Gene Cloning, Expression And Immunological Studies Of L-C3-like From Lampetra Japonica

The third complement component (C3) is the most abundant complement protein inserum. C3plays a central role that supports the activation of all the three pathways ofcomplement activation: the classical, alternative, and lectin pathways. It is regarded as thecore of the complement system for the reason that it connects the natural and adaptiveimmunity especially playing an important role in immune defense, regulation and pathology.Under the stimulus of antigen-antibody complexes or pathogens, C3can be activated andinduced the cascade. Then membrane attack complex is formed and cracked on the surface ofthe target cells eventually. In addition, the cleavage products of C3also have a series ofregulation of the immune system function. Overall, C3plays an important role in innateimmune and adaptive immune system.Lamprey that is always as a representative of the rare jawless vertebrates has anevolutionary status between invertebrates and vertebrates.Its unique evolutionary status andthe special variable VLR variable lymphocyte receptors which mediated adaptive immunesystem are attentioned by immunologists. It has been reported that C3was found in lampreyand it composed with three chains: β、α、γ. However, the homology compared withmammalians C3is less than40%. It is not clear that the related immunological functions oflamprey C3and whether there are other complement molecules in lamprey. As a result, thecomplement system of lampreys has many suspects.In order to study whether lamprey C3molecule was equal to that of mammalians in thesense of its relevant process and characteristics of the immune system, open reading framelength and the3’untranslated region cDNA of lamprey (Lampetra japonica) C3(L-C3-like)were cloned. We have get the whole sequence6274bp, and open reading frame5013bpwhich encoded1671amino acids. According to built the junctional region of α-chain andγ-chain of L-C3-like cDNA from lamprey liver, prokaryotic expressed, and successfullyobtained anti-L-C3-like-2polyclonal antibody. The observation was further confirmed byFACS and laser scanning confocal microscopy in leukocytes and myelocytes. Moreover, theReal-time PCR and western blot analysis indicated that the transcript and protein expressionof L-C3-like was distributed in immune associated tissues. The functional interaction betweenVLRB and L-C3-like were demonstrated by co-immunoprecipitation in parallel with western blot assays. The deposition and detection of L-C3-like on the surface of target cells wereanalysised.This paper has laid a solid foundation for L-C3-like molecular structure and research ofrelated functions through the anti-L-C3-like-2and the related immunological functionidentification. It provides an important tool and support for the further study on lampreyimmune system.