【摘要】： T7噬菌體是一種感染大腸桿菌的烈性噬菌體,基因組為線狀雙鏈DNA,全長(zhǎng)39 936bp。其衣殼蛋白包括頭蛋白P10A、次要頭蛋白P10B、頸圈蛋白P8、尾蛋白P11、P12和尾絲蛋白P17。T7噬菌體作為一種常見的展示系統(tǒng),因其具有載體容量大,插入片段穩(wěn)定,洗滌條件靈活,生長(zhǎng)周期短等優(yōu)點(diǎn)而被廣泛使用。T7噬菌體蛋白芯片是近年來發(fā)展起來的一種新的蛋白質(zhì)檢測(cè)手段,具有高質(zhì)量、高靈敏度、高特異性且微型化特點(diǎn)的一種蛋白質(zhì)分析技術(shù),能使外源蛋白表達(dá)并展示于T7噬菌體表面,再將這些展示外源蛋白的T7噬菌體固化在芯片上,對(duì)其展示的蛋白進(jìn)行檢測(cè)。 本試驗(yàn)根據(jù)GenBank發(fā)表的P11基因序列,設(shè)計(jì)了一對(duì)特異性核苷酸引物,用PCR方法獲得598bp大小的基因片段,并將其克隆到表達(dá)載體上,測(cè)序鑒定,成功構(gòu)建pET-28a(+)/P11表達(dá)載體,轉(zhuǎn)化到大腸桿菌表達(dá)菌株BL21(DE3)中,誘導(dǎo)表達(dá)重組蛋白P11,并進(jìn)一步通過Ni-NTA親合層析柱純化目的蛋白,純化率達(dá)90%。純化產(chǎn)物經(jīng)SDS-PAGE鑒定后免疫Balb/c小鼠,用聚乙二醇PEG介導(dǎo)Balb/c小鼠脾細(xì)胞與SP2/0骨髓瘤細(xì)胞融合,用純化的P11重組蛋白篩選陽(yáng)性雜交瘤細(xì)胞株,對(duì)陽(yáng)性克隆細(xì)胞株經(jīng)3次有限稀釋法克隆化篩選后,最終獲得1株能穩(wěn)定分泌單克隆抗體的雜交瘤細(xì)胞株,命名為2G11。對(duì)單克隆抗體亞類鑒定結(jié)果表明2G11為IgG2b亞類。ELISA試驗(yàn)結(jié)果和Western blot分析表明,單抗能與T7噬菌體蛋白和P11蛋白特異結(jié)合。這為進(jìn)一步研究P11蛋白的結(jié)構(gòu)和功能,提高T7噬菌體蛋白芯片的篩選效率以及建立噬菌體檢測(cè)方法奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

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