead to false-positive reactions in Brucella-free sheep, goats, or cattle. So the antigenic specificity of bp26 protein should be improved. In addition, immunogenic diversity of Brucella bp26 protein of various virulent strains of Brucella spp.exists in different host animals experimented, thus it also would impact usage of Brucella bp26 protein as antigen of differential serodiagnosis in practice.1. Amplification, sequence and bioinformation analysis of bp26 gene of Brucella melitensis M5-90 strainThe remarkable homogeneity was revealed among brucella spp. through sequence of both nucleotide in bp26 gene and amino acid in bp26 protein, and the bp26 protein of Brucella spp. has been identified as an immunodominant antigen in infected cattle, sheep, goats, and humans, so bp26 is hopefully used as a serodiagnosis antigen to develop a novel differential serological test to differentiate animals vaccinated with bp26 mutants of Brucella spp. from those infected naturally and those vaccinated with the parental vaccine strains. According to the paper reported by Rosetti et al (1996), we designed a pair of primers to amplify bp26 gene by PCR with M5-90 DNA as template. The amplified fragment was about 990 bp by electrophoresis of nucleic acids, and the purified PCR product was identified to have an intact open reading frame (ORF) encoding bp26 protein of brucella spp. by sequencing on Beckman CEQTM8000 DNA Analysis System. Bioinformation analysis of bp26 protein encoded by bp26 gene showed that bp26 gene of each representative species of the genus Brucella was extremely conservative, nucleotide sequence homologies ranged more than 99.3% (except for dogs, only 59.4%) and shared deduced amino acid sequence homologies of more than 97.6%. The bp26 protein is with a calculated molecule weight of 26.569 Da and an isoelectric point of 6.62. The first 29 amino acid residues are possibly the signal peptide I. Amino acid residues of the N terminal sequences 29-39,64-69,109-120,171-176 and 204-209 are more likely to be B cell antigen linear epitopes.2. Cloning of intact or truncated bp26 gene of Brucella melitensis M5-90 strain, and construction of recombinant plasmid pQE32-rbp26 and pQE32-rAbp26A specific fragment of the bp26 gene or truncated bp26 gene of Brucella melitensis M5-90 was amplified through PCR, and the recombinant plasmid was constructed through ligating the purified PCR product and pQE32 vector digested with both BamH I and Hind III respectively, then was transformed into competent M15 [pREP4] cells. The competent cells grew on solid LB media containing kanamycin and ampicillin. The positive colonies were selected and then identified by colony PCR, digestion of one or double enzymes, and sequencing. We have gained the recombinant plasmid, named as pQE32-rbp26 or pQE32-rAbp26, and their integrity and the orientation of the amplicons were verified by sequencing the inserted DNA fragments with a Genetic Analysis system, so.3. Selection of antigenic specificity and antigenic comparison of four recombinant bp26 proteins of Brucella melitensis M5-90 strainFour recombinant plasmids, pET30-rbp26,6P-1-rbp26, pQE32-rbp26 and pQE32-rAbp26, were used to transform corresponding E. coli host cells. They expressed the four recombinant bp26 proteins of Brucella melitensis M5-90 strain as a poly-His or GST tagged fusion, named as rbp26-1, rbp26-2, rbp26-3 and rbp26-4. Subsequently, antigenicities of the purified recombinant proteins were analyzed by western blot and iELISA using mouse anti-bp26 protein antibody and reference sera from sheep and goats (90 Brucella-negative sera and 30 Brucella-positive sera verified by RBPT and cELISA) as Abl. The results showed the specificities of the four reconstructed antigens were 99%,98%, 99% and 100%, and their sensitivities were 27%,30%,27% and 23%, respectively. Among them the rbp26-4 showed the best antigenic reactivity and antigenic specificity, named as rAbp26, was suitable as diagnosis antigen for brucellosis marked vaccine (M5-90-26)4. Antigenic analysis, purification of truncated recombinant bp26 protein of Brucella melitensis M5-90 strainRecombinant plasmid pQE32-rAbp26 was transformed into Escherichia coli M15 (pREP4) host cells to express the rAbp26 protein. The rAbp26 protein was then purified by immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography (SEC) on an AKTA explorer system. Its antigenicity was detected by western blot using anti-rAbp26 hyperimmune serum (HIS), Brucella-positive serum from goat or bovid as Ab1 respectively, and analysis of cross-reactions between the rAbp26 protein and other antigens, E.coli. O:157, Salmonella group D, C1, B, and Yersinia enterocolitia 0:9, was also performed using anti-rAbp26 HIS as Ab1. The results showed the purified protein could react with either mouse anti-rAbp26 protein antibody or positive serum from cattle and goats, and only the rAbp26 protein reacted with mouse anti-rAbp26 HIS, and the other antigens of Gram-negative bacteria did not react with HIS. So the rAbp26 protein showed fine antigenic reactivity and antigenic specificity again.5. Development and optimization of reaction conditions of△bp26-iELISA test to detect Brucella bp26 antibodyAn iELISA test to detect Brucella bp26 antidody wae developed through coating the purified rAbp26 protein in 96 microplate as antigen, and its reaction conditions of Abp26-iELISA was optimized. The optimal concentration of the coated r△bp26 protein was 2μg/ml, the optimal dilution of detected sera (Ab1) was 1:50, the optimal reacting time of Ab1 with the r△bp26 protein(Ag) was 60min at 37℃, the optimal reacting time of Ab2 with the Abl-Ag complex was 60min at 37℃, and the optimal time of TMB coloration was 15min at 37℃.6. Determination of the cut-off value of△bp26-iELISA test, and initial application for differential serodiagnosis of brucellosis in sheep and goats△bp26-iELISA test was used to detect 494 reference negative sera and 186 reference positive sera verified by RBPT & Svanova-cELISA tests of sheep and goats from different sites. Among the two groups of data,92 reference negative sera was used to initial determinate the threshold value (Cov) of Abp26-iELISA, and its value was calculated as the mean specific OD450 of control (negative) sera plus threefold standard deviation (SD), and then the two groups of data were analyzed by ROC with Medcalc 9.6.0 software in order to optimize the threshold value of Abp26-iELISA. Sera from sheep and goats inoculated with M28 virulent strain, sera from sheep and goats immunized with vaccine strains, and field sera were detected by Abp26-iELISA test in order to optimize correlating Dsn and /or Dsp with Dsn+Dsp. Results showed the Dsn and Dsp value was equal to 18.6% and 96% when the threshold value (Cov) was 0.7816. The method can distinguish antibodies produced in animals vaccinated with brucellosis marked vaccine (M5-90-26) from those with live attenuated vaccines or infected animals, combined with the results of RBPT, cELISA, attacking test of M28, immunization test of M5-90, M5-90-26 and S2.7. Preparament of ready-to-use△bp26-iELISA kit for detecting Brucella bp26 antibodyNegative and positive reference serum,wash solution, block and protection solution, sample dilution solution, working solution of HRP-Ab2, and one competent TMB solution were respectively prepared and assemblized into a ready-to-use Abp26-iELISA kit for detecting Brucella bp26 antibody. Then sensitivity test, specificity test, repetition test (inter-bulks, and exter-bulks) and storage life test of Abp26-iELISA were accomplished. The results showed the kit’s shelf life could reach up to more than one year.8. Immunogenic analysis of Brucella bp26 proteinAccording to this test’s design, samples of sera from test 1, test 2 and test 3 were detected by Abp26-iELISA, RBPT, and Svanova-cELISA. In the test 1, the immunogenicity of M5-90 in goats and mice was the strongest, the second place is in sheep, that in calfhoods is the worst according to levels of anti-bp26 protein antibodies. In the test 2, the immunogenicity of M5-90 in goats was the strongest among them, and its immunogenicity of S2 in goats was stronger than that of S19 in goats. In the test 3, the immunogenicity of M5-90 in goats under good immunity condition was better than that under poor immunity condition. According to levels and durations of anti-bp26 protein antibodies, anti-’bacteria suspensions’antibodies, and anti-sLPS antibodies, its immunogenicity of S-LPS or tropina (eg,OMPs) was stronger than that of bp26 protein. |
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