新型布氏菌皮肤变态反应制剂的实验及应用研究
|
摘要
布氏杆菌病(Brucellosis)简称布病,是人兽共患的传染变态反应性疾病,布病所具有的迟发型超敏反应为其特异性皮内试验提供了先决条件。皮试诊断方法与血清学诊断方法相结合,对急、慢性布病的诊断具极大价值,同时皮试可使耶尔森09感染引起的布病血清学假阳性反应与布病区分开来。
布氏菌素主要分为两大类:粗制布氏菌素和纯化布氏菌素,统称为变态反应原。粗制布氏菌素主要有强毒菌素、弱毒菌素、犬种菌素、改制菌素、无外源蛋白菌素等。在皮试诊断制品提取过程中,不同菌种、菌体蛋白、脂多糖等因素均影响菌素的质量。本研究采用高速离心法和化学提取法,对布氏菌菌体蛋白、脂多糖、核酸进行提取分析,筛选出三氯醋酸(TCA)和硫酸铵(AS)综合化学反应法,制备布氏菌纯蛋白变应原(Br-PPA),全面开展研究,获得了理想的结果。
材料与对象:
材料:猪种布氏菌Br.S_2,布氏菌104M菌苗,法国纯化布氏菌素(INRA纯化菌素),国产布氏菌素,昆明系小鼠。
观察对象:布病病人,布氏菌感染人群,布氏菌苗免疫人群,结核病人,其它感染病人,非布病疫区健康人。
方法:
制备布氏菌纯蛋白变应原(Br-PPA):猪种布氏菌Br.S_2启开后,接种于土豆葡萄糖琼脂培养基,生化培养箱37℃培养24小时;挑选典型菌落接种于肝琼脂斜面培养48小时;用灭菌生理盐水洗下菌苔,制成浓菌液,浓度为5×10~9菌/ml;15磅20分钟高压灭菌浓菌液;将灭菌液离心,转速4000转/min,分离上清液;取上清液加等量饱和硫酸铵,于4℃过夜进行盐析;将盐析沉淀的蛋白离心,转速10000转/min,取沉淀物;沉淀蛋白用PBS进行溶解,三氯醋酸将溶解蛋白
郑州大学2004年硕士论文
新型布氏菌皮肤变态反应制剂的实验及应用研究
进行沉淀:重复PBS溶解、酸沉淀,用蒸馏水及PBS透析:将蛋白变应原过滤
除菌,真空干燥。
通过测定蛋白、多糖和核酸含量来对Br一P以进行纯度测定;选用18一209昆
明系小鼠进行安全试验:选用300一4009豚鼠用布氏菌104M菌苗免疫来制备致
敏动物;用Br一PPA与参考标准品4IU进行动物标化,进行效价标定。将Br一PPA
稀释为100林g/ml,皮内注射300一4009健康豚鼠,完成致敏效应试验。将稀释的
成品置37℃45天及置4℃半年后取出分别作皮肤试验,并均与参考标准品进行
效价测定,完成稳定性实验。
Br一PpA安全性效果观察:通过对布病病人、布氏菌感染人群、布氏菌苗免
疫人群进行速发型反应发生率比较,确定菌素和Br一PPA对三类人群反应阳性率
差异是否有统计学意义。通过对六类人群进行局部反应和全身反应发生率比较,
确定菌素和Br一PPA对各类人群局部反应和全身反应阳性率差异是否有统计学意
义。
诊断标准研究:通过对菌素和Br一PPA对布病病人、隐性人群和免疫人群的
24h和48h皮内变态反应面积进行比较,确定与菌素组无显著性差异的Br一PPA
面积组,进行敏感性测定。通过菌素和Br一PPA两种变应原对健康人、结核病人
和其他病人等非布病患者变态反应面积比较,确定最佳Br-P队面积组。
所获资料采用统计分析系统SPSS软件处理。
结果:
1.Br一P队纯度测定结果:B卜PPA蛋白含量大于95%,多糖、核酸含量均小于3%,
符合国家生物制品规程要求。
2.安全试验:三批昆明系小鼠腹腔注射一周后,无一死亡,且体重明显增加,
安全性好。
3.效价标定:三批Br一PPA不同预标记量注射豚鼠24h和48h后,其皮肤红肿直
径均随浓度加大而增加;其预标量与参考标准品4JU比值均在1:l士0.2范围内,
达到了国家生物制品规程上的合格要求。
4.致敏效应试验:实验组与对照组皮肤反应未见明显区别,在豚鼠身上24小时
为红肿反应高峰,48小时反应低于24小时。
5.稳定性试验:稀释为100林留ml成品置37℃45天及置4℃半年后取出分别作皮
肤试验,将24h和48h皮肤均径与标准品均径相比,所得结果均在卜1士0.2范围
郑州大学2004年硕士论文
新型布氏菌皮肤变态反应制剂的实验及应用研究
内,符合WHO相关生物制品规程要求。
6.Br-PPA安全性效果观察:对布病病人、布氏菌感染人群、布氏菌苗免疫人群
进行速发型反应发生率比较,菌素和Br一PPA对三类人群的反应阳性率分别是
71.26%和25.22%、50.51%和13.76%、30.48%和9.07%。两者之间对三类人群
的速发型反应差异均有统计学意义(尸灯0.01)。菌素可引起布病病人、隐性感染、
免疫人群不同程度的局部反应,Br一P队未发现局部反应:两者对布病病人、隐
性感染、免疫人群的局部反应差异均有统计学意义(尸丫0.01)。菌素可引起布病
病人不同程度的全身反应,Br一PPA未发现全身反应;两者对布病病人的全身反
应差异有统计学意义(尸灯0.01)。
7.Br一PPA诊断标准研究:Br一PPA多剂量初标化和相近剂量复测结果表明
0.3林创0.lml/人剂量组与参考标准品总均径比值在卜1士0.2范围内。以布病病人对
不同剂量组Br一P队反应均径来确定最佳观察时间为48h。通过对两种变应原皮
内变态反应的面积进行观察,菌素均大于Br一PPA;经厂检验,菌素组与Br一PPA
组1.5xl.5、2.0x2.0、2.5x2.5阳性率差异有显著性(P<0.01);而与Br-PPA组
0.5xo.5、1.0义1.0差异均无显著性(P,0.05)。通过两种变应原对非布病患者进
行观察,菌素组与Br一P队组0.5x0.5、1.0火1.0差异均无显著性(P)0.05),菌素
组与Br一PP
Brucellosis is a kind of infected-metamorphism disease existed in human beings and animals. The characteristics of delayed-type hypersensitivity reaction for Brucellosis provide precondition for its specific skin trial diagnosis. The combination of skin trial and serology has great value for the diagnosis of urgent and tardy brucellosis, and the skin trial can distinguish the artificial positive reaction caused by Yersin 09 in serology test with brucellosis.
Though brucellin includes many kinds, it can be divided into two species, such as rough brucellin and purified brucellin, they all can be intituled allergen. Rough brucellin includes strong-poisoned-brucellin, feeble-poison brucellin, dog-strained brucellin, strypped-down brucellin, and no outer-fountained-protein brucellin. In the course of pick-up for the skin diagnosis products, such as brucellin, the different strain, bacteria proteins, and lipopolysacchride, can all affect the quality of brucellin. The Brucella bacteria proteins, lipopolysacchride and nucleic acid had been extracted and analyzed by the use of the methods such as ultra-speed centrifugation and chemical extraction methods. The trichloroacetic acid (TCA) and ammonium sulfate (AS) synthesis chemical reaction had been filtrated to purify Brucella enduring-heat protein, namingly purified rotein allergen of Brucella (Br-PPA), then it had benn thoroughly studied and ideal results were obtained.
Materials and objects:
Materials: Brucella suis 2,Brucella vaccine strain 104M,Purified brucellin from France,Brucellin saled at marketplace in our courtry,The KunMing series mouse. Observed objects: Brucellosis patientsjnfected crowds of Brucella, The irnmuned crowds, Tuberculoses,other infected patients and healthy crowds. Methods:
The preparation of Br-PPA: The Brucella suis 2 which had been examined eligible were inoculated on murphy-glucose-arar culture medium, and were cultured in biochemistry box at 37 degree for 24 hours. The representative bacterium falls were picked out and inoculated on live agar slant culture medium for 48 hours, the lawn was washed out with sterilized physiological brine and executed strong bacteria loquid, the concentration was 5 multiply 109 numbers per milliliter. The strong bacteria liquid was sterilized at 15 pounds for 20 minutes. The sterilized liquid was centrifuged at 4000g per minute, the upper liquid was extracted and added equal quantity saturated ammonium sulfate, and was salted out at 4 degree for 12 hours. The deposited protein was centrifuged at speed lOOOOg per minute, the centrifuged deposited protein was dissolved with PBS, and the dissolved protein was deposited with trichloroacetic acid. The deposited protein was dissolved with PBS again, and the purity of sample liquid was determined. The deposition with trichloroacetic acid was repeated, and the deposited sample was dialysed with distilled water and PBS. The purified allergen after dialysis was filtered and frozen in vaccum. After the product was examined up to grade, it was diluted and packed out.
The allergen was determined its protein contents by the use of Lowry method, its amylose contens were determined used by anthracene-ketone colorimetry method, its nucleic acid contents were determined by the use of purple-spectrophotometer. The safety trial was completed by the means of observing the alive-keeping situation for KunMing series mouses weighing 18-20g. The causing-sensitive animals were prepared by using guinea-pigs weighing 300-400g which had been immune with Brucella vaccine 104M. The effect-value was determined by the use of Br-PPA and French referenct standard products 4IU. The stability test was completed by the
means that the diluent products were made skin trial, and were used to the determination of effect-value, and were compared with reference standard products after the diluent products being put at 37 degree for 45 days and 4 degree for half a year.
The observation of security effect for Br-PPA: The occurrance rate of fast-type reaction was compared among brecellosis and crowd
布氏菌素主要分为两大类:粗制布氏菌素和纯化布氏菌素,统称为变态反应原。粗制布氏菌素主要有强毒菌素、弱毒菌素、犬种菌素、改制菌素、无外源蛋白菌素等。在皮试诊断制品提取过程中,不同菌种、菌体蛋白、脂多糖等因素均影响菌素的质量。本研究采用高速离心法和化学提取法,对布氏菌菌体蛋白、脂多糖、核酸进行提取分析,筛选出三氯醋酸(TCA)和硫酸铵(AS)综合化学反应法,制备布氏菌纯蛋白变应原(Br-PPA),全面开展研究,获得了理想的结果。
材料与对象:
材料:猪种布氏菌Br.S_2,布氏菌104M菌苗,法国纯化布氏菌素(INRA纯化菌素),国产布氏菌素,昆明系小鼠。
观察对象:布病病人,布氏菌感染人群,布氏菌苗免疫人群,结核病人,其它感染病人,非布病疫区健康人。
方法:
制备布氏菌纯蛋白变应原(Br-PPA):猪种布氏菌Br.S_2启开后,接种于土豆葡萄糖琼脂培养基,生化培养箱37℃培养24小时;挑选典型菌落接种于肝琼脂斜面培养48小时;用灭菌生理盐水洗下菌苔,制成浓菌液,浓度为5×10~9菌/ml;15磅20分钟高压灭菌浓菌液;将灭菌液离心,转速4000转/min,分离上清液;取上清液加等量饱和硫酸铵,于4℃过夜进行盐析;将盐析沉淀的蛋白离心,转速10000转/min,取沉淀物;沉淀蛋白用PBS进行溶解,三氯醋酸将溶解蛋白
郑州大学2004年硕士论文
新型布氏菌皮肤变态反应制剂的实验及应用研究
进行沉淀:重复PBS溶解、酸沉淀,用蒸馏水及PBS透析:将蛋白变应原过滤
除菌,真空干燥。
通过测定蛋白、多糖和核酸含量来对Br一P以进行纯度测定;选用18一209昆
明系小鼠进行安全试验:选用300一4009豚鼠用布氏菌104M菌苗免疫来制备致
敏动物;用Br一PPA与参考标准品4IU进行动物标化,进行效价标定。将Br一PPA
稀释为100林g/ml,皮内注射300一4009健康豚鼠,完成致敏效应试验。将稀释的
成品置37℃45天及置4℃半年后取出分别作皮肤试验,并均与参考标准品进行
效价测定,完成稳定性实验。
Br一PpA安全性效果观察:通过对布病病人、布氏菌感染人群、布氏菌苗免
疫人群进行速发型反应发生率比较,确定菌素和Br一PPA对三类人群反应阳性率
差异是否有统计学意义。通过对六类人群进行局部反应和全身反应发生率比较,
确定菌素和Br一PPA对各类人群局部反应和全身反应阳性率差异是否有统计学意
义。
诊断标准研究:通过对菌素和Br一PPA对布病病人、隐性人群和免疫人群的
24h和48h皮内变态反应面积进行比较,确定与菌素组无显著性差异的Br一PPA
面积组,进行敏感性测定。通过菌素和Br一PPA两种变应原对健康人、结核病人
和其他病人等非布病患者变态反应面积比较,确定最佳Br-P队面积组。
所获资料采用统计分析系统SPSS软件处理。
结果:
1.Br一P队纯度测定结果:B卜PPA蛋白含量大于95%,多糖、核酸含量均小于3%,
符合国家生物制品规程要求。
2.安全试验:三批昆明系小鼠腹腔注射一周后,无一死亡,且体重明显增加,
安全性好。
3.效价标定:三批Br一PPA不同预标记量注射豚鼠24h和48h后,其皮肤红肿直
径均随浓度加大而增加;其预标量与参考标准品4JU比值均在1:l士0.2范围内,
达到了国家生物制品规程上的合格要求。
4.致敏效应试验:实验组与对照组皮肤反应未见明显区别,在豚鼠身上24小时
为红肿反应高峰,48小时反应低于24小时。
5.稳定性试验:稀释为100林留ml成品置37℃45天及置4℃半年后取出分别作皮
肤试验,将24h和48h皮肤均径与标准品均径相比,所得结果均在卜1士0.2范围
郑州大学2004年硕士论文
新型布氏菌皮肤变态反应制剂的实验及应用研究
内,符合WHO相关生物制品规程要求。
6.Br-PPA安全性效果观察:对布病病人、布氏菌感染人群、布氏菌苗免疫人群
进行速发型反应发生率比较,菌素和Br一PPA对三类人群的反应阳性率分别是
71.26%和25.22%、50.51%和13.76%、30.48%和9.07%。两者之间对三类人群
的速发型反应差异均有统计学意义(尸灯0.01)。菌素可引起布病病人、隐性感染、
免疫人群不同程度的局部反应,Br一P队未发现局部反应:两者对布病病人、隐
性感染、免疫人群的局部反应差异均有统计学意义(尸丫0.01)。菌素可引起布病
病人不同程度的全身反应,Br一PPA未发现全身反应;两者对布病病人的全身反
应差异有统计学意义(尸灯0.01)。
7.Br一PPA诊断标准研究:Br一PPA多剂量初标化和相近剂量复测结果表明
0.3林创0.lml/人剂量组与参考标准品总均径比值在卜1士0.2范围内。以布病病人对
不同剂量组Br一P队反应均径来确定最佳观察时间为48h。通过对两种变应原皮
内变态反应的面积进行观察,菌素均大于Br一PPA;经厂检验,菌素组与Br一PPA
组1.5xl.5、2.0x2.0、2.5x2.5阳性率差异有显著性(P<0.01);而与Br-PPA组
0.5xo.5、1.0义1.0差异均无显著性(P,0.05)。通过两种变应原对非布病患者进
行观察,菌素组与Br一P队组0.5x0.5、1.0火1.0差异均无显著性(P)0.05),菌素
组与Br一PP
Brucellosis is a kind of infected-metamorphism disease existed in human beings and animals. The characteristics of delayed-type hypersensitivity reaction for Brucellosis provide precondition for its specific skin trial diagnosis. The combination of skin trial and serology has great value for the diagnosis of urgent and tardy brucellosis, and the skin trial can distinguish the artificial positive reaction caused by Yersin 09 in serology test with brucellosis.
Though brucellin includes many kinds, it can be divided into two species, such as rough brucellin and purified brucellin, they all can be intituled allergen. Rough brucellin includes strong-poisoned-brucellin, feeble-poison brucellin, dog-strained brucellin, strypped-down brucellin, and no outer-fountained-protein brucellin. In the course of pick-up for the skin diagnosis products, such as brucellin, the different strain, bacteria proteins, and lipopolysacchride, can all affect the quality of brucellin. The Brucella bacteria proteins, lipopolysacchride and nucleic acid had been extracted and analyzed by the use of the methods such as ultra-speed centrifugation and chemical extraction methods. The trichloroacetic acid (TCA) and ammonium sulfate (AS) synthesis chemical reaction had been filtrated to purify Brucella enduring-heat protein, namingly purified rotein allergen of Brucella (Br-PPA), then it had benn thoroughly studied and ideal results were obtained.
Materials and objects:
Materials: Brucella suis 2,Brucella vaccine strain 104M,Purified brucellin from France,Brucellin saled at marketplace in our courtry,The KunMing series mouse. Observed objects: Brucellosis patientsjnfected crowds of Brucella, The irnmuned crowds, Tuberculoses,other infected patients and healthy crowds. Methods:
The preparation of Br-PPA: The Brucella suis 2 which had been examined eligible were inoculated on murphy-glucose-arar culture medium, and were cultured in biochemistry box at 37 degree for 24 hours. The representative bacterium falls were picked out and inoculated on live agar slant culture medium for 48 hours, the lawn was washed out with sterilized physiological brine and executed strong bacteria loquid, the concentration was 5 multiply 109 numbers per milliliter. The strong bacteria liquid was sterilized at 15 pounds for 20 minutes. The sterilized liquid was centrifuged at 4000g per minute, the upper liquid was extracted and added equal quantity saturated ammonium sulfate, and was salted out at 4 degree for 12 hours. The deposited protein was centrifuged at speed lOOOOg per minute, the centrifuged deposited protein was dissolved with PBS, and the dissolved protein was deposited with trichloroacetic acid. The deposited protein was dissolved with PBS again, and the purity of sample liquid was determined. The deposition with trichloroacetic acid was repeated, and the deposited sample was dialysed with distilled water and PBS. The purified allergen after dialysis was filtered and frozen in vaccum. After the product was examined up to grade, it was diluted and packed out.
The allergen was determined its protein contents by the use of Lowry method, its amylose contens were determined used by anthracene-ketone colorimetry method, its nucleic acid contents were determined by the use of purple-spectrophotometer. The safety trial was completed by the means of observing the alive-keeping situation for KunMing series mouses weighing 18-20g. The causing-sensitive animals were prepared by using guinea-pigs weighing 300-400g which had been immune with Brucella vaccine 104M. The effect-value was determined by the use of Br-PPA and French referenct standard products 4IU. The stability test was completed by the
means that the diluent products were made skin trial, and were used to the determination of effect-value, and were compared with reference standard products after the diluent products being put at 37 degree for 45 days and 4 degree for half a year.
The observation of security effect for Br-PPA: The occurrance rate of fast-type reaction was compared among brecellosis and crowd
引文