中国恒河猴DNGR-1分子C型凝集素样结构域在大肠杆菌系统表达  

The expression of C-type lectin-like domain of DNGR-1 molecules of Chinese rhesus macaques in E. coli

作　　者：姚文荣[1] 杨贵波[1] 

机构地区：[1]中国疾病预防控制中心性病艾滋病预防控制中心,北京102206

出　　处：《细胞与分子免疫学杂志》2014年第5期489-492,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：十二五传染病防治重大专项(2013ZX10001004-002-001)

摘　　要：目的获得恒河猴C型凝集素结构域家族9的A成员(Clec9A)基因编码区序列;体外表达CLEC9A的C型凝集素样结构域(CTLD)。方法利用Clec9A基因特异引物,反转录PCR扩增中国恒河猴Clec9A基因编码区,亚克隆至pGEM-T载体,获得pGEM-T-DNGR质粒并进行测序。构建CLEC9A分子的CTLD区原核表达质粒pET-32a-CTLD,转化BL21(DE3),IPTG诱导重组蛋白表达,并用SDS-PAGE和Western blot法对目的蛋白进行鉴定。结果 PCR扩增得到726 bp的Clec9A编码区全长,测序和序列比对发现恒河猴CLEC9A编码区的核苷酸序列与人和小鼠的同源性分别为95.0%和73.1%,推测的氨基酸同源性分别为91.7%和57.3%。体外表达获得相对分子质量(Mr)约32 000的CTLD融合蛋白,Western blot方法鉴定发现其与人DNGR-1分子具有相似的抗原性。结论中国恒河猴Clec9A基因与人的高度相似并能编码与人CTLD抗原性相似的蛋白。Objective To obtain the nucleotide sequence and express the C-type lectin-like domain （CTLD） of C-type lectin domain family 9, member A （ClecgA） gene of Chinese rhesus macaques. Methods The complete coding region of rhesus macaque Clec9A gene was amplified by sequence specific RT-PCR and sub-cloned into the pGEM-T vector and sequenced. Recombinant plasmid pET-32a-CTLD was constructed to express the CTLD region of CLECgA in B1_21 （DE3） cells under IPTG induction. The recombinant protein was identified by SDS-PAGE and Western blotting. Results The 726 bp complete coding sequence of ClecgA gene was obtained. Sequencing and sequence alignments showed that rhesus macaque ClecgA gene shared 95.0% and 73.1% nucleotide identities and 91.7% and 57.3% amino acid identities with humans and mice, respectively. In vitro expression of CTLD resulted in a protein with Mt of 32 000. Western blotting revealed that the protein had the similar antigenicity with human DNGR-1. Conclusion The ClecgA gene of Chinese rhesus macaques is highly similar to that of humans and could code a protein with the antigenicity of human CTLD protein.

关 键 词：中国恒河猴 C型凝集素9家族A成员 树突状细胞自然杀伤细胞凝集素受体1 树突状细胞 C型凝集素样结构域 

分 类 号：R392.11[医药卫生—免疫学] Q786[医药卫生—基础医学]