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人源性抗乳腺癌HER2噬菌体单链抗体库的构建与筛选 被引量:1
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作者 卢晓辉 王志文 +4 位作者 蔡颖 黄静 朱丽华 杨清玲 陈昌杰 《浙江大学学报(医学版)》 CAS CSCD 北大核心 2014年第4期434-440,共7页
目的:构建人源性抗乳腺癌噬菌体单链抗体(scFv)库,筛选和鉴定抗乳腺癌人类表皮生长因子受体2(HER2)特异性scFv,为HER2和CXC趋化因子受体4(CXCR4)双靶向融合蛋白的研制提供靶向HER2的高亲和力序列。方法:取已确诊的乳腺癌患者... 目的:构建人源性抗乳腺癌噬菌体单链抗体(scFv)库,筛选和鉴定抗乳腺癌人类表皮生长因子受体2(HER2)特异性scFv,为HER2和CXC趋化因子受体4(CXCR4)双靶向融合蛋白的研制提供靶向HER2的高亲和力序列。方法:取已确诊的乳腺癌患者癌旁淋巴组织,提取总RNA;构建可变区基因的T载体库,将重链可变区(VH)和轻链可变区(VL)基因与pCANTAB连接肽连接,获得pCANTAB-scFv,电转化感受态大肠杆菌TG1;以M13K07辅助噬菌体进行感染,获噬菌体抗体库;利用酶联免疫吸附试验(ELISA)检测筛选后获得的噬菌体抗体活性,以免疫组织化学法检测抗体亲和力,并通过测序进一步鉴定。结果:成功构建大容量pCANTAB-scFv抗体库,获得的scFv长度为750bp,VH和VL长度分别为375和330kb;电转化大肠杆菌TG1后酶切验证插入片段大小正确,得到库容为2.48×10^8的大容量抗体库;通过噬菌体展示和淘洗筛选,富集针对乳腺癌细胞株SKBR-3表面抗原的抗体库;所得抗体对HER2有高度的亲和力和特异性,对乳腺癌组织HER2抗原也具有较高的亲和力;测序结果与人IgG抗体进行对比,所获得的scFv具有高度的人源性。结论:主要构建了大容量人源性抗乳腺癌细胞的噬菌体scFv库,并筛选获得可特异性识别人乳腺癌HER2的高亲和力scFv,为制备以HER2为靶点的抗肿瘤HER2和CXCR4双靶向融合蛋白提供了载体。 展开更多
关键词 乳腺肿瘤/免疫学 DNA 互补/遗传 基因文库 抗体 细菌/遗传 细菌噬菌体/遗传 受体 erbB-2 受体 CXCR4 聚合酶链反应
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Phenotypic and genetic characterization of a novel strain of Acidithiobacillus ferrooxidans(AF2) 被引量:1
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作者 刘倩 周洪波 +2 位作者 杨波 敖敬群 陈新华 《Journal of Central South University》 SCIE EI CAS 2011年第2期386-391,共6页
A comparative study on the phenotypic and genetic characteristics among Acidithiobacillus ferrooxidans (AF2), a typic strain ATCC23270 and a previously isolated strain AF3 was performed. AF2 can use ferrous ion (F... A comparative study on the phenotypic and genetic characteristics among Acidithiobacillus ferrooxidans (AF2), a typic strain ATCC23270 and a previously isolated strain AF3 was performed. AF2 can use ferrous ion (Fe^2+) or elemental sulfur (S^0) as sole energy source, but oxidizes So more effectively than Fe^2+, which is different from ATCC23270 and AF3. The G+C content of AF2 is 51.8% (molar fraction), however, ATCC23270 and AF3 strains have G+C content of 63.7% and 64.8% (molar fraction), respectively. The DNA-DNA hybridization results show that AF2 has 41.53% and 52.38% genome similarity to ATCC 23270 and AF3, respectively, but AF3 has a high genome similarity of 89.86% to ATCC 23270 strain. Rusticyanin (rus) and subunit III of aa3-type cytochrome oxidase (coxC) genes are not detected in AF2, but Fe^2+ oxidase (iro) gene can be detected. To understand the genomic organization of iro gene, a cosmid library of AF2 genome was constructed and iro gene-containing clone was screened. The sequencing result shows that although the nucleotide sequence of iro gene in AF2 is completely identical to that of ATCC 23270 strain, its genomic organization is different from that of ATCC 23270. In AF2, iro is located at downstream ofpurA gene, while it is located at downstream ofpetC-2 gene in ATCC 23270 strain. These results indicate that AF2 is a novel strain ofA. ferrooxidans, and that phenotypic differences among the strains ofA. ferrooxidans are closely correlated with their genetic polymorphisms. 展开更多
关键词 Aeidithiobacillusferrooxidans phenotypic and genetic characterization Iro gene cosmid library iron respiratory chain
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