Cloning Triploid Fish Interferons And Preliminary Study On Their Functions

The immunity can be divided into two categories: the innateimmunity and the adaptive immunity. Innate immunity refers to a kind of natural defensive function endowed with the organs during development and evolution of their germ lines, which is the non-specific defensive function that organs already owned when they were born; Adaptive immunity refers to the defensive functions which are forming through the contact with antigenic properties after their birth. Fish, one of the lowest vertebrates, possess two kinds of immune system: the innate immune system and the adaptive immune system. Fish immune system lies in the intersection of the innate immunity and adaptive immunity evolution in which the innate immunity is of the greater significance for fish disease-resistance.Interferon(IFN) is an important component of the innate immune system in fish and other higher vertebrates,which is a kind of cytokines that could induce gene expression and resist virus. According to the host immune responses caused by interferon receptor after the interferon combined with the receptor,fish IFN can be divided into two categories:type I IFN and type ⅡIFN(IFN-γ).The fish type I IFN gene consists of 5exons and 4 introns,and its receptor contains two subunits: IFNAR1 and IFNAR2. Type ⅡIFN genes have 4 exons and 3 introns and its receptor contains two subunits: IFN-γR1 and IFN-γR2.In this study, we cloned the full-length cDNA sequences of two triploid crucian carp IFN genes andpreliminarily studied their functions.The contents and results in the paper are listed as follows:1.The full length of the 3nIFNa cDNA is 740 bp and consist of a coding region of 552 bp, a 5’ untranslated region(UTR) of 52 bp a nd a 3’UTR of 136 bp. 3nIFNa cDNA was predicted that it can enc ode 183 amino acids. Analysing 3nIFNa protein sequence through E x-PASy, we found that its molecular weight is 2169.1 with the the oretical isoelectric point of 9.35.2.The full length of the 3nIFNb cDNA is 715 bp and consist ofa coding region of 552 bp, a 5’ untranslated region(UTR) of 52 bp and a 3’UTR of 111 bp. 3nIFNb cDNA was predicted that it can e ncode 183 amino acids. Analysing 3n IFNb protein sequence throughEx PASy,we found that its molecular weight is 2162.1 with the th eoretical isoelectric point of 9.65.3. Comparing the protein sequence homology of 3n IFNa and 3n IFNb, we found that their homology with each other are 72.1%. A nalysing bythe phylogenetic tree, we found that 3nIFNa and 3nIFNbshare most similarities with Cyprinid fishIFN and least similarities with mammal IFN.4.Based on the pcDNA5-FRT/TO vector, we constructed the re-co mbinant plasmids expressing 3nIFNa: pcDNA5-FRT/TO-HA-3nIFNaand pcDNA5-FRI/TO-3nIFNa-HA. By transfecting the two plasmids in HEK293 T cells respectively through calcium orthophosphate and conducting the Western blotting assay, we found that 3nIFNa protein can normally express in HEK293 T cells with the molecular weight of 15 KD to 25 KD. 3nIFNa expression was detected after transfection with pcDNA5-FRT/TO-3nIFNa-HA in HEK293 T cells and examiningits cell supernatant, which could indicate that the protein of 3nIFNa is a kind of secretory protein.5. Based on the pcDNA5-FRT/TO vector, we constructed the r ecombinant plasmids expressing 3n IFNb : pcDNA5-FRT/TO-HA-3n IFNb and pcDNA5-FRI/TO-3nIFNb-HA. By transfecting the two plasmids in HEK293 T cells respectively through calcium or thophosphat-e and conducting the Western blotting assay, we found that 3nIF-N b protein can normally express in HEK293 T cells with molecular w eight of 15 KD to 25 KD. However, the expressing protein of the p cDNA5-FRT/TO-HA-3nIFNb and pcDNA5-FRI/TO-3n IFNb-HAcould n ot be detected in supernatantofHEK293 T cells, which indicated th-at3nIFNb is an intracellular protein.6. HEK293 T cells was transfected with pcDNA5-FRT/TO-HA-3n IFNa and pcDNA5-FRT/TO-3nIFNa-HAthrough calcium orthophospha-te then their whole-cell proteins were digested with PNGase F, along with the western blotting assay. We found that glycosylation wa-s detected in HA-3nIFNa,but no glycosylation in 3nIFNa-HA.7. HEK293 T cells was transfected with pc DNA5-FRT/TO-3n IFNb-HA through calcium orthophosphate, then their whole-cell protein swere digested with PNGase F, along with the western blotting assa y.We found that glycosylation was detected in cDNA5-FRT/TO-3n IF Nb-HA, which indicated that 3nIFNb protein are with the capacity for glycosylation modification.In conclusion, in this paper the full length cDNA of two 3nI-F N genes were cloned, and preliminary studies was conducted abo-ut the gene expressions and protein glycosylation of 3n-IFNa and 3nIFNb. The paper has laid a foundation for future systematic stud-ies about IFNs for their disease-resistant function in the innate im-mu nity system in triploid fishes.