水稻L-半乳糖内酯脱氢酶基因的克隆和原核表达及抗体的制备

应用RT-PCR技术从水稻叶片中克隆了水稻L-半乳糖内酯脱氢酶(GLDH)的全长基因.序列分析表明,水稻GLDH基因全长1752bp,亚克隆其部分成熟蛋白编码基因(789bp),利用基因重组技术构建了E.coli/pET30a-GLDHe,经酶切鉴定,DNA测序,证实重组质粒构建正确;将重组质粒转化大肠杆菌Rosseta,IPTG诱导表达,SDS-PAGE分析证实表达出相对分子质量约为36000的蛋白.用原核表达蛋白作为抗原免疫家兔制备抗体,用Westernblot检测抗体的特异性及效价,结果表明,抗体血清效价为1∶1000,在加IPTG诱导后的菌液中可以检测到相应的蛋白条带((相对分子质量为36000)),在水稻叶片样品中能检测到1条相对分子质量为36000的蛋白条带.

Cloning, Prokaryotic Expression of Rice GLDH Gene and Preparation of Anti-GLDH Antibodies

The full length of L-galactono-1,4-lactone dehydrogenase (GLDH) gene was amplified by RT-PCR from rice leaves and sequenced. The amplified gene was about 1752 bp. The coding region for part of mature protein (789 bp) was subcloned into pET-30a( ). The recombinant plasmid pET30a-GLDHe was identified by enzyme digestion and DNA sequencing. Data of SDS-PAGE indicated that a 36 kD protein was expressed. The antiserum against GLDH was generated by immunizing rabbits with the prokaryotic expressed protein. The polyclonal antibody was analyzed by Western blotting for its specificity and titer. The antibody could recognize GLDH protein specifically and its titer was about 1:1000.