马铃薯液泡蔗糖转化酶原核表达及多克隆抗血清的制备

Prokaryotic Expression and Polyclonal Antisera Preparation of Vacuolar Invertase in Potato

马铃薯液泡蔗糖转化酶在马铃薯块茎低温糖化过程中起着关键作用。将马铃薯液泡蔗糖转化酶基因StVIN1的N端部分片段连接到原核表达载体pET-28a,将重组质粒pET-28a-StVIN1-N通过热激的方法转化到宿主大肠杆菌BL21(DE3)中,经0.84 mmol/L IPTG诱导得到带有6×His标签的融合蛋白。以纯化后的蛋白为抗原免疫家兔,获得多克隆抗血清。利用得到的抗血清对纯化后的蛋白进行Western blot鉴定,结果表明所制备的多克隆抗血清具有良好的特异性。

Potato vacuolar invertase plays a key role in potato cold-induced sweetening.In this study,N-terminal fragment of potato vacuolar invertase StVIN1 gene was inserted into the prokaryotic expression vector pET-28a,and the recombinant plasmid pET-28a-StVIN1-N was transformed into the host E.coli BL21(DE3)by heat shock.The fusion protein with 6×His tag was induced by 0.84 mmol/L IPTG.Rabbits were immunized with the purified protein as an antigen to obtain polyclonal antisera.The obtained polyclonal antisera was used to identify the purified protein by Western blot.The results showed that the prepared polyclonal antisera has good specificity.