大肠杆菌血清抗体与硫酸庆大霉素偶联构建体靶向药物-学位论文.doc

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1、 91(2011)e136-e143兽医科学研究 内容列表在科学指南 兽医学研究杂志主页； 大肠杆菌血清抗体与硫酸庆大霉素偶联构建体靶向药物董x伟 陈x军 刘x克 陈x涛 孙x良xx农业大学动物医学学院,长沙,xx410128,中国文章信息 文章历史2009年3月15日收到接受于2011年1月23日关键词: 抗体硫酸庆大霉素共轭 大肠杆菌 摘要轭合物的大肠杆菌血清抗体(Ab)硫酸庆大霉素(GM硫酸)评估的评估能力消除大肠杆菌体外。每个组件的结合在有针对性的药物血清抗体和硫酸庆大霉素的氨基结合羟基PEG6000的氢键,形成稳定的配合物。这个复杂的半衰期4.83 h,无毒副作用,低免疫原性。抗体的

2、结合和庆大霉素之后硫酸,抗体的目标相当好。硫酸庆大霉素的体外抗菌活性增加了1000倍,临床疗效增强100倍,这意味着更高的功效和安全。 1.介绍细菌病控制主要是依靠化学抗生素。由于细菌耐药性的出现,细菌疾病的预防和治疗越来越复杂(球et al .,2002;克雷格,2001;西蒙斯et al .,2006)。为了抵抗细菌,开发新的抗生素是必要的。由于资源有限,新抗生素越来越难以被发现(美林et al .,2006)。所以有必要转换现有的抗生素到一些新类型的药物(Schentag et al .,2007;马丁和拉尔斯,2004;沈和野中郁次郎,2005)。我们的研究集中在药物针对细菌、靶向药物

3、是新型的抗生素是由增加药物的生物利用度有针对性的药物(针对药物系统、TDS或靶向药物传递系统,服务于聋哑人)是一种新型的药物输送系统,可以实现药物损伤组织,器官、细胞或细胞结构的高性能(利玛窦et al .,2006;Ong et al .,2005)。有针对性的药物治疗能明显提高药物浓度在微生物。因此,可以减少剂量和治疗费用,也可以减少药物的副作用。近年来,抗体共轭靶向药物的开发取得了许多突破时被用于肿瘤的治疗。现在,开发了一些靶向药物治疗与癌症和肿瘤squamous-celled癌(Burtness,2005),腺癌,大细胞Carcinom(Raizer,2005),肺癌(Pao和米勒,2

4、005),小细胞癌,胃肠道间质肿瘤(威尔逊et al .,2006)等。但是没有类比目标药物的细菌。接合的抗体药物目标致病性细菌能产生一种新的抗菌药物目标(Iftach et al .,2006)。新抗菌药物显示针对病原体专门体内和体外。,这样就可以避免浪费,造成平均药物体内分布和治疗可以缩短进入病原体的药物将会更快。同时,减少药物剂量和疗程,药物残留也减少,体内病原体耐药性的出现误点。2材料和方法2.1免疫球蛋白的预备纯化制备的抗血清,定容的生理盐水加在10毫升抗血清。掺合料离心机在10000克15分钟和收集上清。5毫升饱和硫酸铵(pH值7.8)的上层清液中添加了外加剂与缓慢地缓慢搅拌30分

5、钟没有泡沫。这种掺合料离心机在10000克15分钟获得沉淀。颗粒在1.37 resuspended硫酸铵溶液和离心机10000克15分钟2次。0.01 PBS的降水量洗治疗1次,然后溶解在0.01 PBS。溶解沉淀转入袋透析的过滤器。透析是前12 h的4 C透析液改变每2小时。透析后的抗血清离心机在1000克15分钟。然后,原油提取免疫球蛋白在上层的收购For ion exchange chromatography,离子交换色谱法、an aliquot of sample was loaded onto a column一个整除的样本加载到一个列(6 mL of bed volume), pa

6、cked with preswollen DEAE-cellulose卷)(6毫升的床上,挤满了preswollen deae纤维素(Whatman, DE-32), and equilibrated with 200 mM citratephosphate(DE-32绘画纸),并与200毫米citrate-phosphate低沉buffer, pH 5.0.缓冲区,pH值5.0。The column was washed 2 times with 9 mL列与9毫升洗2次of 20 mM citratephosphate buffer, pH 5.0, and eluted with20毫米

7、citrate-phosphate缓冲区,pH值5.0,和洗提200 mM citratephosphate buffer, pH 6.4.200毫米citrate-phosphate缓冲区,pH值6.4。The elution profiles of的洗脱概要文件samples were plotted, and fractions that make a peak were pooled样本绘制,分数峰值是汇集and analyzed for antibody activity and purity using enzyme-linked并分析了使用酶联抗体活性和纯度immunosorbe

8、nt assay (ELISA).免疫吸附测定(ELISA)。2.2. ELISAMicro titer 96-well plates (Dynatec Laboratories, McLean, VA)微效价96孔板(弗吉尼亚州McLean Dynatec实验室)were used as the solid support.被用作固体支持。Polystyrene 96-well plates (Nalge96 -板块(Nalge聚苯乙烯Nunc Int., Rochester, NY) were coated with 100 lL of IgG samplesNunc Int,罗彻斯特,纽

9、约)涂上100的免疫球蛋白g样本dissolved in a coating solution (0.05 M carbonate buffer, pH 9.6)溶解在涂层解决方案(0.05碳酸盐缓冲,pH值0.05)and incubated overnight at 4 C. After washing the wells 3 times和孵化隔夜后4 c .洗井3次with PBS-Tween (10 mM phosphate, 0.15 M NaCl, pH 7.2, 0.05%磷酸与PBS-Tween(10毫米,0.15 M氯化钠,pH值7.2,0.05%Tween 20), 300

10、 lL/well of blocking solution 1% BSA solution in二层20),300你/屏蔽解决方案1% BSA的解决方案PBS (10 mM phosphate, 0.15 M NaCl, pH 7.2) was added.PBS(磷酸10毫米,0.15 M氯化钠,pH值7.2)是补充道。After后incubating for 1 h at room temperature, the plate was washed with在室温下孵化1 h,盘子洗了PBST.PBST。To each well of the plate, 100 lL of primar

11、y anti-chicken IgG每个板的,100你的主anti-chicken免疫球蛋白g(1:10,000 solution diluted with 1% BSA conjugated alkaline phosphatase)(1:10,000溶液稀释1% BSA共轭碱性磷酸酶)was added and then incubated for l h.添加,然后孵化l h。After washing with洗后PBS-Tween, 50 lL of p-nitrophenyl phosphate solution was addedPBS-Tween,50对硝基苯磷酸的解决方案了t

12、o each well as a substrate for color development and incubated每个颜色的基质开发和孵化for 30 min. The enzyme reaction was stopped by adding 50 lL of30分钟。这种酶反应是通过添加50会停止3 N NaOH, and the color developed was read on an ELISA plate3 N氢氧化钠,颜色开发阅读ELISA板reader (THERMO max, Molecular Devices Corp., Sunnyvale, CA)读者(热马

13、克斯、分子设备Corp .)、桑尼维尔CA)with a 405-nm filter.与405 nm过滤器。For each plate, 3 controls were prepared: a对于每一个板,3控制准备:一个positive control with reagent grade chicken IgG (SigmaAldrich),积极控制试剂级鸡免疫球蛋白g(Sigma-Aldrich),a nonspecific antigen BSA as another control, and a negative control非特异性抗原BSA作为另一个控制和消极的控制witho

14、ut antigen.没有抗原。All the procedures for ELISA were conductedELISA进行所有程序at room temperature (about 25 C).在室温(25 C)。2.3. BindingGentamycin sulfate-antibody-PEG conjugates were prepared庆大霉素sulfate-antibody-PEG配合准备according to the methods as follows (Yelena et al., 2006; Kevin根据方法如下(Yelena et al .,2006;凯

15、文et al., 2004; Charlotte et al., 2006).et al .,2004;夏洛特et al .,2006)。Purified antibody was bufferexchanged纯化抗体是bufferexchangedinto a solution containing 50 mM potassium phosphate到一个包含50 mM磷酸钾的解决方案and 2 mM EDTA, pH 7.0.和2毫米EDTA,pH值7.0。Gentamycin sulfate was dissolved in硫酸庆大霉素是溶解在dimethylacetamide (DM

16、A) and added to the antibody and PEG二甲基乙酰胺(DMA)和添加到抗体和挂钩solution to make a final gentamycin sulfate/antibody/PEG molar解决方案做出最后硫酸庆大霉素/抗体/ PEG摩尔ratio of 400:2:9.400:2:9的比率。The reaction was allowed to proceed for 24 h at反应被允许继续为24小时4 C with mixing4 C的混合The modified antibody was subsequently purified修改后

17、的抗体被净化on a GE Healthcare HiTrap desalting column (G-25) equilibrated在通用电气医疗集团HiTrap脱盐列(物体)均衡in 35 mM sodium citrate with 150 mM NaCl and 2 mM在35毫米和150毫米氯化钠和柠檬酸钠2毫米EDTA, pH 6.0.EDTA,pH值6.0。Typically, a 4060% yield of antibody was achieved通常,一个抗体的产量达到40 - 60%through this process.通过这一过程。The preparation

18、 was usually greater than的制备通常是大于95% monomeric as assessed by gel filtration and laser light单体的95%作为评估通过凝胶过滤和激光scattering.散射。2,4红外扫描The samples were sprayed over an area of 1 cm-12 on a KBr window.样本喷洒面积达1 cm-12 KBr窗口。This was performed at a very low spraying rate (4.2 mL/min)这是表现在一个非常低的喷涂率(4.2毫升/分钟

19、)under a gentle stream of nitrogen gas using a sample applicator.在温柔的氮气流使用一个示例涂布。All所有spectra were acquired on a Bio-Rad FTS4000 FTIR spectrometer光谱是收购Bio-Rad FTS4000红外光谱分光计(Cambridge, MA) equipped with a broad-band mercury cadmium(Cambridge,MA)配有宽带汞镉telluride detector, cooled with liquid nitrogen.碲

20、化探测器,用液氮冷却。The samples were样本closed by a second KBr window.被第二个KBr关闭窗口。The samples were under continuous在连续的样本dry air purge starting 1 h before the data acquisition.干燥的空气净化数据采集开始前1小时。The spectra的光谱were collected in transmission mode as a coaddition of 256scans at 1 cm-11 resolution over 4 min. The s

21、oftware used was收集256年传输方式作为coaddition扫描1 cm-11决议在4分钟。使用的软件是什么Win-IR pro 3.0 from Bio-Rad.从Bio-Rad Win-IR pro 3.0。The spectra were deconvoluted using光谱是deconvoluted使用a half-width of 5 cm-11 and an enhancement factor of 2 (Todd半角5 cm-11和2(Todd的增强因子et al., 2000; Gooris, 2007).et al .,2000;Gooris,2007)

22、。.2.5紫外线The samples, including conjugate, BSA, gentamycin sulfate,样品,包括共轭、BSA、硫酸庆大霉素、antiserum and PEG6000, were diluted in different concentration在不同浓度抗血清和PEG6000稀释with 10 mM PBS, pH 7.4; then the samples were incubated at与10毫米PBS,pH值7.4;然后样本孵化4 C for 5 h, then scanned with ultraviolet.4 C 5 h,然后用紫

23、外扫描。All spectras that were所有的光谱,acquired on a Bio-Rad FTS4000 FTIR spectrometer (Cambridge,获得一个Bio-Rad FTS4000红外光谱谱仪(剑桥,MA) had been equipped with a broad-band mercury cadmium tellurideMA)已经配备了宽带碲化镉汞detector, and cooled with liquid nitrogen.探测器,并与液态氮冷却。Deionized water去离子水was selected as control.被选为控

24、制。2.6. Colloidal goldA stock of 0.01% colloidal gold was made by a slight modification胶体金是由0.01%的股票略有修改method described by Frens (1973).方法被顺便(1973)。Ten milligrams of chloroauric10毫克的氯金acid (Sigma) was added to 100 mL of boiling distilled酸()加入100毫升的沸腾蒸馏deionized water in a silicified triangular glass

25、 bottle.去离子水在硅化的三角玻璃瓶子。This solution这个解决方案was boiled and stirred vigorously.煮,搅拌。After 3 min, 2.5 mL of 1% trisodium3分钟后,三钠的2.5毫升的1%citrate dihydrate (Sigma) was rapidly dropped into the boiling柠檬酸二水合物()迅速下降到沸腾solution.解决方案。While still being heated, the color of the solution同时被加热,溶液的颜色changed from g

26、old to faint blue within about 40 s, after that it改变从黄金到微弱的蓝色在40年代,在那之后turned to dark blue.变成了深蓝色。After 2 min, the color of the solution changed2分钟后,溶液的颜色变化to a clear brilliant red, indicating the formation of monodisperse清晰的红色,表明单分散的形成spherical particles.球形颗粒。The solution was boiled for another 10

27、min to解决方案是煮10分钟reach the reaction endpoint and then slowly cooled at room temperature.达到反应在室温下端点,然后慢慢冷却。It was stored at 4 C in a tightly sealed silicified glass bottle.这是存储在4 C密封硅化的玻璃瓶中。The conjugate solution was added dropwise with stirring to the共轭的解决方案是添加一滴一滴地激动人心的gold colloid and the mixture g

28、ently stirred for 30 min at room temperature.金胶体和混合物在室温下轻轻地搅拌30分钟。At the end of the reaction period, 100200 lL 1% polyethylene结束时的反应期,100 - 200年1%的聚乙烯glycol compound and 100 lL of 10% NaCl in distilled water醇化合物和100年10%氯化钠的蒸馏水were added and the mixture were stirred for an additional 1为一个额外的添加和混合搅拌1

29、-2 min.2分钟。The mixture was then divided evenly and added to Escherichia混合均匀划分,添加到大肠coli solution.杆菌的解决方案。The mixture was embedded and negatively混合物是嵌入式和消极stained with Phosphotungstic acid, and then scanned by electron沾染了磷钨酸,然后通过电子扫描microscopy.显微镜。2.7.Determination of minimum inhibitory concentration

30、s (MICs) and测定(中等收入国家)和最低抑制浓度minimal bactericidal concentration (MBCs)最小杀菌浓度(MBCs)MICs were determined according to the National Committee for中等收入国家都根据国家委员会决定Clinical Laboratory Standards (NCCLS) criteria for E. coli.临床实验室标准(NCCLS)大肠杆菌的标准。Enteropathogenic致肠病的E. coli was tested and E. coli ATCC25922

31、was selected大肠杆菌是测试和大肠杆菌ATCC25922选中to be the standard quality control strain (Arthur et al., 1999).是标准的质量控制应变(Arthur et al .,1999)。2.8. Determination of the PAETwenty milliliters of 1:20 diluted overnight culture (OD of 0.1 at20毫升的1:20稀释一夜之间文化(OD 0.1600 nm = 106 cfu) was incubated for 2 h at 37 C wi

32、th or without600海里= 106 cfu)孵化为2 h 37 C有或没有the conjugate to be tested, at concentrations of 10 MIC.被测试的共轭,10麦克风的浓度。In order为了to remove the conjugate, exposed bacteria were washed twice with消除共轭接触细菌洗两次phosphate-buffered saline (pH 7.2) by centrifugation for 10 min atphosphate-buffered盐水(pH值7.2)离心10分钟

33、7000g; controls were treated similarly.7000克;控制同样对待。The pellets were resuspended球团矿是resuspendedin 20 mL of LB liquid medium after being incubated at孵化后磅20毫升的液体介质37 C and samples (1 mL) were obtained at 2 h (2 h of exposure37 C和样品(1毫升)得到2 h(2 h的接触to the conjugate) for corrections that were made to en

34、sure that修正的共轭),以确保all the cultures would start with the same bacterial count所有的文化将开始使用相同的细菌数Samples样品were obtained at time 0 (immediately after washing and after correction)洗后立即得到在时间0(修正后)and then hourly up to 7 h and OD values determined然后每小时7 h和OD值确定(reflecting bacterial growth).(反映细菌生长)。The OD v

35、alues were converted intoOD值被转换成cfu (bacterial growth) by using a standard curve which wascfu(细菌生长)通过使用标准曲线constructed relating the number of bacteria to the OD.细菌的数量OD相关的构造。The PAE was defined according to Craig & Gudmundsson6 asPAE是根据克雷格& Gudmundsson6定义PAE =TC, where T refers to the time required

36、for the viable countsPAE =温度系数,T是指可行的计算所需的时间of the exposed bacteria to increase by 1 log10 above the counts observed暴露在细菌的增加1 log10以上观察到的数量immediately after washing and C means the corresponding洗后立即和C意味着相应的time for the conjugate unexposed controls.的共轭未曝光控制。2.9. Treatment of colibacillosis in animal

37、model with the conjugateTo determine the LD50 of swine E. coli, mice model was used.确定猪大肠杆菌的LD50,小鼠模型使用。Sixty mice were divided into 10 groups, 2022 g of each one.60小鼠分为10组,20 g的每一个。Six六个mice were randomly divided into each group.小鼠随机分为两组。The 10 groups were10组recorded as group 1, group 2, group 3, g

38、roup 4, group 5, group 6,记录为组1、组2组3组4组5、6组,group 7, group 8, group 9, and group 10, respectively.集团7日8日9,分别和组10。Inoculation of E. coli diluents: E. coli liquid culture is diluted大肠杆菌的接种稀释剂:大肠杆菌液体文化是稀释with ordinary broth in a 10-fold serial dilution to be 101, 102,与普通肉汤系列稀释10倍到101岁,102年,103, 104, 105

39、, 106, 107, 108, 109, 1010, 10 gradient dilution103,104,105,103,104,108,109,1010,10梯度稀释of E. coli liquid culture, then these 10 groups mice were inoculated大肠杆菌的液体培养,那么这十组小鼠接种respectively, and 0.2 mL per mice was injected intraperitoneally.分别和0.2毫升/小鼠腹腔注射。The value was calculated using Karber method:

40、 Log CCID50使用Karber值的计算方法:日志CCID50(LD50) = Ld (S0.5).(LD50)=记为(s - 0.5)。To determine the effect of conjugate against colibacillosis确定共轭对大肠杆菌病的影响in vivo, we used mice as an animal model.体内,我们使用小鼠作为动物模型。One hundred eighty一百八十年mice were divided into 6 groups, 2022 g of each one.老鼠被分为6组,20 g的每一个。Thirty

41、mice三十个老鼠were randomly divided into each group.被随机分为两组。Animals were intramuscularly动物肌内injected with five LD50 dose of enteropathogenic E. coli.五LD50注射剂量的大肠杆菌致肠病的。Drug药物delivery methods were shown in Table 1.交货方法如表1所示。We evaluated the therapeutic我们评估了治疗effect in the following facts: Healing-a course

42、 of medication影响以下事实:正在治疗的药物treatment when all the mice were breathing normally in good spirits;当所有的治疗小鼠正常呼吸精神抖擞;Effectiveness-a course of medication treatment when the mice当老鼠效力在药物的治疗had difficulties in breathing recovery, but were in good spirits;呼吸困难的复苏,但精神抖擞;Ineffectiveness-a course of medicatio

43、n treatment when the medicationIneffectiveness-a药物治疗时药物的进程treatments were ineffective to eliminate or alleviate the治疗无效的消除或缓解symptoms, or all of the animals died.症状,或所有的动物死亡。2.10.Determinate the pharmacokitetics, safety and immunogenic of定pharmacokitetics,安全性和免疫原性conjugate共轭Inoculating conjugate and

44、 determination of the plasma concentration接种共轭和决心的血浆浓度of gentamicin sulfate: Rabbits with urethane anesthesia与氨基甲酸乙酯麻醉的硫酸庆大霉素:兔子and then, make the common carotid artery.然后,使常见的颈动脉。Before drugs injecting,药物注射之前,6 ml blood was collected as blank control.6毫升血液收集空白控制。Then each time intramuscularly然后每次肌内

45、rabbits were injected with serum antibody, PEG6000 and兔子注射血清抗体,PEG6000和gentamicin sulfate 1.88 mg/kg.硫酸庆大霉素1.88毫克/公斤。After injection, at 0.25, 0.5, 0.75, 1,注射后,在0.25,0.5,0.75,1,2, 4, 8, 10, 12, 14, 16, 24 h time points, 1 ml blood sample was taken2、4、8、10、12、14、16、24小时时间点,1毫升血液样本from the rabbits.从兔子

46、。After that, 0.2 ml heparin was injected into the在那之后,0.2 ml肝素注入catheter.导管。Next time firstly dispose about 5 drops of blood sample,下次首先处理5滴血液样本,heparin was added into samples as anticoagulant.肝素和抗凝剂添加到样本。Blood samples血液样本were centrifuged at 2000g for 10 min, and in order to control the在2000 g离心10分钟,为了控制plasma concentration within a linear range, dilute blood sam