Preparation,purification and characterization of the polyclonal antibody against α1-AT

Objective To prepare the polyclonal antibody against α1-antitrypsin(α1-AT)by immunizing rabbits with α1-AT,and to purify and characterize the antibodies produced from rabbits in order to establish the production process of the polyclonal antibody against α1-AT.Methods The healthy rabbits were routinely immunized with α1-AT to prepare polyclonal antibody against α1-AT.The serum antibody titers were determined by ELISA.The rabbits with qualified antibody titers were killed to collect blood.The antisera,obtained by centrifugalizing the whole blood,were purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography.The purified polyclonai antibodies were analyzed by 15%SDS-PAGE,and antibody titers weIe measured by ELISA.Results ELISA assay showed that the titers of antisera were over 105.The polyclonal antibodies,purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography,had good purity and immunological activity.Conclusion The production and purification processes of polyclonal antibody against α1-AT are successfully established.     

丙型肝炎病毒多表位抗原的融合表达及抗原性分析

目的 表达HCV核心蛋白Core、非结构区NS3、NS4和NS5区的部分优势表面抗原的融合基因,为丙型肝炎病毒的检测提供合适抗原.方法 通过KT-PCK从HCV全长基因组中分别克隆Core、NS3、NS4和NS5区的部分优势表面抗原的基因片段,运用重叠延伸PCR技术将它们拼接成融合基因,将融合基因再克隆到表达裁体pBVIL-2中.转化大肠杆菌JM109,阳性克隆经42℃热诱导融合蛋白的表达,表达产物经SDS-PAGE及Western blot鉴定.重组蛋白经阳离子交换和分子筛纯化.结果 42℃诱导可高表达分子量约为43 kD的融合抗原,重组蛋白主要以包涵体形式存在.经过纯化目的 蛋白纯度达90%以上的,表达量约为886μg/mL.ELISA结果表明纯化蛋白具有良好的抗原性.结论 HCV复合多表位抗原融合蛋白可作为HCV诊断抗原提供了靶抗原.     

Preparation,purification and characterization of the polyclonal antibody against α1-AT

Objective To prepare the polyclonal antibody against α1-antitrypsin(α1-AT)by immunizing rabbits with α1-AT,and to purify and characterize the antibodies produced from rabbits in order to establish the production process of the polyclonal antibody against α1-AT.Methods The healthy rabbits were routinely immunized with α1-AT to prepare polyclonal antibody against α1-AT.The serum antibody titers were determined by ELISA.The rabbits with qualified antibody titers were killed to collect blood.The antisera,obtained by centrifugalizing the whole blood,were purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography.The purified polyclonai antibodies were analyzed by 15%SDS-PAGE,and antibody titers weIe measured by ELISA.Results ELISA assay showed that the titers of antisera were over 105.The polyclonal antibodies,purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography,had good purity and immunological activity.Conclusion The production and purification processes of polyclonal antibody against α1-AT are successfully established.     

Preparation,purification and characterization of the polyclonal antibody against α1-AT

Objective To prepare the polyclonal antibody against α1-antitrypsin(α1-AT)by immunizing rabbits with α1-AT,and to purify and characterize the antibodies produced from rabbits in order to establish the production process of the polyclonal antibody against α1-AT.Methods The healthy rabbits were routinely immunized with α1-AT to prepare polyclonal antibody against α1-AT.The serum antibody titers were determined by ELISA.The rabbits with qualified antibody titers were killed to collect blood.The antisera,obtained by centrifugalizing the whole blood,were purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography.The purified polyclonai antibodies were analyzed by 15%SDS-PAGE,and antibody titers weIe measured by ELISA.Results ELISA assay showed that the titers of antisera were over 105.The polyclonal antibodies,purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography,had good purity and immunological activity.Conclusion The production and purification processes of polyclonal antibody against α1-AT are successfully established.     

Preparation,purification and characterization of the polyclonal antibody against α1-AT

Objective To prepare the polyclonal antibody against α1-antitrypsin(α1-AT)by immunizing rabbits with α1-AT,and to purify and characterize the antibodies produced from rabbits in order to establish the production process of the polyclonal antibody against α1-AT.Methods The healthy rabbits were routinely immunized with α1-AT to prepare polyclonal antibody against α1-AT.The serum antibody titers were determined by ELISA.The rabbits with qualified antibody titers were killed to collect blood.The antisera,obtained by centrifugalizing the whole blood,were purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography.The purified polyclonai antibodies were analyzed by 15%SDS-PAGE,and antibody titers weIe measured by ELISA.Results ELISA assay showed that the titers of antisera were over 105.The polyclonal antibodies,purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography,had good purity and immunological activity.Conclusion The production and purification processes of polyclonal antibody against α1-AT are successfully established.     

抗α1-抗胰蛋白酶多克隆抗体的制备、纯化和鉴定

目的 用α1-抗胰蛋白酶(α1-antitrypsin,α1-AT)免疫家兔以产生多克隆抗体,并对产生的抗体进行有效地纯化和效价鉴定,建立抗α1-AT多克隆抗体的生产工艺.方法 按照常规方法免疫健康家兔进行抗α1-AT多克隆抗体的制备,用ELISA检测血清抗体效价.处死抗血清效价合格的家兔,取血、离心,获取抗血清.联合应用辛酸-硫酸铵沉淀法和A蛋白亲和层析法对抗血清进行纯化,纯化的多克隆抗体经15%十二烷基硫酸钠.聚丙烯酰胺凝胶电泳分析纯度后,用ELISA测定效价.结果 ELISA检测表明,家兔产生的抗α1-AT多克隆抗体效价大于105.联合应用辛酸-硫酸铵沉淀法和A蛋白亲和层析法进行纯化获得了纯度高、活性好的抗α1-ATT多克隆抗体.结论 成功建立了抗α1-AT多克隆抗体的生产和纯化工艺.     

洞庭湖洲滩开沟沥水控制钉螺繁殖的研究

目的 目的 评价洞庭湖区有螺洲滩实施开沟沥水工程后对钉螺孳生繁殖的影响。方法 方法 2009年11月-2012年11 月, 选择位于东洞庭湖区的岳阳监狱和君山区有螺外洲滩, 分别设为干预区和对照区2组研究现场观察区。在干预区洲 滩实施开沟沥水工程, 采用挖掘机开挖 “井” 字沟, 观察该工程对洲滩土壤含水量和相对地下水位、 以及对钉螺孳生繁殖 的影响。对照区洲滩不采取任何措施。结果 结果 开沟沥水工程实施前, 2组观察区在枯水期不同高程的洲滩土壤平均含 水量均在35.56%左右。工程实施后, 干预区洲滩土壤平均含水量26.53%, 而对照区则为35.56%, 两者差异有统计学意 义 （F = 6.53,P < 0.05）; 干预区不同高程相对地下水位明显低于对照区, 差异有统计学意义 （χ2 = 33.33,P < 0.05）。工 程实施前, 干预区和对照区钉螺自然死亡率分别0.98%和0.89%, 差异无统计学意义 （P > 0.05）; 工程实施后的2012年干 预区未再查见钉螺和幼螺,而对照区活螺平均密度和幼螺则仍分别为29.37只/0.1 m2 和 213±108.45只/0.1 m2 。结论 结论 在 湖区有螺洲滩开沟可迅速沥干洲滩积水, 显著降低洲滩土壤的含水量, 改变钉螺繁殖和孳生的生态条件, 从而控制钉螺 的孳生繁殖。     

Preparation,purification and characterization of the polyclonal antibody against α1-AT

Objective To prepare the polyclonal antibody against α1-antitrypsin(α1-AT)by immunizing rabbits with α1-AT,and to purify and characterize the antibodies produced from rabbits in order to establish the production process of the polyclonal antibody against α1-AT.Methods The healthy rabbits were routinely immunized with α1-AT to prepare polyclonal antibody against α1-AT.The serum antibody titers were determined by ELISA.The rabbits with qualified antibody titers were killed to collect blood.The antisera,obtained by centrifugalizing the whole blood,were purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography.The purified polyclonai antibodies were analyzed by 15%SDS-PAGE,and antibody titers weIe measured by ELISA.Results ELISA assay showed that the titers of antisera were over 105.The polyclonal antibodies,purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography,had good purity and immunological activity.Conclusion The production and purification processes of polyclonal antibody against α1-AT are successfully established.     

弹性蛋白样多肽融合胸腺肽α1的原核表达方法

目的 建立一种低成本生产胸腺肽α1(Tα1)的方法.方法 采用融合表达方式表达Tα1基因,通过人工合成Tα1和弹性蛋白多肽(ELP)基因,构建Tα1-G-ELPn/pET41a(+),同时构建RimJ-pACYCDuet1载体,共转B121,采用自诱导表达培养基进行蛋白表达,表达蛋白通过变温自动聚合沉淀获得高纯度融合蛋白,将融合蛋白羟胺裂解,变温沉淀,收集上清,最终获得目标蛋白.结果 1L培养基最终获得纯度为98%的目标蛋白29.5 mg.结论 建立了一种不需色谱和亲和纯化的生产胸腺肽α1的方法.     

丙型肝炎病毒核心区多肽的抗原表位分析和原核表达

目的 分析HCV核心区多肽的抗原表位,选择具有优势抗原表位的多肽用原核表达载体表达,并获得该表达载体的稳定表达菌株. 方法 根据软件分析选取带有优势抗原表位的HCV核心区多肽,找出对应DNA序列,逆转录PCR扩增目的基因,克隆到原核表达载体中进行诱导表达.表达的目的蛋白用His Bind Columns纯化,用ELISA法检测其免疫活性. 结果 获得了序列正确的HCV-core基因和表达良好的该基因原核表达载体pET-28a-core,该表达载体表达的目的蛋白可被很好地纯化并具有良好的HCV抗原活性. 结论 成功地重组了HCV核心区蛋白,获得了HCV抗原性良好的HCV核心区抗原.