【摘要】： 目的真核重組表達輪狀病毒(rotavirus,RV)SA11株VP7衣殼蛋白并制備、純化其卵黃抗體(IgY),并鑒定其活性。方法將SA11標(biāo)準(zhǔn)株在MA104細胞中增殖,收集病毒液。從病毒液中提取RNA,通過逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)擴增獲得SA11株衣殼蛋白VP7的長度為978 bp的編碼基因,將PCR產(chǎn)物連接到pMD18-T載體上,進行測序。將重組體亞克隆到分泌表達載體pPICZαB中,再將重組體轉(zhuǎn)化入大腸桿菌Top10中,用BstXⅠ線性化酶切重組質(zhì)粒后電轉(zhuǎn)入畢赤酵母X-33中,進行測序。轉(zhuǎn)染成功的菌落再用甲醇誘導(dǎo)表達,親和層析法純化重組蛋白抗原,十二烷基硫酸鈉聚丙烯酰胺凝膠電泳(SDS-PAGE)和蛋白免疫印跡(Western blotting)鑒定,用重組蛋白免疫羅曼母雞,收集雞蛋,Western blotting鑒定、純化IgY。體外中和實驗鑒定IgY作用。結(jié)果細胞增殖液中提取的RNA銀染可見11個條帶,成功構(gòu)建了含pPICZαB-SA11 VP7的畢赤酵母X-33,畢赤酵母X-33分泌的融合蛋白被收集、純化。畢赤酵母X-33表達的SA11 VP7的衣殼蛋白經(jīng)West blotting驗證與預(yù)測蛋白相對分子量40 200相符。從雞蛋中提取的雞的IgY驗證為抗VP7的抗體。純度可達到95%,1個雞蛋能產(chǎn)10.2 mg的IgY。在體外實驗中證明免疫后的IgY與蝕斑相對減少有關(guān)。結(jié)論抗重組RV SA11衣殼蛋白VP7和IgY的成功制備為疫苗和診斷試劑的開發(fā)奠定了基礎(chǔ)。
[Abstract]:Objective to express and prepare VP7 capsid protein of rotavirus (rotavirus,RV) SA11 strain, purify its yolk antibody (IgY), and identify its activity. Methods the standard strain of SA11 was proliferated in MA104 cells and the virus solution was collected. RNA, was extracted from the viral fluid and amplified by reverse transcription-polymerase chain reaction (RT-PCR) to obtain the 978 bp encoding gene of capsid protein VP7 of SA11 strain. The PCR product was ligated to pMD18-T vector and sequenced. The recombinant was subcloned into secretory expression vector pPICZ 偽 B, then transformed into Escherichia coli Top10. The recombinant plasmid was digested with BstX 鈪，

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