粟酒裂殖酵母SpTrz2蛋白全长和N端的原核表达与多克隆抗体制备  

Prokaryotic expression and antibody preparation of full-length and N-terminal half of SpTrz2 protein in Schizosaccharomyces pombe

作　　者：刘金玉 黄鹰[2] LIU Jinyu;HUANG Ying(College of Life Science, Yangtze University, Jingzhou 434025, China;College of Life Sciences, Nanjing Normal University, Nanjing 210023, China)

机构地区：[1]长江大学生命科学学院,湖北荆州434025 [2]南京师范大学生命科学学院,江苏南京210023

出　　处：《浙江农业学报》2021年第1期34-42,共9页Acta Agriculturae Zhejiangensis

基　　金：湖北省教育厅科学技术研究计划指导性项目(B2019036);长江大学科研发展基金(801100010142)。

摘　　要：采用生物信息学方法分析粟酒裂殖酵母SpTrz2蛋白的生物学特性,利用PCR方法扩增粟酒裂殖酵母yAS56菌株SpTrz2的全长和N-末端一半的编码基因,通过NdeⅠ和XhoⅠ酶切位点将SpTrz2的全长和N-末端一半编码基因定向插入pET-28a(+)原核表达载体中,并转化大肠埃希菌BL21(DE3);经过IPTG诱导表达后采用SDS-PAGE电泳切胶纯化法获得重组蛋白,以此为抗原免疫新西兰大白兔获得多克隆抗体,通过Western blotting检测抗体特异性。结果表明,SpTrz2是一个跨膜蛋白,含有3个跨膜结构域,具有多个B细胞抗原表位。SDS-PAGE电泳分析发现,粟酒裂殖酵母SpTrz2的全长和N-末端一半(SpTrz2和SpTrz2N)都以包涵体形式高效表达,其理论相对分子质量分别约为75.99 ku和44.77 ku。Western blotting检测显示,制备的兔抗SpTrz2和SpTrz2N多克隆抗体可以特异性识别天然的SpTrz2蛋白。本研究成功诱导表达、纯化重组蛋白SpTrz2和SpTrz2N,并制备了多克隆抗体,为SpTrz2蛋白的生物学功能相关研究奠定了基础。To bioinformatically analyse the biological characteristics of Schizosaccharomyces pombe SpTrz2 protein,both the full-length and the N-terminal half of S.pombe sptrz2 gene were amplified by PCR and inserted into pET-28a(+)expression vector through NdeⅠand XhoⅠrestriction enzyme sites.The recombinant plasmids were transformed into Escherichia coli BL21(DE3)and the transformants were induced by IPTG.Then recombinant proteins were purified by the way of SDS-PAGE gel slices.Polyclonal antibodies were prepared by immunizing New Zealand white rabbit using the purified recombinant proteins.The specificity of antibodies was tested by Western blotting.The results showed that SpTrz2 was a transmembrane protein with three transmembrane domains and contained multiple B cell antigen epitopes.SDS-PAGE analysis suggested that the full-length and the N-terminal half of SpTrz2 protein(SpTrz2 and SpTrz2N)with a relative molecular mass of about 75.99 ku and 44.77 ku,respectively,mainly existed in a form of inclusion body.The Western blot analysis showed that polyclonal antibodies of SpTrz2 and SpTrz2N could specifically recognize natural SpTrz2 protein.In this study,recombinant SpTrz2 and SpTrz2N protein were successfully expressed and purified,and their polyclonal antibodies were prepared.All above results could provide experimental evidence for the study of biological function of SpTrz2 protein.

关 键 词：粟酒裂殖酵母 SpTrz2蛋白 原核表达 多克隆抗体 

分 类 号：Q939.9[生物学—微生物学]