本文關(guān)鍵詞： 蜜蜂螺原體 幾丁質(zhì)脫乙酰酶 定點突變 克隆表達(dá) 酶學(xué)性質(zhì)　出處：《南京農(nóng)業(yè)大學(xué)學(xué)報》2016年03期 　論文類型：期刊論文

【摘要】：[目的]通過表達(dá)載體p ET-28a(+)在大腸桿菌中克隆表達(dá)蜜蜂螺原體Spiroplasma melliferum CH-1中可能與致病相關(guān)的幾丁質(zhì)脫乙酰酶(chitin deacetylase,CDA)基因chid,并測定其部分酶學(xué)性質(zhì),為進(jìn)一步研究該基因在蜜蜂螺原體致病過程中的作用奠定基礎(chǔ)。[方法]利用Overlap Extension PCR(OE-PCR)定點突變技術(shù),在引物設(shè)計時插入目的突變,通過3次PCR獲得突變后的目的片段,測序驗證后構(gòu)建重組質(zhì)粒p ETchid,挑取BLchid表達(dá)菌株。IPTG誘導(dǎo)表達(dá)目的蛋白,NTA-Ni2+柱純化,Western blotting驗證,紫外分光光度法測定其酶活力和酶學(xué)性質(zhì)。[結(jié)果]DNA測序結(jié)果表明:chid基因中待突變位點已由TGA突變?yōu)門GG,成功克隆到chid基因全長,并得到外源表達(dá)的完整目的蛋白。該基因編碼區(qū)為672 bp,編碼的氨基酸構(gòu)成相對分子質(zhì)量約28×103的蛋白,同源性比較發(fā)現(xiàn)其與S.melliferum KC3/BC3/IPMB4A菌株序列相似性為97%。測得外源表達(dá)的該酶活力最高可達(dá)10.14 U·m L-1,最適溫度為50℃左右,最適p H值為7.0~7.5。[結(jié)論]首次克隆了螺原體幾丁質(zhì)脫乙酰酶基因chid,并得到具有活性的外源目的蛋白,為后續(xù)研究螺原體與宿主蜜蜂的相互作用及其致病機(jī)制提供了重要信息。
[Abstract]:[Objective] to study the expression vector pET-28a (pET-28a). Cloning and expression of chitinase from Spiroplasma melliferum CH-1 in Escherichia coli. Chitin deacetylase. CDA) gene chidand some enzymatic properties were determined, which laid a foundation for further study on the role of CDA gene in the pathogenesis of bee spiroplasma. [Methods] the aim mutation was inserted into the primer design using Overlap Extension PCR- OE-PCR site-directed mutation technique. The target fragment was obtained by three times of PCR, and the recombinant plasmid pETchid was constructed after sequencing. The target protein was induced by BLchid expression strain. The enzyme activity and enzymatic properties were determined by UV spectrophotometry. [Results DNA sequencing showed that the mutation site in the DNA gene had been transformed from TGA to TGG, and the chid gene was cloned successfully. The encoding region of the gene is 672 BP, and the amino acids encoded constitute a protein with a molecular weight of about 28 脳 10 ~ 3. Homology analysis showed that the sequence similarity between S.melliferum KC3/BC3/IPMB4A and S.melliferum KC3/BC3/IPMB4A was 97%. The highest activity of the enzyme expressed by exogenous was 10.14%. U 路mL ~ (-1). The optimum temperature is about 50 鈩，

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