摘要
为利用斑马鱼动物模型进一步研究bHLH转录因子家庭重要成员hand2基因在心脏发育中的功能,采用生物信息学结合RT-PCR的方法获得了斑马鱼hand2基因.将所得片段插入原核表达载体pGEX-4T-1中,并通过IPTG诱导表达出GST-Hand2融合蛋白,经GST亲和层析法纯化后,免疫新西兰大白兔制备了多克隆抗体,利用western b lotting和免疫组化的方法对抗体进行分析.分析表明:斑马鱼hand2基因成熟肽编码区含有627 bp,编码208个氨基酸,与人Hand2蛋白的同源性达到83%;IPTG诱导表达后,表达的融合蛋白占菌体总蛋白的71%,经GST纯化获得SDS-PAGE电泳下单一条带.融合蛋白相对分子质量约为49 000.分析表明,制备的抗体具有很好的特异性.
The hand2 gene in zebrafish is cloned by bioinformatics combined with RT-PCR technique,then hand2 fragment is inserted into pGEX-4T-1 vector.The GST-Hand2 fusion protein is induced by IPTG.After purification by GST affinity chromatography,GST-Hand2 fusion protein is used to immune the New Zealand white rabbits to prepare polyclonal antibody.The antibody is assayed by western blotting and immunohistochemistry.The results show that zebrafish hand2 gene coding region has 627 bp,which encods 208 amino acids,has 83% identification with human Hand2 gene.The GST-Hand2 fusion protein expressed in BL21 accounts for 71% of the total proteins.The GST-Hand2 fusion protein displays a single strap in SDS–PAGE after purification,whose molecular mass is 49 000.The report demonstrates that the antibody has high sensitivity and specificity.
出处
《湖南师范大学自然科学学报》
CAS
北大核心
2010年第1期108-113,共6页
Journal of Natural Science of Hunan Normal University
基金
国家自然科学基金资助项目(30671137)
湖南省教育厅优秀青年资助项目(06B053)
