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目的分离鉴定犬钩虫C型凝集素AcaCTL-1基因,并在大肠埃希菌中表达。方法根据EST序列设计引物,用RT-PCR及3′(RACE技术扩增获得AcaCTL-1全长cDNA序列,并对其进行初步生物信息学分析;将AcaCTL-1成熟肽编码序列克隆、连接到表达载体pET32a,构建重组质粒pET32a/AcaCTL-1;在大肠埃希菌BL21(DE3)中诱导表达重组蛋白,SDS-PAGE分析表达情况,Ni亲和层析纯化可溶性蛋白。结果成功克隆了AcaCTL-1全长cDNA序列,其最大开放阅读框由525 bp组成,预测编码174个氨基酸残基组成的蛋白,含17个氨基酸组成的信号肽,为C型凝集素家族蛋白成员;构建pET32a/AcaCTL-1重组质粒,并在大肠埃希菌中高效表达。表达产物多以包涵体形式存在,小部分为可溶性蛋白。结论成功克隆并表达了犬钩虫C型凝集素AcaCTL-1基因,为进一步了解AcaCTL-1的功能奠定了基础。
OBJECTIVE: To isolate and characterize CloAcL-1 gene of canine hookworm and express it in Escherichia coli. Methods Primers were designed according to the EST sequence. The full-length cDNA sequence of AcaCTL-1 was amplified by RT-PCR and 3 ’(RACE), and the preliminary bioinformatics analysis was carried out. The mature coding sequence of AcaCTL-1 was cloned and linked to The expression vector pET32a was constructed and the recombinant plasmid pET32a / AcaCTL-1 was constructed.The expression of recombinant protein was induced in Escherichia coli BL21 (DE3), the expression of recombinant protein was analyzed by SDS-PAGE and the soluble protein was purified by Ni affinity chromatography.Results AcaCTL- 1 full-length cDNA sequence, the largest open reading frame of which consists of 525 bp and is predicted to encode a protein consisting of 174 amino acid residues and a signal peptide of 17 amino acids, which is a member of the C-type lectin family protein. Constructing the pET32a / AcaCTL- 1 recombinant plasmid was expressed highly in E.coli.Expression products mostly existed in the form of inclusion bodies and a small amount of soluble protein.Conclusion Cloning and expression of Acinetobacter C-type lectin AcaCTL-1 gene was carried out in order to further understand the relationship between AcaCTL -1 features laid the foundation.