發(fā)布時間：2019-02-21 14:34

【摘要】：目的構(gòu)建人偏肺病毒融合蛋白亞單位F1、F2原核表達(dá)系統(tǒng),初步獲得抗F1、F2重組蛋白的多克隆抗體,為相關(guān)疫苗的研究奠定基礎(chǔ)。方法聚合酶鏈反應(yīng)(PCR)法擴(kuò)增F1、F2基因片段,經(jīng)T-A克隆確保其準(zhǔn)確性,雙酶切后插入原核表達(dá)載體p ET32a(+)中,轉(zhuǎn)化大腸埃希菌BL21(DE3),按優(yōu)化后條件大量誘導(dǎo)表達(dá),純化后經(jīng)Western Blot檢測特異性。取純化蛋白免疫BALB/c小鼠制備多克隆抗體,ELISA法檢測效價,間接免疫熒光法(IFA)檢測多克隆抗體是否能與人偏肺病毒發(fā)生特異性反應(yīng)。結(jié)果 F1、F2基因均正確插入pET32a(+)中,并具有正確的讀碼框架。0.5 mmol/L IPTG 37℃誘導(dǎo)培養(yǎng)5 h可獲得大量帶His標(biāo)簽的重組蛋白,純化后濃度分別達(dá)到200μg/ml和300μg/ml。Western Blot結(jié)果顯示,重組蛋白可被抗His標(biāo)簽抗體特異識別;免疫小鼠獲得多克隆抗體效價分別為抗pET32a-F1最高1∶640,抗pET32a-F2最高1∶40 960。IFA顯示抗p ET32a-F1、抗pET32a-F2多克隆抗體均可與人偏肺病毒作用產(chǎn)生特異性熒光。結(jié)論成功構(gòu)建了F1、F2的原核表達(dá)質(zhì)粒,并在大腸埃希菌中獲得高效表達(dá),該蛋白具有良好的抗原性;免疫小鼠獲得特異性多克隆抗體,有利于人偏肺病毒的深入研究。
[Abstract]:Objective to construct the prokaryotic expression system of fusion protein subunit F1 / F 2 of human metapulmonary virus, and to obtain the polyclonal antibody against the recombinant protein F1 / F 2, so as to lay a foundation for the study of the related vaccine. Methods the F1F 2 gene fragment was amplified by polymerase chain reaction (PCR), and its accuracy was ensured by T-A cloning. After double enzyme digestion, it was inserted into prokaryotic expression vector p ET32a () and transformed into Escherichia coli BL21 (DE3). The expression was induced in large quantities according to the optimized conditions, and the specificity was detected by Western Blot after purification. The purified protein was used to immunize BALB/c mice to prepare polyclonal antibody. The titer of polyclonal antibody was detected by ELISA and indirect immunofluorescence (IFA) was used to detect whether the polyclonal antibody could react specifically with human metapulmonary virus. Results the F1F 2 gene was inserted into pET32a () correctly and had the correct frame of reading code. A large number of recombinant proteins with His tag could be obtained after 5 h culture at 37 鈩，

本文編號：2427589