β-伴大豆球蛋白α′-亚基的基因克隆及原核表达

发布时间：2018-05-16 14:29

  本文选题：β-伴大豆球蛋白 + 亚基　； 参考：《西北农业学报》2017年02期

【摘要】：为制备N-连接糖基缺失的重组β-伴大豆球蛋白α′-亚基,以‘鲁96150’大豆种子为原料提取总RNA,经RT-PCR一步法获得‘鲁96150’大豆的全长cDNA,采用自行设计的引物F1/F2扩增得到目的基因α′,与pGEM-T easy载体相连构建重组克隆载体pGEM-α′,经XhoⅠ/EcoRⅠ双酶切得到目的基因与载体pET-28a连接构建重组原核表达载体pET-28a-α′,将经菌落PCR、双酶切及测序鉴定正确的表达载体转入感受态细胞E.coli BL21(DE3),经异丙基硫代半乳糖苷(IPTG)诱导表达重组蛋白α′-亚基。对重组α′-亚基的诱导表达条件进行筛选,发现在菌液OD600值为0.8、诱导温度30℃、IPTG浓度为0.2mmol/L的诱导条件下诱导9h后α′-亚基的表达量较高,重组α′-亚基的分子质量大小约为70ku;工程菌pET-28a-α′-BL21经超声破碎、离心后发现重组α′-亚基部分存在于上清液中,部分形成包涵体蛋白。重组α′-亚基的克隆及表达为β-伴大豆球蛋白结构及功能特性的研究奠定基础。
[Abstract]:In order to prepare recombinant 尾 -concomitant glycosylin 伪 -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 伪 -pGEM- 伪 was amplified by self-designed primer F1/F2, and the recombinant clone vector pGEM- 伪 was constructed by ligation with pGEM-T easy vector. The recombinant prokaryotic expression vector pET-28a- 伪 was constructed by digesting the target gene with the vector pET-28a by double enzyme digestion of Xho 鈪，

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