【摘要】： 目的:采用基因工程技術(shù)大量表達(dá)重組幽門螺桿菌(Helicobac ter pylori,H.pylori)鞭毛粘附素(HpaA),用此重組蛋白作為抗原,制備高效價(jià)的抗HpaA的雞蛋黃抗體IgY,為研制H.pylori預(yù)防性抗體制劑奠定基礎(chǔ)。 方法: 1.誘導(dǎo)重組質(zhì)粒pQE30-hpaA在大腸桿菌DH5a中表達(dá)rHpaA,SDS-PAGE分析其表達(dá)形式。 2.重組蛋白經(jīng)Ni~(2+)-NTA樹脂純化后,采用Bradford法測(cè)定蛋白濃度。 3.用rHpaA免疫30周齡立克體雞,以水稀釋法聯(lián)合鹽析法提取HpaA-IgY,純化濃縮后用Bradford法測(cè)定其含量,SDS-PAGE電泳檢測(cè)純度,Western blot鑒定抗原特異性。 4. ELISA測(cè)定效價(jià)和巴氏消毒后的活性。 結(jié)果: 1. SDS-PAGE電泳顯示pQE30-hpaA-DH5a表達(dá)產(chǎn)物相對(duì)分子量為30 000。 2. SDS-PAGE分析表達(dá)形式,rHpaA主要存在于細(xì)菌裂解液上清中,約占70%,部分存在于包涵體中,經(jīng)Ni~(2+)-NTA樹脂對(duì)上清進(jìn)行純化后,純度約為90%,Bradford法測(cè)得蛋白質(zhì)含量為0.95mg/ml。 3. HpaA-IgY提純后,Bradford法測(cè)得含量為24.6mg/ml,純度約為90%,Western blot顯示在相對(duì)分子量30 000處出現(xiàn)單一條帶,ELISA效價(jià)為1:12800。
[Abstract]:Aim: to prepare the high titer HpaA yolk antibody IgY, for the preparation of H.pylori prophylaxis antibody preparation using the flagellin (HpaA), of recombinant Helicobacter pylori (Helicobac ter pylori,H.pylori) expressed in large quantities by genetic engineering technique and the recombinant protein was used as antigen to prepare the high titer chicken egg yolk antibody (IgY,). Methods: 1. The expression of recombinant plasmid pQE30-hpaA in Escherichia coli DH5a was analyzed by rHpaA,SDS-PAGE analysis. 2. 2. The recombinant protein was purified by Ni~ (2) -NTA resin, and the protein concentration was determined by Bradford method. RHpaA was used to immunize 30 week-old ricket-type chickens. HpaA-IgY, was purified and concentrated by water dilution method and salt-out method. The content of HpaA-IgY, was determined by Bradford method. SDS-PAGE electrophoresis was used to detect the purity and the specificity of the antigen was identified by Western blot. 4. The titer and pasteurized activity were determined by ELISA. Results: 1. SDS-PAGE electrophoresis showed that the relative molecular weight of the pQE30-hpaA-DH5a expression product was 30000.2. The expression form of SDS-PAGE was mainly found in the supernatant of bacterial lysate, accounting for about 70%, and partly in the inclusion body. The supernatant was purified by Ni~ (2) -NTA resin. The protein content measured by Bradford method was 0.95 mg / ml 路ml. 3. After purification by HpaA-IgY, the content was 24.6 mg / ml, and the purity was about 90%. Western blot showed that a single band Elisa titer was 1: 12800 at the relative molecular weight of 30 000.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392

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本文編號(hào)：2236470