十二指肠钩虫脂肪酸与视黄醇结合蛋白(Ad-FAR-1) 基因的克隆及原核表达

目的克隆十二指肠钩虫脂肪酸与视黄醇结合蛋白(Ad-FAR-1)基因并在大肠杆菌中表达。方法运用3′RACE及RT-PCR技术分别扩增Ad-FAR-1 cDNA部分片段,获得序列经拼接后利用在线BLAST程序检索GenBank中相似的核酸序列及推导的氨基酸序列;设计引物,将Ad-FAR-1成熟肽编码序列克隆、连接到原核表达载体pET32a,构建重组表达质粒并转化到大肠杆菌BL21(DE3)中用IPTG诱导表达,SDS-PAGE分析表达情况。结果成功克隆到Ad-FAR-1全长cDNA序列并构建了pET32a/Ad-FAR-1原核表达重组质粒,Ad-FAR-1融合蛋白在大肠中得到了高效表达。结论成功获得并表达了十二指肠钩虫脂肪酸与视黄醇结合蛋白(Ad-FAR-1)基因,为进一步研究该蛋白的特性与功能奠定了基础。

Cloning and Prokaryotic Expression of Fatty Acid and Retinol Binding Protein (Ad-FAR-1) from Ancylostoma duodenale

Objective To clone and express the fatty acid and retinol binding protein(Ad-FAR-1) from Ancylostoma duodenale.Method The full length cDNA encoding Ad-FAR-1 was amplified by 3'RACE and RT-PCR.The sequence encoding the mature protein of Ad-FAR-1 was obtained through RT-PCR and then ligated into pET32a to construct the recombinant plasmid-pET32a/Ad-FAR-1.The recombinant Ad-FAR-1 was expressed in E.coli BL21(DE3) by inducing with IPTG and analysed by SDS-PAGE.Result The full-length cDNA of Ad-FAR-1 was obtained from A.duodenale and deposited in GenBank(accession numbers: EF667295).The 543 bp cDNA,encoding the Ad-FAR-1 precursor of 181 amino acids,was expressed in E.coli BL21((DE3) after inducing by IPTG.Conclusion Ad-FAR-1 was cloned from A.duodenale.A recombinant plasmid(-PET-32a/Ad-FAR-1 was constructed and the Trx fusion protein was expressed by IPTG inducing.