猪乳转铁蛋白N端活性区的表达和多克隆抗体的制备

Preparation of polyclonal antibodies with recombinant N terminus of porcine lactotransferrin

在采用亲和层析技术筛选猪嗜血霉形体表面黏附蛋白MSG1相关受体的过程中,发现猪乳转铁蛋白(LTF)可能是MSG1蛋白的潜在受体。为了对其受体功能进行确认,对截短的猪乳转铁蛋白进行了原核表达和多克隆抗体的制备。首先,以人工合成的猪乳转铁蛋白全基因为模板,通过PCR扩增猪乳转铁蛋白基因N端762个碱基后连接入pET-32a(+)载体中。其次,将重组质粒导入大肠杆菌BL-21感受态细胞中用IPTG进行蛋白诱导表达,对表达的重组蛋白用His单抗进行特异性鉴定,并对鉴定后的蛋白应用包涵体洗液进行纯化。最后,以纯化的蛋白通过背部多点皮内注射免疫小鼠和家兔制备多克隆抗体。结果,重组蛋白的分子质量在44ku左右,能够与His单抗发生特异性反应。Western-blot反应证明,制备的鼠和家兔多克隆抗体具有高度的特异性。猪乳转铁蛋白鼠和家兔多抗的制备为猪乳转铁蛋白受体功能的确认和激光共聚焦对其定位提供了技术储备。

Porcine lactotransferrin（LTF） was found to be a potential receptor protein during screening receptors that reacted with MSG1 of by affinity chromatography.In order to further confirm the receptor function,the truncated LTF was expressed and polyclonal antibodies were prepared.Firstly,a 762 bp fragment at the N-terminus of porcine lactotransferrin was amplified from synthesized full-length gene of porcine LTF and cloned into the plasmid pET-32a（＋）.Secondly,the recombinant plasmid was transformed into Escherichia coli competent cells BL-21 and induced by IPTG.The recombinant protein was identified specifically by His monoclonal antibody and purified by inclusion body lotion.Finally,mice and rabbits polyclonal antibodies were prepared by back multi-intracutaneous injection with purified protein.The results showed that the recombinant protein was approximately 44 ku and specifically reacted with His monoclonal antibody.Western-blot result showed the mouse and rabbit polyclonal antibodies were specifically reacted with recombinant LTF.The preparation of mice and rabbits polyclonal antibodies will provide technical assist for the confirmation of porcine LTF receptor function and the location of porcine LTF by confocal laser technology.