Cloning, Expression Of Caprine INF-γ And Identification For Its Antiviral Activity Against ORFV

Many bacteriosis and virosis do serious harm to goats, while there was not effectivemedicine for treating these diseases. IFN-γ is a kind of multi-effective cytokine, possessingthe ability of anti-bacteria, anti-virus, immune surveillance for tumor, immunomodulation.Recombinant IFN-γ produced by prokaryotic expression has been used to treat humnan andanimal clinical diseases, however, it hasn’t been used for goat diseases. Active recombinantgoat IFN-γmay be a kind of safe and effective medicine for goat diseases.In order to obtain active recombinant goat IFN-γ, primers were designed according to thegoat IFN-γmRNA in GenBank. PBMCs were isolated and used to abstract total RNA afterstimulated and cultured. cDNA was synthsised through RT-PCR. Goat IFN-γORF wasobtained by PCR to construct cloning vector. After identification, primers for expressionvector were designed and goat IFN-γwithout signal peptide sequence was obtained by PCR.Expression vector was induced to produce recombinant IFN-γafter identification.In order toobtain polyclonal antibody, rabbits were immuned with purified recombinant IFN-γ.Prokaryotic expression IFN-γ was assayed for biology activity. Results obtained were asbelow:Sequencing analysisi shows that the gene is goat IFN-γ，and shares more than99%similarity with goat IFN-γgenes accessed in GenBank. The IFN-γgene is501bp long, with asignal peptide sequence encoded by69bp and a mature peptide encoded by432bp. Thewhole protein is about15.9ku, consisiting of2protential N-Gly sites.Recombinant expression strain was induced at different conditions, and results showedthat a amount of soluble protein could be produced after induced for6h with0.3mmol/LIPTGat30℃. Recombinant IFN-γwas used to immuned rabbits after purified, and antibody couldbe detected by ELISA in a titter of1:106.In order to study the activity of recombinant protein, purified protein was used in CPEinhibition. The titter of ORFV in control and treatment group was detected by RT-PCR.Results showed that the titter of ORFV in treatment group was far less than that of positivecontrol(P<0.001)。The titter of recombinant IFN-γwas7.65×25U/mg. Results above showed, recombinant cloning vector and expression vector wereconstructed successfully and active recombinant protein could be obtained. Theseachievements made the idea that recombinant goat IFN-γcould be used in clinical treatmentfor goat diseases possible.