In order to detect the protein delivery mediated by the PTD (protein transduction domain) of TAT Protein, a expression vector, named pT7460-GFP, was constructed by insert the PTD DNA Sequence, followed by a GFP (green...In order to detect the protein delivery mediated by the PTD (protein transduction domain) of TAT Protein, a expression vector, named pT7460-GFP, was constructed by insert the PTD DNA Sequence, followed by a GFP (green fluorescent protein) gene fused in-frame, into the pT7450 vector. The TAT-GFP fusion protein was expressed in the E.coli ER2566. Most of the fusion protein was presented in the inclusion body. The protein was purified by Ni 2+ affinity chromatography under denature conditions, then by a Sepharose Q column to remove urea. The soluble denatured protein was added directly to medium containing the Myeloma Cell SP2/0. It came out that the fusion protein could be detected delivered into the cells under fluorescent microscope in a short time.展开更多
目的制备甲型副伤寒沙门菌菌体抗原单克隆抗体,并对其进行鉴定及初步应用。方法用甲醛灭活的甲型副伤寒沙门菌免疫BALB/c小鼠,取脾细胞与Sp2/0骨髓瘤细胞融合,筛选出稳定分泌甲型副伤寒沙门菌特异性单克隆抗体的杂交瘤细胞株,对克隆纯...目的制备甲型副伤寒沙门菌菌体抗原单克隆抗体,并对其进行鉴定及初步应用。方法用甲醛灭活的甲型副伤寒沙门菌免疫BALB/c小鼠,取脾细胞与Sp2/0骨髓瘤细胞融合,筛选出稳定分泌甲型副伤寒沙门菌特异性单克隆抗体的杂交瘤细胞株,对克隆纯化后的阳性细胞扩大培养后,免疫BALB/c小鼠,收获腹水,间接ELISA法检测抗体效价,并进行Ig类和亚类鉴定;腹水经辛酸-硫酸铵盐析法纯化后,采用间接ELISA法、玻片凝集试验、SDSPAGE法、免疫扩散试验进行检测。将纯化后的2株单克隆抗体用于双抗体夹心ELISA法的建立,并用自制配对抗体检测甲型副伤寒患者血浆、乙型副伤寒患者血浆、伤寒患者血浆和正常人血浆。结果共筛选出2株持续分泌单克隆抗体的杂交瘤细胞株,命名为1H4和2A9,腹水抗体效价为1∶10~7和1∶10~6,分别为IgG_1和Ig G_3亚类,轻链均为κ型。纯化后的单克隆抗体1H4、2A9效价分别为1∶10~7和1∶10~3,1H4纯度大于85%,2A9纯度小于60%。2株单克隆抗体与甲型副伤寒沙门菌50503、50973可产生凝集反应,与伤寒沙门菌50096不产生反应;1H4在稀释度为1∶8时可与甲型副伤寒沙门菌菌体特异多糖(organism specific polysaccharide,OSP)产生凝集反应,而2A9在每个稀释度与OSP均不发生反应。将1H4和2A9用于双抗体夹心ELISA检测法建立,确定1H4/HRP-2A9的组合为最佳配对,用该配对抗体检测阳性样本检出率为100%,正常人血浆样本检出率为0。结论获得2株甲型副伤寒沙门菌菌体抗原单克隆抗体,可用于甲型副伤寒沙门菌的快速鉴定。展开更多
文摘In order to detect the protein delivery mediated by the PTD (protein transduction domain) of TAT Protein, a expression vector, named pT7460-GFP, was constructed by insert the PTD DNA Sequence, followed by a GFP (green fluorescent protein) gene fused in-frame, into the pT7450 vector. The TAT-GFP fusion protein was expressed in the E.coli ER2566. Most of the fusion protein was presented in the inclusion body. The protein was purified by Ni 2+ affinity chromatography under denature conditions, then by a Sepharose Q column to remove urea. The soluble denatured protein was added directly to medium containing the Myeloma Cell SP2/0. It came out that the fusion protein could be detected delivered into the cells under fluorescent microscope in a short time.
文摘目的制备甲型副伤寒沙门菌菌体抗原单克隆抗体,并对其进行鉴定及初步应用。方法用甲醛灭活的甲型副伤寒沙门菌免疫BALB/c小鼠,取脾细胞与Sp2/0骨髓瘤细胞融合,筛选出稳定分泌甲型副伤寒沙门菌特异性单克隆抗体的杂交瘤细胞株,对克隆纯化后的阳性细胞扩大培养后,免疫BALB/c小鼠,收获腹水,间接ELISA法检测抗体效价,并进行Ig类和亚类鉴定;腹水经辛酸-硫酸铵盐析法纯化后,采用间接ELISA法、玻片凝集试验、SDSPAGE法、免疫扩散试验进行检测。将纯化后的2株单克隆抗体用于双抗体夹心ELISA法的建立,并用自制配对抗体检测甲型副伤寒患者血浆、乙型副伤寒患者血浆、伤寒患者血浆和正常人血浆。结果共筛选出2株持续分泌单克隆抗体的杂交瘤细胞株,命名为1H4和2A9,腹水抗体效价为1∶10~7和1∶10~6,分别为IgG_1和Ig G_3亚类,轻链均为κ型。纯化后的单克隆抗体1H4、2A9效价分别为1∶10~7和1∶10~3,1H4纯度大于85%,2A9纯度小于60%。2株单克隆抗体与甲型副伤寒沙门菌50503、50973可产生凝集反应,与伤寒沙门菌50096不产生反应;1H4在稀释度为1∶8时可与甲型副伤寒沙门菌菌体特异多糖(organism specific polysaccharide,OSP)产生凝集反应,而2A9在每个稀释度与OSP均不发生反应。将1H4和2A9用于双抗体夹心ELISA检测法建立,确定1H4/HRP-2A9的组合为最佳配对,用该配对抗体检测阳性样本检出率为100%,正常人血浆样本检出率为0。结论获得2株甲型副伤寒沙门菌菌体抗原单克隆抗体,可用于甲型副伤寒沙门菌的快速鉴定。
