本文選題：β-伴大豆球蛋白 + 亞基��； 參考：《西北農(nóng)業(yè)學(xué)報(bào)》2017年02期

【摘要】：為制備N-連接糖基缺失的重組β-伴大豆球蛋白α′-亞基,以‘魯96150’大豆種子為原料提取總RNA,經(jīng)RT-PCR一步法獲得‘魯96150’大豆的全長(zhǎng)cDNA,采用自行設(shè)計(jì)的引物F1/F2擴(kuò)增得到目的基因α′,與pGEM-T easy載體相連構(gòu)建重組克隆載體pGEM-α′,經(jīng)XhoⅠ/EcoRⅠ雙酶切得到目的基因與載體pET-28a連接構(gòu)建重組原核表達(dá)載體pET-28a-α′,將經(jīng)菌落PCR、雙酶切及測(cè)序鑒定正確的表達(dá)載體轉(zhuǎn)入感受態(tài)細(xì)胞E.coli BL21(DE3),經(jīng)異丙基硫代半乳糖苷(IPTG)誘導(dǎo)表達(dá)重組蛋白α′-亞基。對(duì)重組α′-亞基的誘導(dǎo)表達(dá)條件進(jìn)行篩選,發(fā)現(xiàn)在菌液OD600值為0.8、誘導(dǎo)溫度30℃、IPTG濃度為0.2mmol/L的誘導(dǎo)條件下誘導(dǎo)9h后α′-亞基的表達(dá)量較高,重組α′-亞基的分子質(zhì)量大小約為70ku;工程菌pET-28a-α′-BL21經(jīng)超聲破碎、離心后發(fā)現(xiàn)重組α′-亞基部分存在于上清液中,部分形成包涵體蛋白。重組α′-亞基的克隆及表達(dá)為β-伴大豆球蛋白結(jié)構(gòu)及功能特性的研究奠定基礎(chǔ)。
[Abstract]:In order to prepare recombinant 尾 -concomitant glycosylin 偽 -N-linked glycosylated subunit, Total RNAs were extracted from soybean seeds of'Lu 96150'. The full-length cDNA of 'Lu96150' soybean was obtained by one-step RT-PCR method. The target gene 偽 -pGEM- 偽 was amplified by self-designed primer F1/F2, and the recombinant clone vector pGEM- 偽 was constructed by ligation with pGEM-T easy vector. The recombinant prokaryotic expression vector pET-28a- 偽 was constructed by digesting the target gene with the vector pET-28a by double enzyme digestion of Xho 鈪，

本文編號(hào)：1897229