β-伴大豆球蛋白α′-亚基的基因克隆及原核表达

Cloning and Prokaryotic Expression of Soybean β-conglycininα′-Subunit Gene

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。

In order to build the E.coli expression system for the α′ subunit of soybean β-conglycinin,the total RNA were extracted from the seeds of soybean variety ‘Lu 96150’. The α′ coding sequence was amplified by one step assay of RT-PCR,and then inserted into the vector of pGEM-T easy. After digested by XhoI/EcoRI,the α′ fragment was inserted into the prokaryotic expression vector pET-28a containing His-tag. The constructed vector pET-28a-α′ was verified by the colony PCR,restriction endonuclease digestion and DNA sequencing. Then the pET-28a-α′ vector was transformed into E.coli host strain BL21 （DE3） for IPTG induction expression. A recombinant protein about 70 ku was well expressed by inducing with the OD600 value 0.8,IPTG 0.2 mmol/L at 30 ℃ for 9 h. After ultrasonication and centrifuge the target protein α′ subunit was found in the supernatant not in the form of inclusion body,more convenient to the further purification work. This study could provide a theoretical basis for the structure and function relationship study of soybean β-conglycinin.