摘要
lbe是已经证明的心脏标记基因。为了深入研究lbe在心脏发育中的功能,需要获得lbe蛋白并制备其抗体。首先提取野生型成体果蝇的总RNA,反转录获得其cDNA文库,通过PCR克隆出lbe编码区序列,将其连接到pET-28a原核表达载体上。经酶切及测序鉴定后,质粒构建成功。将重组质粒(pET-28a-lbe)转化大肠杆菌菌株Rosseta,用IPTG诱导表达出融合蛋白,经Ni-IDA凝胶柱纯化后,最后将纯化的融合蛋白免疫新西兰大白兔制备lbe多克隆抗体,并用Western blotting检测抗体的效价和特异性。结果显示获得了lbe原核表达重组融合蛋白及高效价的特异性兔抗lbe多克隆抗体,为lbe功能的进一步研究奠定了基础。
lbe is proven to be a cardiac marker gene. Obtianing of the 1be protein and it＇ s antibody is the first step to detect the function of lbe in heart development. First, reverse transcriptional PCR after isolated the total RNA from adult Drosophila wild type and use it as the template, The part of encoding sequence of zebrafish lbe was amplified with RT- PCR, and inserted into pET-28a vector in frame. The recombinant plasmid was identified with enzyme digestion and sequencing. The recombinant expression plasmid（pET-28a-lbe） was transformed into Rosseta and fusion protein was induced by IPTG. The purity protein obtained by treating the lysates with Ni-IDA gel column purification. Then the New Zealand white rabbits was immunized with purified recombinant protein to generate antibody. The antibody titer and specificity was identified by Western blotting. All these results showed that His-lbe fusion protein was successfully purified and the high sensitivity and high specificity anti-lbe polyclonal antibody was generated, which provided a powerful approach for the further studies of lbe function.
出处
《激光生物学报》
CAS
CSCD
2012年第2期162-165,161,共5页
Acta Laser Biology Sinica
基金
国家自然科学基金资助项目(30930054)
关键词
lbe
果蝇
融合蛋白
多克隆抗体
lbe
Drosophila
fusion protein
polyclonal antibody
