Cloning And Expression In E.coli Of Yak Type â…  Interferon Genes

Interferons(IFNs) are a group of pleiotropic cytokines that are originally discovered as a result of their antiviral activity. IFNs play a very important role in resistance to viruses, intracellular bacterial and parasitical infections, anti-tumor proliferation and immunomodulatory activities. There are two classes of IFNs, type Ⅰand type Ⅱ. IFN-α and IFN-β are the main members of type Ⅰ . They are produced by leucocyte and fibroblast respectively. IFN-γ is the only type II IFN. They both have broad-spectrum antivirus and stronger immunomodulatory activities, and both can restrain DNA virus and RNA virus. After gene engineering products IFN-α 2a and IFN-α 2b are selled in marker in 1986, engineering product IFN-β and IFN-γ also confirmed to sale in 1990 and 1993 respectively. In order to study and empolder yak interferon and make use of them to prevent and therapy correlative diseases, we cloned and expressed yak interferon -α -A (IFN-α -A), interferon -α- Ⅱ (IFN-α- Ⅱ) and interferon -β(IFN-β) genes.Total RNA was isolated from yak peripheral blood lymphocytes stimulated with PHA. The yak IFN-α -A, IFN- α -Ⅱ and IFN-β cDNA were amplified by RT-PCR respectively, and then cloned and sequenced. The ORF of the cloned yak IFN-α -A gene was 570bp in length, and the homologies between the yak IFN- α -A and the IFN- α-B gene of cattle was 96. 8%, and the homologies between the yak IFN-α -A and the IFN-α -A gene of cow, water buffalo, pig, horse and human were 94.4%, 62. 6%, 78. 1%, 77. 1% and 71. 4%, respectively, while the homologies between the yak IFN-α -A gene and the IFN-α -A gene of dog, laboratory mouse house mouse, chicken and domestic goose were between 65. 4% and 30. 3%. The ORF of the yak IFN-α -Ⅱ gene was 588bp in length, and the homologies between the yakIFN-α-Ⅱ gene and the IFN-α-1 gene of cattle was 95. 3%, and the homologies between the yakIFN-α-Ⅱ gene and the IFN-α-Ⅱ gene of cow, water buffalo, horse, pig and human were 94. 2%, 92. 8%, 66. 7%, 63. 2% and 64. 4%, while the homologies between the yak IFN-α -Ⅱ gene and the IFN-α -Ⅱ gene of dog, laboratory mouse, house mouse, chicken and domestic goose were between 59. 2% and 30. 7%. The ORF of the cloned yak IFN-β gene was 498bp in length, and the homologies between the yak IFN-β and the IFN-β gene of cow, pig, horse, cat and human were 98. 6%, 68. 9%, 66. 1% and 63. 5%, respectively, wihile the homologies between the yak IFN-P gene and the IFN-P gene of mice, dog and chicken were all less than 25%. Phylogenetic analysis based on the amino sequence from the IFN-a -A, IFN- a - II and IFN-Pgene were all species-specific.After being sequenced, the sub-cloned IFN- a -A , IFN- a - II and IFN- P genes were inserted successfully to the expression vector pGEX-4T-l respectively, then stimulated with IPTG to express recombinant proteins. SDS-PAGE analysis showed that the fusion proteins of yak IFN-a -A and IFN-a -II were approximately 45ku in the form of inclusion bodies, and the fusion proteins of yak IFN-|3 were was approximately 41ku in the form of inclusion bodies. Western-blotting results showed that the three recombinant proteins were all expressed successfully.