Cloning, Prokaryotic Expression And Immune Function Analysis Of CFH Gene Of Branchiostoma Belcheri

The complement system is a key component of the innate immune system, which plays a central role in the elimination of microbes, clearing of immune complexes, destroying self cells and modulating the adaptive immune response. There are three major pathways to initiate complement activation, including the classical pathway, the lectin pathway and the alternative pathways. Complement factor H (CFH) is the central fluid-phase regulatory protein of the alternative pathway, initially identified as a glyprotein composed of 20 conserved complement protein (CCP) domains. Each CCP domain, also known as’sushi’domain or short consensus repeats (SCRs), contains a consensus sequence spanning about 60 residues with four invariant cysteine residues, a highly conserved tryptophan, and relatively conserved glycine, proline, and hydrophobic residues. CFH can accelerate the decay and cleavage of C3 convertase by binding to C3b and also inactivate C3b on self surfaces as a cofactor of factor I.Amphioxus, as atransitional species from invertebrates to vertebrates in evolutionary history, belongs to the ancient chordate lineage. Both morphological and molecular evidences show that amphioxus is a crucial organism for the understanding of chordate evolution including the origin of the immune system. In recent years, with the development of genome sequencing of a large number of different species finished, especially to complete the whole genome sequencing of Branchiostoma ftoridae, as well as the rapid development of comparative immunology, the attention was focus on amphioxus immune increasingly, further understand the issue about vertebrate evolution as well as the origin of the immune system. Therefore, Our study cloned the full-length of CFH cDNA through RACE, and made a bioinformatic analysis on the sequence, detected the expression in different tissues. In addition, the preliminary researched the antibacterial function of amphioxus. The result is as follows:(1) A full-length amphioxus cDNA of 1295 bp was cloned, and the amphioxus cDNA revealed a 855bp open reading frame (ORF), a 181bp 5’-untranslated region (UTR) and a 259bp 3’-UTR.(2) By the bioinformatic analysis, the ORF encodes a putative AmphiCFH protein with 284 amino acids. The result revealed that it had a signal peptide of 24aa and two conserved CCP domains, but no N-glycosylation site was found, and the molecular weight of AmphiCFH was 31.12KDa and the theoretical PI was 4.29.(3) The AmphiCFH was positioned between invertebrates and vertebrates, and is consistent with the classical taxonomy.(4) A specific protein bands was expressed in E.coli by Prokaryotic expression, and its molecular weight was consistent with the predicted size of the CFH protein theoretical molecular size.(5) The AmphiCFH gene expression was detected in muscle, gill, intestine, hepatic cecum and notochord, but varied expression levels were observed among the different tissues with the highest level in notochord. The expression of AmphiCFH gene in notochord, hepatic cecum and muscle are higher than those in gill and intestine.(6) The AmphiCFH gene expression at different time points (2 h,4 h,6 h,8 h,10 h, 12 h,24 h,48 h) after LPS stimulation have changed. The expression level of AmphiCFH mRNA was up-regulated at 8h and 24 h, and the peak of AmphiCFH mRNA transcript was observed at 8 h post-injection. Our findings displays that CFH of Branchiostoma belcheri involve in innate immunity responses and provide an insight into the immune mechanise of CFH in amphioxus.In conclusion, our study elaborates the CFH gene in amphioxus and provides an insight into the innate immunity and the evolution of the CFH protein family.