目的构建具有免疫学活性的抗小鼠细胞毒性T淋巴细胞相关抗原4(CTLA-4)单链抗体ScFv(single chain fragment of variety region),为进一步将其应用到动物体内奠定基础。方法采用RT-PCR技术,从分泌抗小鼠CTLA-4单克隆抗体(mAb)的杂交瘤细...目的构建具有免疫学活性的抗小鼠细胞毒性T淋巴细胞相关抗原4(CTLA-4)单链抗体ScFv(single chain fragment of variety region),为进一步将其应用到动物体内奠定基础。方法采用RT-PCR技术,从分泌抗小鼠CTLA-4单克隆抗体(mAb)的杂交瘤细胞中扩增mAb的VH、VL基因,并进一步将其组装成VH-Linker-VL型的ScFv片段。将ScFv片段亚克隆至pGMET载体,测序正确后将其克隆至真核分泌型表达载体pSect2-GFP,将pSect2-GFP-ScFv纯化质粒转染中国仓鼠卵巢细胞(CHO)并进行表达,同时经Zeocin筛选稳定表达株。用Western blot方法检测ScFv融合蛋白的表达,采用ELISA检测ScFv的亲和活性。结果经Zeocin筛选2周后,CHO细胞株可稳定表达GFP-ScFv蛋白。Western blot法证实GFP-ScFv蛋白在细胞上清和细胞裂解液中均有表达,大小约55kD。ELISA检测表明,CHO培养上清中的GFP-ScFv蛋白经浓缩初纯后能与重组小鼠CTLA-4纯化抗原结合,并具有抑制亲本单抗与其结合的能力。结论成功构建了抗小鼠CTLA-4的ScFv真核表达载体,并能有效表达出具有免疫学活性的ScFv融合蛋白。展开更多
目的构建具有免疫学活性的抗人甲胎蛋白单链抗体ScFv(single chain fragment of variety region,ScFv),为进一步的研究奠定实验基础。方法采用RT-PCR技术,从分泌抗人AFP单克隆抗体的杂交瘤细胞中扩增mAb的VH、VL基因,并进一步将其组装成...目的构建具有免疫学活性的抗人甲胎蛋白单链抗体ScFv(single chain fragment of variety region,ScFv),为进一步的研究奠定实验基础。方法采用RT-PCR技术,从分泌抗人AFP单克隆抗体的杂交瘤细胞中扩增mAb的VH、VL基因,并进一步将其组装成VH-Linker-VL型的ScFv片段。将ScFv片段亚克隆至原核分泌型表达载体pUC19,将pUC19-ScFv纯化质粒转染大肠杆菌BL21并用IPTG诱导表达ScFv蛋白。用SDS-PAGE方法检测ScFv的表达,采用ELISA检测ScFv的亲和活性。结果大肠杆菌BL21可被诱导表达ScFv蛋白。SDS-PAGE法证实ScFv蛋白在细胞包涵体有表达,大小约24 kD。ELISA检测表明,ScFv蛋白经能与重组人AFP纯化抗原结合,并具有抑制亲本单抗与其结合的能力。结论成功构建抗人AFP的ScFv表达载体,并能有效表达出具有免疫学活性的ScFv蛋白。展开更多
OBJECTIVE:To collect and screen for ethnopharmacological properties(antileishmanial,antibacterial and brine lethality assays) of medicinal plant Ocimum basilicum from Peshawar region(34.008 latitude and 71.57 altitude...OBJECTIVE:To collect and screen for ethnopharmacological properties(antileishmanial,antibacterial and brine lethality assays) of medicinal plant Ocimum basilicum from Peshawar region(34.008 latitude and 71.57 altitudes).METHODS:In the present study a general antileishmanial activity against Leishmania tropica strain was carried out.The antibacterial potential of the plant was performed against 06 gram positive and 06 gram negative bacteria.Brine shrimp cytotoxicity assay at different concentrations were investigated.RESULTS:The anti-promastigotes profile of the plant showed good antileishmanial activity exhibited LC_(50) value 21.67 μg/mL.The result for gram positive antibacterial activity revealed that the O.basilicum leaves extract possesses significant inhibitory activity at highest two concentrations ranging from20.66 ± 0.31 to 31.86 ± 0.80 for Clostridium perfringens type C and Bacillus subtitilis,respectively,as compared to the gentamycin(27.36 ± 0.55 and21.80 ± 0.72,respectively).For gram negative bacteria good activity was observed.A highest zone of inhibition was recorded for Pseudomonas aeroginosa(28.83 ± 0.28) atthe highest concentration(10 mg/mL).The LC_(50) value obtained for brine shrimp lethality assay was 91.56 μg/mL.CONCLUSION:The herb basil possesses effective cidal activities which make this plant a good candidate for the isolation of antiprotozoal and antibacterial compounds which may lead to the development of novel drug.展开更多
文摘目的构建具有免疫学活性的抗小鼠细胞毒性T淋巴细胞相关抗原4(CTLA-4)单链抗体ScFv(single chain fragment of variety region),为进一步将其应用到动物体内奠定基础。方法采用RT-PCR技术,从分泌抗小鼠CTLA-4单克隆抗体(mAb)的杂交瘤细胞中扩增mAb的VH、VL基因,并进一步将其组装成VH-Linker-VL型的ScFv片段。将ScFv片段亚克隆至pGMET载体,测序正确后将其克隆至真核分泌型表达载体pSect2-GFP,将pSect2-GFP-ScFv纯化质粒转染中国仓鼠卵巢细胞(CHO)并进行表达,同时经Zeocin筛选稳定表达株。用Western blot方法检测ScFv融合蛋白的表达,采用ELISA检测ScFv的亲和活性。结果经Zeocin筛选2周后,CHO细胞株可稳定表达GFP-ScFv蛋白。Western blot法证实GFP-ScFv蛋白在细胞上清和细胞裂解液中均有表达,大小约55kD。ELISA检测表明,CHO培养上清中的GFP-ScFv蛋白经浓缩初纯后能与重组小鼠CTLA-4纯化抗原结合,并具有抑制亲本单抗与其结合的能力。结论成功构建了抗小鼠CTLA-4的ScFv真核表达载体,并能有效表达出具有免疫学活性的ScFv融合蛋白。
文摘目的构建具有免疫学活性的抗人甲胎蛋白单链抗体ScFv(single chain fragment of variety region,ScFv),为进一步的研究奠定实验基础。方法采用RT-PCR技术,从分泌抗人AFP单克隆抗体的杂交瘤细胞中扩增mAb的VH、VL基因,并进一步将其组装成VH-Linker-VL型的ScFv片段。将ScFv片段亚克隆至原核分泌型表达载体pUC19,将pUC19-ScFv纯化质粒转染大肠杆菌BL21并用IPTG诱导表达ScFv蛋白。用SDS-PAGE方法检测ScFv的表达,采用ELISA检测ScFv的亲和活性。结果大肠杆菌BL21可被诱导表达ScFv蛋白。SDS-PAGE法证实ScFv蛋白在细胞包涵体有表达,大小约24 kD。ELISA检测表明,ScFv蛋白经能与重组人AFP纯化抗原结合,并具有抑制亲本单抗与其结合的能力。结论成功构建抗人AFP的ScFv表达载体,并能有效表达出具有免疫学活性的ScFv蛋白。
文摘OBJECTIVE:To collect and screen for ethnopharmacological properties(antileishmanial,antibacterial and brine lethality assays) of medicinal plant Ocimum basilicum from Peshawar region(34.008 latitude and 71.57 altitudes).METHODS:In the present study a general antileishmanial activity against Leishmania tropica strain was carried out.The antibacterial potential of the plant was performed against 06 gram positive and 06 gram negative bacteria.Brine shrimp cytotoxicity assay at different concentrations were investigated.RESULTS:The anti-promastigotes profile of the plant showed good antileishmanial activity exhibited LC_(50) value 21.67 μg/mL.The result for gram positive antibacterial activity revealed that the O.basilicum leaves extract possesses significant inhibitory activity at highest two concentrations ranging from20.66 ± 0.31 to 31.86 ± 0.80 for Clostridium perfringens type C and Bacillus subtitilis,respectively,as compared to the gentamycin(27.36 ± 0.55 and21.80 ± 0.72,respectively).For gram negative bacteria good activity was observed.A highest zone of inhibition was recorded for Pseudomonas aeroginosa(28.83 ± 0.28) atthe highest concentration(10 mg/mL).The LC_(50) value obtained for brine shrimp lethality assay was 91.56 μg/mL.CONCLUSION:The herb basil possesses effective cidal activities which make this plant a good candidate for the isolation of antiprotozoal and antibacterial compounds which may lead to the development of novel drug.
