【作者】 陈亮；

【导师】 高云英；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2008， 硕士

【摘要】 T7噬菌体是一种感染大肠杆菌的烈性噬菌体,基因组为线状双链DNA,全长39 936bp。其衣壳蛋白包括头蛋白P10A、次要头蛋白P10B、颈圈蛋白P8、尾蛋白P11、P12和尾丝蛋白P17。T7噬菌体作为一种常见的展示系统,因其具有载体容量大,插入片段稳定,洗涤条件灵活,生长周期短等优点而被广泛使用。T7噬菌体蛋白芯片是近年来发展起来的一种新的蛋白质检测手段,具有高质量、高灵敏度、高特异性且微型化特点的一种蛋白质分析技术,能使外源蛋白表达并展示于T7噬菌体表面,再将这些展示外源蛋白的T7噬菌体固化在芯片上,对其展示的蛋白进行检测。本试验根据GenBank发表的P11基因序列,设计了一对特异性核苷酸引物,用PCR方法获得598bp大小的基因片段,并将其克隆到表达载体上,测序鉴定,成功构建pET-28a(+)/P11表达载体,转化到大肠杆菌表达菌株BL21(DE3)中,诱导表达重组蛋白P11,并进一步通过Ni-NTA亲合层析柱纯化目的蛋白,纯化率达90%。纯化产物经SDS-PAGE鉴定后免疫Balb/c小鼠,用聚乙二醇PEG介导Balb/c小鼠脾细胞与SP2/0骨髓瘤细胞融合,用纯化的P11重组蛋白筛选阳性杂交瘤细胞株,对阳性克隆细胞株经3次有限稀释法克隆化筛选后,最终获得1株能稳定分泌单克隆抗体的杂交瘤细胞株,命名为2G11。对单克隆抗体亚类鉴定结果表明2G11为IgG2b亚类。ELISA试验结果和Western blot分析表明,单抗能与T7噬菌体蛋白和P11蛋白特异结合。这为进一步研究P11蛋白的结构和功能,提高T7噬菌体蛋白芯片的筛选效率以及建立噬菌体检测方法奠定了基础。更多还原

【Abstract】 Bacteriophage T7 is a lysogeny phage capable of infecting most strains of Escherichia coli, a double standed DNA of genome molecular is 39 936 bp. The capside protein of T7 include major head protein P10A, head assemble protein P10B, neck protein P8, Tail protein P11, P12 and Tail fibers protein P17. T7 phage display system is easy to use and has the capacity to display peptides up to about 1200 amino acids, including stability of insert gene, varity of elution conditions and short of growth cycle. T7 phage protein chip is fixed the bacteriophage T7 which expressed and displayed the exogenous proteins on the chip, the chip could be used for the detection the exogenous proteins. P11 gene was inserted into plasmid pET-28a(+) to construct recombinant plasmid pET-28a(+)/P11. Recombinant P11 protein was expressed under the introduction of 0.1mol/L IPTG and well purified by nickel ion affinity chromatogagh method. SDS-PAGE analysis showed that the molecular weight of the expressed protein was approximately 27 kDa. The Balb/c mice were immunized with the purified protein P11 mixed adjuvant. The monoclonal antibodys (2P11) were identified with indirect immunized fluresent assay and Western blot assay. Indirect ELISA was used to detect the Ig isotype.We acquired the recombinant P11 protein and prepared a strain of hybridoma cell secreting the MAbs against P11 protein, the MAbs are useful material in vitro diagnosis.更多还原