Cloning Of Interferon-γ And Interleukin-2 Genes, And Expresion And Activity Determination Of Interferon-γ Of Ten Kinds Of Guangxi Chickens

The interferon- Y and interleukin-2 of ten kinds of Guangxi chickens were amplified by RT-PCR from total RNA extracted from blood lymphocytes at first time, which were cultured in RPMI 1640 medium and stimulated with 10ug/ml ConA in vitro for 16 hours. After cloning into the pMD18-T vetor, the nucleotide acid sequences of genes were identified by PCR and restrictive endonucleases digestion. The results showed that the coding region of interferon- y encode 145 amino acids of predicted mature protein and weigh at 16.8 kD, and the coding region of interleukin-2 encode a predicted mature protein of 143 amino acids with a molecular weight at 16.3 kD. The sequence homology of interferon- Y was 38.8%-40.6% and 99.2%-100% in nucleotide acids, 23.6%-29.1% and 97.6%-99.4% in amino acids,and the homology of interleukin-2 was 25.2%-30.3% and 98.6%-99.8% in nucleotide acids, 14.6%-16.7% and 96.5%-99.3% in amino acids, Compared with mammals and other chickens.A pair of primer was designed to clone the interferon- Y genes protein(mIFN- γ ). After being inserted into pGEX-4T-1 vector , the recombinant plasmid of pGEX- mIFN- γ was digested by EcoR I and Sal I to identify whether the aim fragment was inserted into the plasmid in correct orientation or not. The recombinant was then transformed into E.coli BL21 competent cells induced by IPTG . Protein expression of the recombinants was assayed by SDS-PAGE and Western blot. A new protein band was found in SDS-PAGE and Western blot with molecular mass of about 42.4kD. It indicates that the protein of mIFN- y gene was correctly transcribed and translated in the transformed bacteria. F48E9 Newcastle disease virus was used to detect the bioactivity of mIFN- Y at first time, and the result showed that the bioactivity of mIFN- γ was 1.33 × 10~4U/ml. These results proved that IFN-γ and its recombinant protein could be further applied to develop effective immunoadjuvants and enhance the immunity of animals against infectious diseases.