T7噬菌体衣壳蛋白P11的表达纯化及单克隆抗体的制备
发布时间：2018-10-07 17:52
【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。
[Abstract]:T7 phage is a potent bacteriophage infected with Escherichia coli. Its genome is a linear double-stranded DNA, with a length of 39 936 BP. Capsid proteins include head protein P10A, minor head protein P10B, collar protein P8, tail protein P11P12 and tail filament protein P17.T7 phage as a common display system. The phage protein chip of .T7 has been widely used in recent years because of its advantages of short growth cycle. It is a new protein detection technique with high quality, high sensitivity, high specificity and miniaturization. The foreign proteins can be expressed and displayed on the surface of T7 phage, and then the T7 phages which display the foreign proteins are solidified on the microarray to detect the displayed proteins. According to the sequence of P11 gene published by GenBank, we designed a pair of specific nucleotide primers, obtained the 598bp size gene fragment by PCR method, cloned it into the expression vector, sequenced and identified, successfully constructed the pET-28a () -p11 expression vector. The recombinant protein P11 was induced to express in E. coli strain BL21 (DE3), and the target protein was purified by Ni-NTA affinity chromatography. The purification rate reached 90%. The purified product was identified by SDS-PAGE and immunized with Balb/c mice. The spleen cells of Balb/c mice were fused with SP2/0 myeloma cells mediated by polyethylene glycol PEG, and the positive hybridoma cell lines were screened by purified P11 recombinant protein. A hybridoma cell line which could secrete monoclonal antibody stably was obtained after three times of cloning screening by limited dilution method, and it was named 2G11. The results of monoclonal antibody subclass identification showed that 2G11 was a IgG2b subclass. Elisa assay and Western blot analysis showed that the McAb could specifically bind to T7 phage protein and P11 protein. This will lay a foundation for further studying the structure and function of P11 protein, improving the screening efficiency of T7 phage protein chip and establishing phage detection method.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
本文编号：2255118
[Abstract]:T7 phage is a potent bacteriophage infected with Escherichia coli. Its genome is a linear double-stranded DNA, with a length of 39 936 BP. Capsid proteins include head protein P10A, minor head protein P10B, collar protein P8, tail protein P11P12 and tail filament protein P17.T7 phage as a common display system. The phage protein chip of .T7 has been widely used in recent years because of its advantages of short growth cycle. It is a new protein detection technique with high quality, high sensitivity, high specificity and miniaturization. The foreign proteins can be expressed and displayed on the surface of T7 phage, and then the T7 phages which display the foreign proteins are solidified on the microarray to detect the displayed proteins. According to the sequence of P11 gene published by GenBank, we designed a pair of specific nucleotide primers, obtained the 598bp size gene fragment by PCR method, cloned it into the expression vector, sequenced and identified, successfully constructed the pET-28a () -p11 expression vector. The recombinant protein P11 was induced to express in E. coli strain BL21 (DE3), and the target protein was purified by Ni-NTA affinity chromatography. The purification rate reached 90%. The purified product was identified by SDS-PAGE and immunized with Balb/c mice. The spleen cells of Balb/c mice were fused with SP2/0 myeloma cells mediated by polyethylene glycol PEG, and the positive hybridoma cell lines were screened by purified P11 recombinant protein. A hybridoma cell line which could secrete monoclonal antibody stably was obtained after three times of cloning screening by limited dilution method, and it was named 2G11. The results of monoclonal antibody subclass identification showed that 2G11 was a IgG2b subclass. Elisa assay and Western blot analysis showed that the McAb could specifically bind to T7 phage protein and P11 protein. This will lay a foundation for further studying the structure and function of P11 protein, improving the screening efficiency of T7 phage protein chip and establishing phage detection method.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
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