猪新基因P58^IPK的分子克隆、原核表达及其多克隆抗体的制备

Cloning,prokaryotic expression of novel swine gene P58^(IPK) and its polyclonal antibody preparation

目的:分子克隆出猪P58IPK新基因,制备其多克隆抗体,为下一步研究流感病毒与宿主相互作用机制提供条件。方法:根据猪EST数据库,利用生物信息学方法,电子克隆猪P58IPK新基因;采用RT-PCR方法从猪淋巴组织分子克隆P58IPK基因完整开放阅读框(ORF),全长1 518 bp,编码505个氨基酸(GenBank登录号:HQ287801),并对其进行初步生物信息学分析;构建P58IPK原核表达载体pET-P58IPK,诱导表达并纯化重组的his-P58IPK融合蛋白,后免疫兔子制备多克隆抗体。结果:成功制备P58IPK多克隆抗体,通过双向琼脂扩散实验检测抗体有活性,ELISA鉴定血清中多抗效价达到1∶20 000,Western blot与免疫荧光检测反应特异性良好。结论:成功克隆猪新基因P58IPK,并制备了其多克隆抗体。

AIM： To clone a novel swine gene P58IPK[58-kDa（inhibitor of protein kinase） protein] and prepare its polyclonal antibody for further research of influenza and host interaction.METHODS： The swine P58IPK gene was first identified in silico through homology searching in the swine EST database.Then this gene was amplified by reverse transcription polymerase chain reaction（RT-PCR）.The cDNA of the gene contained the complete open reading frame（ORF） of 1 518 bp,and encoded 505 amino acid residues（Accession No.HQ287801）.The gene was first analyzed using bioinformatics methods.Then P58IPK was cloned into a prokaryotic expression vector pET-32a to construct a recombinant plasmid named as pET-P58IPK.The fusion protein his-P58IPK was expressed in E.coli BL21 and purified using a his-tag protein purification column.Subsequently rabbits were immunized with the purified protein.RESULTS： Specific polyclonal antibody against the fusion protein his-P58IPK was obtained.The activity of the antibody was determined through double-immunodiffusion test.The titer of the antibody was 1∶20 000 as shown by ELISA.specifically recognized the protein P58IPK by Western blot and immunofluorescence assay.CONCLUSION： The novel swine gene P58IPK has been successfully cloned and its polyclonal antibody has been prepared.