| Cyprinus carpio,a species from yellow river,is an important economic fish in Henan province.However,with the increase of breeding density,the immunity of C.carpio is decreased,and the diseases occur frequently,which bring a great threat to the development of aquaculture.In order to control the disease,it is of great theoretical and practical significance to study the immune defense mechanism of C.carpio.The adaptive immunity of C.carpio is not perfect,and their innate immunity plays an important role in defending against pathogens.As an important part of innate immunity,complement system plays an irreplaceable role in resisting pathogen infection and regulating immunity.Therefore,the study of related components in complement system will help us understand the immune defense mechanism of C.carpio.C1q DC(C1q-domain-containing)proteins refer to a series of proteins containing C1 q domain at the C terminal,which are important pattern recognition receptors and play an important role in the complement activation reaction in defending against pathogen infection.In orde to study the expression response and immune recognition characteristics of C1 q DC to pathogen analogues(LPS and Poly I:C)in C.carpio.The full-length c DNA sequence of C1 q DC gene of C.carpio,which named C.c C1 q DC,was cloned by reverse transcription-polymerase chain reaction(RT-PCR)and rapid amplification of c DNA ends(RACE)method.The full-length c DNA of C.c C1 q DC was 958 bp,and the open reading frame(ORF)is 723 bp encoding a deduced protein with 241 amino acids.C1 q DC indicated the highest similarity in cyprinid fishes based on the amino acid sequence homology and phylogenetic analysis.The expression levels of C.c C1 q DC in different tissues of C.carpio and the expression profiles of C.c C1 q DC in liver,spleen,kidney and head kidney of C.carpio after LPS and Poly I:C infection were examined by quantitative real-time PCR(RT-q PCR),respectively.The results showed that C.c C1 q DC was ubiquitously expressed in different tissues,with the highest expression in liver and gill.The m RNA expressions of C.c C1 q DC in liver and spleen were significantly up-regulated(p<0.05),and significantly down-regulated in kidney and head kidney after LPS and Poly I:C infection(p<0.05).The agglutination activities of recombinant C1q-domain protein(r C1 q DC)to Aeromonas hydrophila,Vibrio anguillarum,Streptococcus agalactiae and Bacillus subtilis in vitro were tested and showed the obvious agglutination activities against Aeromonas hydrophila and Bacillus subtilis.The binding activities of r C1 q DC to LPS and PGN were tested by ELISA and exhibited the apparent binding activities against LPS and PGN.These results suggested that the C.c C1 q DC could play an important role in pathogen recognition and immune defense.CFH(Complement factor H)family proteins are important complement regulatory proteins,which play the key roles in regulating complement activation response and maintaining homeostasis.In order to study the role of CFH protein in preventing pathogen infection in C.carpio,the open reading c DNA sequence of CFH gene of C.carpio,which named C.c CFH,was cloned by RT-PCR method.The results showed that the ORF of C.c CFH is 2817 bp encoding a deduced protein with 939 amino acids.Based on amino acid sequence homology and phylogenetic analysis,it was revealed that C.c CFH was the highest similarity with the other fish in cyprinid.The expressions of C.c CFH were ubiquitous in different tissues,with the highest expression in liver.The expression profiles of C.c CFH in spleen,kidney and head kidney after LPS and Poly I:C infection were examined by RT-q PCR,and the results showed that the expressions of C.c CFH in spleen and kidney were significantly up-regulated(p<0.05),and significantly down-regulated in head kidney after LPS and Poly I:C infection(p<0.05),respectively.These findings indicated that the C.c CFH could play an important role in pathogen recognition and immune response. |
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