Wenrong Yao1, Guibo Yang1. 1. National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
Abstract
OBJECTIVE: To obtain the nucleotide sequence and express the C-type lectin-like domain (CTLD) of C-type lectin domain family 9, member A (Clec9A) 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 CLEC9A in BL21 (DE3) cells under IPTG induction. The recombinant protein was identified by SDS-PAGE and Western blotting. RESULTS: The 726 bp complete coding sequence of Clec9A gene was obtained. Sequencing and sequence alignments showed that rhesus macaque Clec9A 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 Mr; of 32 000. Western blotting revealed that the protein had the similar antigenicity with human DNGR-1. CONCLUSION: The Clec9A gene of Chinese rhesus macaques is highly similar to that of humans and could code a protein with the antigenicity of human CTLD protein.
OBJECTIVE: To obtain the nucleotide sequence and express the C-type lectin-like domain (CTLD) of C-type lectin domain family 9, member A (Clec9A) gene of Chinese rhesus macaques. METHODS: The complete coding region of rhesus macaqueClec9A 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 CLEC9A in BL21 (DE3) cells under IPTG induction. The recombinant protein was identified by SDS-PAGE and Western blotting. RESULTS: The 726 bp complete coding sequence of Clec9A gene was obtained. Sequencing and sequence alignments showed that rhesus macaqueClec9A 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 Mr; of 32 000. Western blotting revealed that the protein had the similar antigenicity with humanDNGR-1. CONCLUSION: The Clec9A gene of Chinese rhesus macaques is highly similar to that of humans and could code a protein with the antigenicity of human CTLD protein.