Cloning,characterization And Expression Analysis Of The Tumor Necrosis Factor-α1Genomic DNA From Common Carp

With the development of molecular biology techniques and gene cloningtechnology, the non-mammalian vertebrate immune system research is becomingincreasingly in-depth and system, especially the cytokines has a certain values in theincrease fish farming disease-resistant. Cytokines were found in the study of theimmune system of higher animals, it is plays a vital role the in regulation reaction ofthe immune system. The study found, the cytokines molecules in the evolutionaryprocess is highly conserved in the regulation and control of immune responses. Inmammals, inflammation can cause a cascade of cytokines, the TNFα usually the firstcytokines to be released, followed is the IL-1β. So, TNFα plays a key role in the earlyinflammatory response. TNFα is secreted by several immune cells: monocytes ormacrophages, NK cells, nerve cells and activated B cells. TNFα is responsible for aseries of signaling between cells. In addition to as resistance to parasites, bacterial andviral infections of the important media, TNFα also has the effect of treatment in thebody, including anti-tumor, shock, and embryo development, but at the same timeexcessive secretion will of lethal pathological effects. TNFα plays an important role inthe fish innate immunity, it is a proinflammatory, When excessive secretion will leadto the shaking and tissue damage, Too low secretion will cause the insulin ofresistance state.In this study,the carp TNFα1cDNA fragments with the nucleic acid probe ofobtained by EST sequencing, after digoxigenin labeled, application of nucleic acidhybridization screening mitogen stimulated peripheral blood leukocyte cDNA libraryof carp, the full-length sequence of carp TNFα1cDNA was acquired. According tosequence non-translated region of TNFα1cDNA the pair of primers were designed,using PCR the TNFα1genomic DNA sequences was obtained.Finally,on the basis of the TNFα1cDNA sequences designed the pair of primers, by Real time quantitativePCR method, analysis the transcription of the TNFα1gene in the normal and mitogenstimulation leucocytes at different times. The results showed that: the carp TNFα1cDNA full-length of1319bp, containing the open reading frame size of768bp,deduced encoding255amino acids and the5′and3′untranslated region and3′poly(A) polyadenylation signal (AATAAA).Genomic DNA length is2748bp,sequenceand phylogenetic analysis,the gene evolutionary is conservation, as with most otherspecies contain4exons and3introns, number of bases of each exon close to themammalian exons correspondingly, exon and intron splice sites are complied withGT-AG rules. Quantitative PCR analysis showed that the TNFα1transcription wasincreased stimulated by mitogen,especially the result of joint stimulation by LPS andPHA is particularly enhanced significant, with the time continued to not improve butgradually decline.It suggests TNFα1transcription is different sensitivities to differentinducers,also shows that TNFα play a critical role in the early stimulating.