本文關(guān)鍵詞： 包蟲病 細(xì)粒棘球絳蟲 EgM 蛋白表達(dá) 疫苗　出處：《中國病原生物學(xué)雜志》2017年01期 　論文類型：期刊論文

【摘要】：目的構(gòu)建細(xì)粒棘球絳蟲成蟲相關(guān)基因EgM123基因原核表達(dá)載體,誘導(dǎo)表達(dá)并鑒定EgM123蛋白。方法提取pET41b-EgM123重組質(zhì)粒,以此為模板PCR擴(kuò)增EgM123基因片段,克隆至原核表達(dá)載體pET28a中,經(jīng)限制性內(nèi)切酶雙酶切和測序鑒定。將重組菌質(zhì)粒轉(zhuǎn)化大腸埃希菌BL21,經(jīng)異丙基-β-D-硫代半乳糖苷(IPTG)誘導(dǎo)目的基因表達(dá),采用SDS-PAGE對表達(dá)蛋白進(jìn)行分析,Western blot鑒定其免疫反應(yīng)性。結(jié)果 PCR擴(kuò)增EgM123基因片段長度為594bp,經(jīng)酶切分析和測序鑒定證明pET28a-EgM123原核表達(dá)載體構(gòu)建正確。重組質(zhì)粒轉(zhuǎn)化BL21在37℃條件下,經(jīng)IPTG(終濃度為0.4mmol/L)誘導(dǎo)3h,獲得分子質(zhì)量單位約為29ku的EgM123蛋白,與理論值相符。該蛋白能被EgM123-GST免疫兔抗血清識別。結(jié)論成功構(gòu)建了pET28a-EgM123原核表達(dá)載體,表達(dá)蛋白具有免疫反應(yīng)性,為研制包蟲病疫苗奠定了基礎(chǔ)。
[Abstract]:Objective to construct the prokaryotic expression vector of the EgM123 gene associated with Echinococcus granulosus and to express and identify the EgM123 protein. Methods the recombinant plasmid of pET41b-EgM123 was extracted. The EgM123 gene fragment was amplified by PCR and cloned into prokaryotic expression vector pET28a. The recombinant plasmid was transformed into Escherichia coli BL21 and induced by isopropyl- 尾 -D- thiogalactoside (IPTG). The immunoreactivity of the expressed protein was identified by SDS-PAGE and Western blot. Results the length of EgM123 gene fragment amplified by PCR was 594bp. The construction of pET28a-EgM123 prokaryotic expression vector was confirmed by restriction endonuclease analysis and sequencing. The recombinant plasmid was transformed into BL21 at 37 鈩，

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