目的 评价不同消毒方式对布鲁菌的消毒效果,为建立高等级生物安全实验室及疫苗生产车间中布鲁菌的消毒标准操作规范提供实验证据.方法 筛选粗糙型及光滑型布鲁菌作为模式菌.将模式菌的高浓度菌液和低浓度菌液分别经紫外线、有效氯含量为2 000 mg/ml三氯异氰尿酸及75％乙醇,经过15 min、30 min、60 min杀菌处理,将处理后的细菌置于37℃培养5d,观察其存活情况,进行消毒效果评价.结果 紫外线能在15 min内完全杀灭生物安全柜中的高浓度及低浓度布鲁菌.2 000 mg/ml三氯异氰尿酸在15 min内不能完全杀灭较高浓度布鲁菌,但对低浓度布鲁菌的杀灭效果较好.75％乙醇能在15 min内完全杀灭高浓度及低浓度布鲁菌.结论 紫外线、75％乙醇对布鲁菌有较好的消毒效果,可用于高等级生物安全实验室及布鲁菌活疫苗生产车间的常规消毒.

Objective To evaluate the immune response of a new plague component vaccine,which composed of extracted F1 antigen and recombinant V antigen.Methods Mice were randomly divided into four experimental groups,and were tested at five weeks after the first immunization.The blood was collected from each animal by inner canthus.Bronchoalveolar lavage (BAL) was obtained by exposing the trachea and making a small incision.Through this cannula the lungs were repeatedly lavaged by slowly injecting and withdrawing phosphate buffered saline.Individual serum and BAL samples were examined for total anti-F1 and anti-rV IgG and IgA antibodies using ELISA.Results ELISA results showed that plague component vaccine could induce high titer of anti-F1 and anti-rV IgG in the serum,and low titer of anti-F1 IgA could be detected.Low titer of anti-F1 and anti-rV IgG rather than IgA could be detected in BAL.Conclusion The new plague vaccine can cause significant humoral immunity response in mice,which would be a new generation of plague vaccine.

Objective:To develop a method of 1oop-mediated isothermal amplification(LAMP) for detection of Yersinia pestis.Methods: According to conserved F1 gene of Yersinia pestis and principle of LAMP,a set of LAMP reaction system containing four primers were designed.Strains of Yersinia pestis and non-Yersinia pestis were detected with LAMP to evaluate the specificity of the method.The dilution of the Yersinia pestis was detected with LAMP and PCR to evaluate the sensitivity of the method.Results: 10 non-Yersinia pestis strains were found all negative by LAMP,performing high specificity of this method.The detection limit of this LAMP assay was about 20 yersinia pestis strains per reaction.The sensitivity of this LAMP assay was 10-fold higher than regular PCR.A visual detection approach with addition of dye was feasible to interpret the positive or negative results.Conclusion: The genetic diagnostic method of Yersinia pestis based on LAMP is rapid,sensitive and specific.This method may be the potential effective way for detection of Yersinia pestis on the scene.

Objective To determine the F1 and rV antigen contents in plague component vaccine by double antibody sandwich ELISA.Methods The antigens in bulk of plague component vaccine were identified with monoclonal antibodies(McAbs)against F1 rV antigens,and determined quantitatively by double antibody sandwich ELISA.The completeness of antigen adsorbed onto aluminium hydroxide adjuvant was verified.The final product of vaccine was stored at 4 ℃ for 18 months,from which samples were taken 1 and 18 months after storage respectively and determined for F1 and rV antigen contents by the developed double antibody sandwich ELISA method to analyze the stability of antigen.The three-step purification procedure for three batches of bulk rV antigen of Yersiria pestis were verified.Results The F1 and rV antigens in bulk of plague component vaccine showed good reactogenicity.The F1 and rV antigen contents in four batches of bulks were determined by the developed method,of which the results showed an error of not more than 20% with those determined by Lowry method.No F1 or rV antigen was detected in the centrifuge supernatant of four batches vaccine,indicating that the antigens were completely adsorbed by aluminium hydroxide adjuvant.The F1 and rV antigen contents in vaccine were stable after storage at 4 ℃ for 18 months.After three batches of bulk were purified by anion exchange,hydrophobic and gel filtration chromatography,both the purity and content of rV antigen increased.Conclusion The developed double antibody sandwich ELISA method may be used for the quantitative determination of F1 and rV antigens in plague component vaccine.

Objective To establish a sandwich ELISA in detection of the concentration of rV antigen of Yersinia pestis.Methods The McAbs against rV antigen were prepared by the hybridoma technology and Sandwich ELISA was developed on the basis of the epitope analysis and McAb specificity,in which specificity,accuracy,precision and linear range were validated.Results The diagnostic kit for Yersinia pestis rV antigen was successfully set up.The minimum detection concentration of rV antigen is 10 ng/mL.Conclusion The kit is used in detection of the concentration of rV antigen in stock solution of plague component vaccine and in monitoring of antigeneicity in the preparing process.It is a important method for quality control in preparation of plague component vaccine,this method also provides a foundation for a further developing the plague diagnostic reagents and other related investigations.

Objective To develop a stable and effective procedure for fermentation of recombinant Yersinia pestis and purification of LcrV antigen.Methods The regularity of growth of recombinant E.coli strain pET-V / BL21(DE3) in tube and in conical flask as well as expression of LcrV antigen were investigated,based on which the medium,time point and duration for induction with IPTG and IPTG concentration were optimized and scaled up to a 30 L fermenter to develop a stable fermentation procedure.The harvested bacteria were subjected to freeze-thawing,from which protein solution was collected by centrifugation and treated by high pressure homogenization,the purified by Q.Sepharose HP anion exchange chromatography,Phenyl Sepharose 6 FF(hs) hydrophobic chromatography and Superdex 75 pg gel filtration chromatography,based on which a stable purification procedure was developed.Three consecutive batches of test samples were purified by the developed procedure and subjected to overall control tests according to the requirements in Chinese Pharmacopoeia(VolumeⅢ,2010 edition).Results After culture and induction under the optimized condition,the density of harvested bacteria(A600 value) reached 34,while the expression level and amount of LcrV antigen reached were more than 36% and 1.6 g / L respectively.After purification by column chromatography,the yield,purity and total recovery rate of LcrV antigen were more than 140 mg,more than 95% and more than 8.5% respectively.All the quality indexes of purified LcrV antigen met the requirements in Chinese Pharmacopoeia(VolumeⅢ,2010 edition).Conclusion A stable procedure suitable for large-scale production of LcrV antigen was developed,which laid a foundation of preparation of novel Y.pestis component vaccine.

Objective To optimize the condition for transport of live attenuated anthrax vaccine and the determination method for viable count.Methods The viable counts of live attenuated anthrax vaccine for percutaneous scarification submitted by the manufacturers under various transport conditions were determined by plate counting.The vaccine transported at normal temperature for 3 d was determined by sampling using measurable pipette.However,even batches of vaccine transported at low temperature for 6 h were determined using measurable pipette and pipettor separately,and one batch using the two tools for 5 times separately.The precisions of determination results by using measurable pipette and pipettor were compared.Results All the seven batches of vaccines transported at normal temperature were unqualified in viable counts.However,the viable counts of vaccines transported at low temperature were qualified and significantly higher than those transported at normal temperature(P 0.01).The determination results by using measurable pipette and pipettor showed no significant difference(P 0.05),of which the coefficients of variation were 11.3% and 23.0% respectively.Conclusion Live attenuated anthrax vaccine shall be transported at low temperature during submission to control tests.Sampling by pipettor for viable counting was precise and easy to handle,which was benefit to the quality control of vaccine.

PA (protective antigen) is a major immunogen of the vaccine against anthrax. In the present study, a new expression system, Escherichia coli strain Rosetta 2(DE3), was used for high-level expression of rPA (recombinant PA) whose gene contains 66 rare E. coli codons (9.0% of 733 total PA gene codons). The rPA-formed inclusion bodies were washed with Triton X-100 and 2 M urea and solubilized in 5 M urea, followed by a 60%-satd.-ammonium sulfate precipitation. Finally, the untagged rPA was efficiently and rapidly purified by single-step hydrophobic-interaction chromatography using Phenyl-Sepharose High Performance resin on an AKTA Purifier 10 system. The yield was approx. 13 mg of high-purity (>99%) biologically active rPA per litre of culture, which can be used for the detection of anthrax and as a potential component of a vaccine against anthrax.

Objective To analyze the safety of recombinant plague vaccine and provide experimental support for clinical research.Methods Acute toxicity test was carried out.Mice were observed carefully after vaccination.Abnormal toxicity test was carried out in mice and guinea pigs.They were observed and weighed seven days after administration.Local stimulation was evaluated,through pathologic examination of local subcutaneous tissue of rabbits.Systematic anaphylaxis test was carried out in guinea pigs to evaluate the safety of recombinant plague vaccine.Results No abnormal symptom and death happened to mice in acute toxicity test.The maximum tolerable dose of recombinant plague vaccine for mice was more than 10 000 μg/kg.There wasn't any abnormal reaction happened in abnormal toxicity test.The weight of every animal increased seven days after administration.No pathologic change appeared after injection with recombinant plague vaccine.There was no abnormal reaction appeared in systematic anaphylaxis test.Conclusion The results of acute toxicity test,abnormal toxicity test,local stimulation test and systematic anaphylaxis test showed that subcutaneous injection of recombinant plague vaccine is safe,which provide a foundation for further clinical research.