Objective:To optimize the BALB/c mouse rhinitis model sensitized by Artemisia annua pollen allergen, and explore the humoral and cellular immune indicators that can be used for the evaluation of allergic reactions. Methods:Using BALB/c mice as experimental animals, using Artemisia annua pollen allergen extract as sensitizing protein, through different content of the main allergen Art a1 and different sensitization times, different immunization programs were set to immunize mice subcutaneously, One week and five weeks after the last immunization, Artemisia annua pollen allergen extract containing 50 μg/ml and 500 μg/ml Art a1 was used for nasal stimulation, once a day, for 1 week each time.Observe the allergic reaction of mice, detect the pathological changes of nasal tissues, determine the levels and dynamic changes of antigen-specific IgE, IgG1, IgG2a and other antibodies in the serum of each group of mice. and detect the changes in the number of antigen-specific IL-4, IL-5, IL-2, IFN-γ and other lymphocytes in the spleen of mice. Results:Sensitized mice showed obvious scratching and sneezing reactions after being stimulated by antigen; obvious allergic inflammation appeared in nasal tissue; The increase in serum level of Artemisia annua pollen-specific IgE antibody was significantly correlated with the challenge antigen; The antigen-specific IL-4 lymphocytes in the spleen of the sensitized mice were significantly increased, but the IFN-γ-specific lymphocytes did not change significantly. Conclusions:The successful establishment of a mouse model of Artemisia annua pollen allergen allergy is the first domestic use of ELISPOT technology to detect an increase in the number of antigen-specific IL-4 lymphocytes in Artemisia annua allergy mice, laying a foundation for the subsequent evaluation of the efficacy of preparations for desensitization treatment basis.

目的 分析屋尘螨变应原制剂外观异常对其质量的影响.方法 依据进口药品注册标准(JS20130104)对1批(T6469)屋尘螨变应原制剂进行全面质量检定,同时与检定合格样品(A3016、A3064及A3110批)进行相应比对,Western blot鉴定主要变应原组分,纳米粒径颗粒分析仪检测制剂中铝佐剂-蛋白吸附物的中值粒径(D50).选取检定合格样品进行冷冻模拟验证,观察低温冷冻对制剂外观性状的影响.结果 T6469批制剂外观不均一(外观性状异常与正常样品),混悬液浊度有差异,部分样品出现摇不散的贴壁颗粒,外观性状异常样品与正常样品比较,浑浊度降低,沉降速度增快,铝佐剂吸附物液面高度明显低于外观正常样品.经检定,T6469、A3016、A3064及A3110批制剂pH、铝含量、苯酚含量及游离上清活性均在质量标准范围内,在相对分子质量25 000及14 000处均出现特异性条带,但T6469批外观性状异常样品条带变弱.T6469外观性状异常与正常样品之间D50差异有统计学意义(P＜0.05);检定合格样品冻存前后D50差异有统计学意义(P＜0.05).T6469批外观性状异常样品与正常样品和检定合格样品比较,总生物学活性明显降低,差异有统计学意义(P＜0.05).检定合格样品放置低温环境至冻结后重现了上述外观异常性状,生物活性与未处理样品相比显著降低(P＜0.05).结论 该批屋尘螨变应原制剂的外观异常可能是由于样品贮存及运输中出现低温冻结,引起铝佐剂-蛋白凝胶颗粒出现聚集,形成较大颗粒,最终导致其生物活性显著降低.

In the past ten years, the research and application of microbiome has continued to increase. The microbiome has gradually become the research focus in the fields of life science, environmental science, and medicine. Meanwhile, many countries and organizations around the world are launching their own microbiome projects and conducting a multi-faceted layout, striving to gain a strategic position in this promising field. In addition, whether it is scientific research or industrial applications, there has been a climax of research and a wave of investment and financing, accordingly, products and services related to the microbiome are constantly emerging. However, due to the rapid development of microbiome sequencing and analysis related technologies and methods, the research and application from various countries have not yet unified on the standards of technology, programs, and data. Domestic industry participants also have insufficient understanding of the microbiome. New methods, technologies, and theories have not yet been fully accepted and used. In addition, some of the existing standards and guidelines are too general with poor practicality. This not only causes obstacles in the integration of scientific research data and waste of resources, but also gives related companies unfair competition opportunity. More importantly, China still lacks national standards related to the microbiome, and the national microbiome project is still in the process of preparation. In this context, the experts and practitioners of the microbiome worked together and developed the consensus of experts. It can not only guide domestic scientific research and industrial institutions to regulate the production, learning and research of the microbiome, the application can also provide reference technical basis for the relevant national functional departments, protect the scale and standardized corporate company's interests, strengthen industry self-discipline, avoid unregulated enterprises from disrupting the market, and ultimately promote the benign development of microbiome-related industries.

目的 建立重组金黄色葡萄球菌(Staphylococcus aureus,SA)疫苗特异性人血清IgG抗体的Luminex系统多重检测方法,并进行验证.方法 以重组SA疫苗所含的5种抗原(mHla、IsdB-N2、SpA5、mSEB、MntC)作为磁珠偶联蛋白,分别与相应磁珠偶联,加人参考血清(第3次免疫重组SA疫苗后21 d的志愿者血清,筛选出抗5种抗原特异性高效价血清8份,混合),37℃孵育;加入山羊F(ab')2抗人IgG-Fc (PE),37℃孵育;用Luminex系统进行多重检测,并对偶联抗原浓度、孵育时间、二抗稀释度进行优化.同时分析5种抗原间的干扰情况,并验证方法学的特异性、准确性及精密性.结果 mHla、IsdB-N2、SpA5、mSEB、MntC最适偶联浓度分别为30、150、7.5、2.5、7.5μg/2.5 mil,最适一抗及二抗孵育时间均为30 min,最适二抗稀释度为1∶5000.5种抗原间不存在明显的交叉表位,与特异的抗体反应过程无明显相互干扰.游离的重组蛋白对偶联抗原与抗体间反应的抑制率随着浓度的增加而增加,3种浓度的参考血清的回收率介于87.0％～120.8％之间,批内和批间CV值均＜10％.结论 成功建立并优化了重组SA疫苗特异性抗体的Luminex系统多重检测方法,且该方法具有良好的特异性、准确性及稳定性.

目的 研究1株微生态活菌制品生产用粪肠球菌的安全性.方法 采用目前肠球菌安全性评价主要方法,测定粪肠球菌GMCC 0460.3株的全基因组序列并分析毒力和耐药性相关基因;以生物化学方法测定其耐药性和有毒代谢产物的产生情况;经口灌胃小鼠测定其动物体内毒力.结果 该株肠球菌对卡那霉素和磺胺类药物耐药,耐药谱窄;基因组存在多种肠球菌毒力基因;生化试验表明其溶血性阴性、氨基脱羧酶活性阴性、硝基还原酶活性较低、细胞表面疏水性较低、生物膜形成能力较弱;动物实验结果表明该株菌在小鼠体内无明显毒性作用.结论 粪肠球菌GMCC0 460.3株实验评价的结果显示了良好的安全性.

目的 筛选一种简便、易行的苯酚含量测定方法,作为螨变应原制品中苯酚含量测定的通用方法.方法 建立比色法测定苯酚含量的标准曲线,并确定线性及测定范围;用比色法测定4种螨变应原制品中的苯酚含量,进行精密性和准确性验证;将比色法与企业标准规定方法测定结果进行比较.结果 比色法6次检测结果显示,苯酚浓度在0～40 μg/mL范围内与吸光度值呈良好的线性关系,R2均≥0.999 8;比色法测定4种螨变应原制品苯酚含量,日内精密性相对标准偏差(relative standard deviation,RSD)不超过2.3％,日间精密性RSD不超过3.5％;准确性验证回收率为98％～104％.比色法测定3家企业螨变应原制品苯酚含量,结果全部符合质量标准规定,且与各自企业标准中规定方法的测定结果一致,差异无统计学意义(P＞0.05).结论 利用比色法测定螨变应原制品中苯酚含量,重复性好,准确度高,操作简便,可用作该制品苯酚含量测定的通用方法.

Objective To investigate the dynamic changes of antibodies induced by leptospiral vaccines. Methods Antigens for antibody detection were screened out. ELISA was used to analyze antibody responses induced at different time points after immunizing guinea pigs with different batches of leptospiral vaccines from different manufacturers. To investigate the relationship between antibody responses induced by leptospiral vaccines and their protective effects in animal model, guinea pigs were challenged with Leptospira after immunization. Results There was no significant antigen-antibody reaction between the LigA protein or Patoc Ⅰ antigen and the serum samples of guinea pigs immunized with leptospiral vaccines. Notable IgG and IgM antibody reactions were observed in all vaccination groups when using bacterial proteins from seven Lep-tospira reference strains which were used for the preparation of leptospiral vaccines as envelope antigens. An-tigen-specific IgG antibodies peaked at 35 d after the last immunization, and the highest peak of antigen-spe-cific IgM antibodies was reached 11 d after the last immunization. Results of the challenge test showed that non-diluted leptospiral vaccines induced significant IgG and IgM antibody reactions in guinea pigs as com-pared with those diluted three or nine times, showing good protective effects. Conclusions Analysis of the dynamic changes of antibodies induced by leptospiral vaccines revealed that there was correlation between the induced serum antibody responses and the protective effects. This study provided reference for further study on alternative methods for evaluating leptospiral vaccine potency.

目的 制备甲型副伤寒沙门菌(Salmonella paratyphiA,SPA)O特异多糖(O-specific polysaccharide,OSP)-霍乱毒素B亚单位(cholera toxin B subunit,CTB)结合疫苗,并初步探讨其理化及生物学特性.方法 SPA OSP多糖原液(简称OSP多糖)经溴化氰活化,己二酰肼(ADH)衍化,在碳二亚胺(EDAC)作用下与CTB偶联,结合物经Sepharose4FF柱纯化,获得多糖-蛋白结合物,对其理化指标、血清特异性、免疫原性及血清体外杀菌力进行检测.结果 制备的SPA OSP-CTB结合物(简称OSP-CTB结合物)衍化度为(2.63±0.13)％、多糖回收率为(74±0.2)％;鉴别试验显示OSP-CTB结合物与SPA O诊断血清、CTB抗体均有阳性沉淀线产生;免疫小鼠血清中OSP-CTB结合物抗体滴度显著增长,阳转率达100％,血清体外杀菌抗体滴度为1∶64.结论 用溴化氰活化多糖制备的SPA OSP-CTB结合物具有良好免疫原性,可进一步进行疫苗研发.

肠球菌目前作为益生菌的研究热点,被应用于多种微生态制剂,在胃肠道疾病等多种疾病的治疗和预防过程中发挥着重要作用.但肠球菌在某些条件下可能成为致病菌,在临床报告的数据中,有一部分院内感染就是由肠球菌引起.鉴于肠球菌的益生性和潜在的致病风险,迫切需要对肠球菌的安全性进行评价.目前国内外的研究主要从耐药性和毒力因子方面评价肠球菌的安全性,也有部分研究通过动物试验考察肠球菌的安全性,结果均表明,尚未发现益生性肠球菌的安全问题.

目的 探索建立Der.f 1含量测定抗原、 抗体国家标准品的可行性.方法 首先对粉尘螨1组主要变应原(Der.f 1)含量测定用的3种不同企业抗原标准品(ST-H011、MS-14、EL-UAS)进行比较分析,筛选适用性好的抗原标准品;进一步对3种抗体系统(5-17/Der1-BD、14-1/12G2、6A8/4C1)进行比较,筛选最佳检测抗体系统;最后再进一步利用两种抗原标准品和3种抗体系统对不同企业粉尘螨变应原制品中的Der.f 1进行含量测定.结果 ST-H011和MS-14用于测定Der.f 1含量具有较好的通用性,EL-UAS用于Der.f 1含量测定结果与前两者差异较大;3种Der.f 1特异性抗体检测系统具有较好的通用性;利用上述两种Der.f 1抗原标准品和3种抗体系统测定各企业粉尘螨变应原制品中的Der.f 1含量时,结果均符合质量标准规定.结论 两种Der.f 1抗原标准品及三种抗体系统测定粉尘螨变应原中Der.f 1含量,均具有较好的通用性和适用性,其中14-1/12G2抗体测定结果的一致性最好,可作为Der.f 1含量测定用抗体国家标准品的候选.

目的:针对铝佐剂吸附的螨变应原注射液缺乏有效的生物效力评价方法的现状,探索用基于ImmunoCAP仪的荧光酶联免疫分析方法(ImmunoCAP法)作为该制品体外效力评价方法可行性,以促进该制品质量标准的提高.方法:利用ImmunoCAP法测定螨变应原注射液对过敏病人血清中IgE的抑制率(又称为变应原活性),作为该制品效力评价的检测指标.研究中分析了该方法的特异性;探索了孵育时间、温度、血清稀释度、变应原浓度等条件对抑制率的影响;验证了该方法的精密性;测定了13批维持剂量螨变应原注射液的总变应原活性和上清游离变应原活性.结果:应用ImmuonCAP法测定维持剂量螨变应原注射液对过敏病人血清IgE抑制率,具有较好的特异性;维持剂量制品对血清中特异性IgE的抑制率与变应原浓度具有明显的量效反应关系;该法具有很好的精密性,RSD最大不超过5％;13批维持剂量制品总变应原对血清中IgE抑制率均大于50％;上清中游离变应原对血清中IgE抑制率均不高于20％.结论:ImmunoCAP法可用于铝佐剂吸附的螨变应原注射液的体外效力评价,重复性好,精密度高,操作简便.

目的 构建壳聚糖(chitosan,CS)介导的甲型副伤寒沙门菌nmpC和pagC的DNA疫苗,并探讨该疫苗的免疫效果.方法 按哺乳动物的密码子偏好性对nmpC和pagC的基因序列进行优化,并将优化后的序列克隆至真核表达载体provax中,构建provax-nmpC和provax-pagC DNA疫苗.采用复凝聚法制备载DNA疫苗的CS纳米粒,并对CS/DNA纳米粒的特性进行检测.将provax-nmpC和provax-pagC DNA疫苗及其相应的CS纳米粒体外转染BHK细胞,采用Western blot检测目的蛋白的表达.采用活体电击法分别用不同组分的蛋白免疫小鼠,ELISA法检测血清抗体水平及抗体类型.结果 provax-nmpC和provax-pagC DNA疫苗经双酶切鉴定,构建正确.疫苗可与CS发生有效结合,纳米粒形似球形,粒径均一,包封率均达90％以上,且CS可防止DNA被DNase Ⅰ酶降解.provax-nmpC及CS/provax-nmpC均能诱导小鼠产生有效的免疫反应,两组间差异无统计学意义(P＞0.05);provax-pagC与CS/provaxpagC均能诱导小鼠产生有效的免疫反应,且provax-pagC组的抗体水平明显高于CS/provax-pagC(P＜0.05).provax-nmpC和provax-pagC组的IgG1及IgG2a抗体水平均高于相应的CS组(P＜0.05),且IgG1和IgG2a间差异无统计学意义(P＞0.05).结论 DNA疫苗provax-nmpC和provax-pagC均可诱导有效的免疫反应;载基因CS纳米粒并未增强DNA疫苗的免疫效果;CS未改变DNA疫苗的免疫应答类型,但具有调节Th1和Th2平衡的作用.

目的:探索益生菌在过敏性疾病的预防和治疗中的作用.方法:通过查阅近3年来关于益生菌预防和治疗过敏性疾病方面的文献,主要从动物实验、人体临床试验以及可能的治疗机制等方面详述了益生菌与过敏性疾病防治的相关性.结果:动物实验相关文献表明:益生菌对过敏性疾病的预防和治疗有一定的疗效.临床研究相关文献调研结果也表明:益生菌对过敏性鼻炎、特应性皮炎、哮喘和食物过敏等疾病具有一定的治疗效果.对过敏性疾病有治疗效果的益生菌为鼠李糖杆菌、嗜酸乳杆菌、双歧杆菌等,益生菌单一菌种和混合益生菌都能起到治疗作用.结论:益生菌能够对过敏性疾病起到预防和治疗的作用,尤其是在生命早期,或者降低宿主患过敏性疾病的概率,或者使患者的过敏症状减轻甚至消除.但依然存在着诸多问题,需要继续投入大量工作进行更深入的研究.

近年来,DNA重组技术在生物医药领域获得了迅速发展[1-2].《中国药典》三部(2015版)中,对人用重组DNA蛋白制品进行了定义:人用重组DNA蛋白制品是采用重组DNA技术,对编码所需蛋白质的基因进行遗传修饰,利用质粒或病毒载体将目的基因导入适当的宿主细胞,表达并翻译成蛋白质,经过提取和纯化等步骤制备而成的具有生物学活性的蛋白质制品,用于疾病的预防和治疗[3].同时还规定,工程细胞的来源、管理及检定应符合《生物制品生产检定用菌毒种管理规程》和《生物制品生产检定用动物细胞基质制备及检定规程》的相关要求;对于生物制品生产用重组菌种均应建立种子批系统,通常包括主种子批和工作种子批,从原始种子传代和扩增后保存的为主种子批,从主种子批传代和扩增后保存的为工作种子批,工作种子批用于生产.

目的 建立一种稳定的屋尘螨点刺液生物活性测定方法,以替代现用的放射性吸附抑制测定方法(radio allergosorbent test,RAST).方法 建立定量ELISA竞争抑制法,对屋尘螨点刺液进行生物活性测定,并对该法进行精密性、准确性、适用性验证,确定线性测定范围.应用RAST和定量ELISA竞争抑制法平行测定3批屋尘螨点刺液生物活性,对结果进行比较.结果 定量ELISA竞争抑制法测定屋尘螨点刺液制品生物活性,其最佳测定范围为0.052 ～0.125 HEP/mL;高、中、低3个浓度的日内精密性≤10.3％,日间精密性≤11％,回收率为87％ ～ 106％,表明该方法具有较好的精密性;低、中、高3个加标样本的回收率均在73.18％～ 99.02％之间,表明该方法具有较好的准确性;3批屋尘螨点刺液生物活性最佳测定范围的稀释度为1/48 ～1/192,所有测定样本的相对活性在80％～110％范围内,表明该方法适用于屋尘螨点刺液生物活性检测.两种方法测定屋尘螨点刺液生物活性,结果差异无统计学意义(P＞0.05),且均符合质量标准规定.结论 定量ELISA竞争抑制法测定屋尘螨点刺液的生物学活性,重复性好,准确性高,操作简便,可作为现用生物活性测定RAST法的替代方法.

疫苗发展简史 在人类历史进程中,为了保护自身健康,人类一直在寻求摆脱各种瘟疫的方法.直到一百多年前,传染病的各种病原体被逐步确认以后,对传染病的控制才逐步被规范为控制传染源、切断传播途径和保护易感人群三部分,而接种疫苗,正是保护易感人群工作中最重要的内容.

目的 探讨壳聚糖(chitosan,CS)纳米粒作为甲型副伤寒沙门菌重组外膜蛋白NmpC和PagC的佐剂对小鼠的免疫效果.方法 采用离子交联法将甲型副伤寒沙门菌重组外膜蛋白NmpC和PagC与CS结合制成CS纳米粒,扫描电镜观察纳米粒外观,激光粒度分析仪测定纳米粒粒径和多分散系数,BCA法测定其包封率.将CS纳米粒经肌肉注射免疫BALB/c小鼠,ELISA法检测小鼠血清特异性抗体IgG、IgG1和IgG2a水平.结果 载蛋白CS纳米粒粒径约为300 nm,包封率达85％以上.NmpC和PagC的CS免疫组小鼠血清IgG抗体滴度远高于铝佐剂组,产生的IgG1和IgG2a抗体滴度也均高于相应的铝佐剂免疫组,且差异均有统计学意义(P＜0.05);IgG1和IgG2a之间差异无统计学意义(P＞0.05).结论 CS纳米粒作为新型疫苗佐剂可用于甲型副伤寒蛋白疫苗的研究.

目的 建立国产伤寒甲型副伤寒结合疫苗的质量控制标准.方法 参照《中国药典》三、四部(2015版)、《预防用疫苗临床前研究技术指导原则》、《结合疫苗质量控制和临床研究技术指导原则》及《世界卫生组织生物制品规程续编Ⅱ》(1995年版)等中的相关原则及方法,对4批国产伤寒甲型副伤寒结合疫苗的各项质量指标进行检测.结果 4批国产伤寒甲型副伤寒结合疫苗的各项质量检测指标均符合国家拟定的质量标准.结论 建立的国产伤寒甲型副伤寒结合疫苗的质量控制标准适用于该疫苗工艺的质量控制.

目的 验证电位滴定法用于螨变应原注射液氯化钠含量测定的可行性.方法 利用电位滴定法和硫氰酸铵滴定法检测螨变应原注射液中氯化钠含量,确定两种方法的线性范围,并进行精密性、准确性验证.应用两种方法测定13批螨变应原注射液中氯化钠含量.结果 电位滴定法测定螨变应原注射液氯化钠含量的线性检测范围(0.10～1.50 mL)较硫氰酸铵滴定法(0.50 ～ 1.50 mL)广;精密性验证结果表明,电位滴定法检测氯化钠含量的RSD不高于1.87％,硫氰酸铵滴定法不高于1.97％;准确性验证结果表明,硫氰酸铵滴定法检测氯化钠含量的加标回收率为95.20％ ～ 112.66％,电位滴定法为92.88％～99.44％.两种方法检测13批(共27个亚批)螨变应原注射液氯化钠含量,结果基本一致,差异无统计学意义(P＞0.05),且全部符合该制品注册检验标准.结论 电位滴定法可作为硫氰酸铵滴定法的补充方法,用于螨变应原注射液氯化钠含量的测定.

Objective To prepare,identify and preliminarily monoclonal antibody (McAb) against Salmonella paratyphi A.Methods BALB/c mice were immunized with S.paratyphi A inactivated with formaldehyde,of which the spleen cells were fused with Sp2/0 myelomas cells.The hybridoma cell strains stably secreting McAb against S.paratyphi A were screened and purified,with which BALB / c mice were immunized.The ascites of immunized mice was collected,determined for antibody titer by indirect ELISA,and identified for class and subclass of Ig.The ascitcs was further purified by salting out with caprylic acid-ammonium sulphate,then subjected to indirect ELISA,slide agglutination test,SDS-PAGE and immunodiffusion test.The purified McAbs secreted by two hybridoma cell strains were used for development of a double antibody sandwich ELISA for determination of plasma from patients with paratyphoid A,paratyphoid B and typhoid as well as normal human plasma.Results Two hybridoma cell strains stably secreting McAb were obtained and named as 1H4 and 2A9 respectively.The secreted McAbs,at titers of 1 ∶ 107 and 1 ∶ 106,were IgG1 and IgG3 respectively,both of which were of κ chain.The titers of purified McAbs 1H4 and 2A9 were 1 ∶ 107 and 1 ∶ 103,while the purities were more than 85％ and less than 60％,respectively.Both the McAbs showed agglutination with S.paratyphi A 50503 and 50973 while no agglutination with S.typhi 50096.McAb 1H4 at a dilution of 1 ∶ 8 showed agglutination with organism specific protein (OSP) of S.paratyphi A.However,McAb 2A9 at any dilution showed no agglutination with the OSP.The combination 1H4/HRP-2A9 was optimal to the development of double antibody sandwich ELISA,by which the positive rate of positive samples was 100％ while that of normal human plasma was 0.Conclusion The prepared McAbs may be used for the rapid identification of S.paratyphi A.