黄瓜S期激酶相关蛋白Skp1的原核表达及其多克隆抗体的制备

Prokaryotic Expression of Cucumber Skp1 in Escherichia coli and Preparation of Antiserum

S期激酶相关蛋白1(Skp1)是SCF型E3泛素连接酶途径的核心蛋白,在真核生物的细胞周期、转录调控、信号传导等细胞进程中发挥关键作用。为了深入研究Skp1的基因功能,通过反转录PCR扩增黄瓜Skp1基因的全长阅读框,Skp1基因全长阅读框由468个核苷酸组成,共编码155个氨基酸,将其通过酶切连接的方式克隆到大肠杆菌表达载体pET-28a中,获得重组载体pETSkp1,PCR验证及克隆测序确定开放阅读框的正确性。将重组质粒pETSkp1转化大肠杆菌BL21菌株,通过IPTG诱导表达,SDS-PAGE分析表明,在37℃条件下,经IPTG(1 mmol/L)分别诱导3,6,9 h,Skp1基因在大肠杆菌BL21中均得到高效表达,6,9 h对表达量的影响不明显,因此,可用3 h作为后期诱导表达蛋白的时间。将纯化的蛋白作为抗原,免疫新西兰大耳白兔,采集第5次后血清用ELISA测定效价范围为1∶128000~512 000。提取黄瓜叶片的总蛋白,利用获得的抗血清进一步通过Western Blot检测特异性,结果表明,抗血清在500倍和1 000倍稀释时均能特异性地检测到黄瓜叶片中的Skp1蛋白,条带大小在20 k Da左右,与预期的Skp1蛋白大小一致。

Skp1 is a key protein of ubiquitin E3 ligase SCF complex.In order to further identify Skp1 function,the open reading frame of Skpl gene which is composed of 468 nucleotide in length encoding 155 amino acids was amplified by PCR using PGADT7-Skp1 as template,and cloned into expression vector pET-28 a to generate recombinant plasmid pETSkp1.After verification by colony PCR and sequencing,the recombinant plasmid was transformed into Escherichia coli strain BL21 for protein expression.SDS-PAGE analyses showed that under the condition of 37 ℃,Skp1 protein was efficiently expressed after IPTG treatment for 3,6,and 9 h respectively.Treatment for6,9 h showed no significant impact on the expression of Skp1,thereby treatment of 3 h could be used for further protein expression.The expressed Skp1 protein was purified and used for producing the antiserum in rabbit.After collecting the fifth anti-serum the sensitivity of the anti-serum was analyzed using ELISA assay and showed the sensitivity between 1 ∶ 128 000-512 000.Western Blot analysis was further used to detect Skp1 protein using total protein extracted from cucumber leafs,and a specific band at 20 k Da was detected which was corresponding to theexpected size of Skp1.