Recognition Of HMGB1 Homologous Molecule Of Clonorchis Sinensis And Its Promotion Of NF-κB Activation In Mouse RAW264.7 Macrophage Line

Objective:High mobility group protein 1 homologous molecules（Cs HMGB1）from dead Clonorchis sinensis may be involved in the development and progression of Clonorchis sinensis associated Intrahepatic cholangiocarcinoma（ICC）through activation of specific pattern recognition receptor signaling pathways.The objective of this study was to identify CDS of Cs HMGB1 molecule and to study the effect of this encoded protein on the activation of NF-κB in mouse RAW264.7macrophage cell line in vitro,so as to lay a foundation for further study on the mechanism of its mediating the development of ICC related to Clonorchiasis sinensis.Methods:The m RNA of O.viverrini HMGB1 was amplified by RACE method and RT-PCR method based on the prediction sequence of C.sinensis HMGB1 m RNA.The amplified products were cloned and sequenced,and sequence alignment and splicing were performed.ORF was analyzed,and the c DNA of adult C.sinensis was amplified by PCR to verify CDS.The molecular evolution of HMGB1 CDS and coding protein sequences of different species were analyzed by constructing evolutionary tree.ORF was inserted into prokaryotic expression vector for protein expression,purification and identification.Mouse RAW264.7macrophage cell line was stimulated with purified Cs HMGB1 in vitro,and the activation level of NF-κB was determined by detecting the activity of p Ni Fty2-SEAP plasmid.Results:（1）The HMGB1 CDS of 381 bp and 477 bp of C.sinensis were identified and verified to be transcripted in adult C.sinensis,and the products were uniform.（2）According to the identified homologous protein CDS of Cs HMGB1,20 and 23.5 k D fusion proteins（encoding 127 and 159 amino acids of the target protein,respectively）were expressed in the prokaryotic cells.（3）Cs HMGB1（CDS and protein）are highly homologous with H.comorchiasis HMGB1 molecules,and highly homologous with S.japonicum and S.mansoni HMGB1 molecules,but very low homologous with crustaceans and vertebrates including human.（4）Cs HMGB1 stimulated mouse RAW264.7 macrophage line in vitro,and SEAP activity was detected by transfection of p Ni Fty2-SEAP plasmid.OD₆₂₀value was significantly increased compared with that of unstimulated control group（P<0.01）,and a blue purple reaction was observed in the cells after stimulation.Conclusions:（1）The encoding sequence of Cs HMGB1 was identified,and the transcripts of Cs HMGB1 in adult Clonorchis sinensis were heterogeneous,with at least 381 bp and 477 bp of ORF transcripts.（2）Cs HMGB1 have high homology with T.emorchiasis and Schistosoma,but low homology with humans and vertebrates.（3）Prokaryotic expression of Cs HMGB1 homologous protein can stimulate the efficient activation of NF-κB in mouse macrophage cell RAW264.7.