Method for cloning and expressing recombinant chicken interferon (IFN)-gamma genes and detecting activity of recombinant chicken IFN-gamma genes

[0015] Embodiment 1: A method for cloning expression and activity detection of recombinant chicken IFN-γ gene of the present invention, the steps are as follows:

[0016] Step 1: Use the recombinant plasmid PMD-18T-preIFN-γ with signal peptide as a template to amplify the ChIFN-γ gene fragment without signal peptide, then purify and recover the PCR product and connect it into the cloning vector pMD18-T to construct the cloning vector pMD18-T-ChIFN-γ, sequencing showed 100% homology with the chicken IFN-γ gene sequence provided by Genebank;

[0017] Step 2: Subcloning the sequenced product into the expression vector pProEX TM HT a On, the expression vector pProEX TM HT a - ChIFN-γ was transformed into the expression host E.coli DH5α, induced with 0.1mmol / L IPTG at 30°C for 4 hours, and a specific induced expression band with the expected molecular weight of 20.0kD was produced. Western blot analysis proved that the fusion protein ChIFN-γ successfully obtained expression;

[0022]Example 2: The present invention clones, expresses and detects the relevant biological activity of the gene encoding the mature protein of ChIFN-γ, so as to provide a theoretical basis for ChIFN-γ in the prevention and control of diseases in poultry breeding industry and the development of green feed. The difference between the present invention and the prior art is that the present invention constructs the gamma-interferon mature protein gene (remove the signal peptide) into the expression vector (pProEX TM A soluble protein obtained on HTa), which avoids renaturation and retains the natural nature of the protein. The attenuated Newcastle disease NDV (preserved by the Animal Nutrition Institute of Northeast Agricultural University) was used in the detection of protein activity. The operation is simpler, and it only needs to be operated between ordinary cells, and there is no need to operate between stricter cells (P3). When detecting the anti-heat stress activity of recomb