Study On The Construction, Expression And Bioactivity Of Chicken Interferon Alpha/Chicken Interleukin-2 And ChIFN-α-Linker-ChIL-2 Fusion Protein

Virosis of chicken is characteristic of its variety and harmness. There has been lack of medicine with high-performance and broad-spectrum to apply at present. In order to search for the genetic antiviral drug with high-performance, broad-spectrum, no-residue and no-drug-tolerance, and solve the technical difficulty of the lack of such drug clinically, this research studies the construction, fusion, expression and bioactivity of chicken Interferon Alpha/ chicken Interleukin-2 mosaic gene.First, design a primer with double enzyme, using RT-PCR method to enlarge, clone the mature gene of chicken interleukin-2 from the spleen RNA of roman chicken; then using the splicing by overlap extension-PCR method to link the mature gene of chicken interferon alpha and ChIL-2 with flexible genetic linker to construct a mosaic gene ChIFN-α-linker-ChIL-2 and put it in the cloning carrier pGEM-T easy, pick out those positive recombined plastids of ChIFN-α-linker-ChIL-2,ChIL-2 and cut them with restriction enzyme and link them with the expression carrier pQE-30, which were also cut with the same method, to construct the expression carrier pQE-30/ChIFN-α-linker-ChIL-2,pQE-30/ChIL-2, shift the above recombined expression carriers and the pQE-30/ChIFN-αinto E. coli. JM109 and induct its expression with IPTG. Denaturize, renaturize and dialyze the expressed rChIFN-α, rChIL-2, rChIFN-α-linker- ChIL-2 respectively with carbamide, self-innovative renaturation buffer and PBS buffer. Use cell pathological changes restrain method to test respectively the activity of rChIFN-α-linker-ChIL-2, rChIFN-αin resisting VSV; Use MTT method and ChIL-2 ELISA testing method to test respectively the immunocompetence of rChIFN-α-linker-ChIL-2, the activity by ChIL-2 of ConA stimulated chicken T-lymphoiesis and the activity of ChIL-2 in resisting specific immunity. Test respectively the resisting activity of rChIFN-α-linker-ChIL-2 protein over NDV and AIV H9N2 on SPF chicken embryo, and its antivirus activity in the chicken embryo. Test respectively the immune strengthen effect of rChIFN-α-linker-ChIL-2 protein over the NDV live vaccine and inactivated vaccine and its antivirus activity in chicken's body and the bacterin immune strengthen effect of ChIL-2 protein. The result showed that the cloned mature gene covers 363bp and edcoded 121 amino acid; the molecular weigh of the expressed rChIL-2 protein is about 16.3 kD; after purifying, the pure degree of protein is above 95%.the chimeric gene of chIFN-α-linker-chIL-2 was constructed and cloned into pGEM-T Easy vector, respectively. The rchIFN-α-linker-chIL-2 protein was abundantly fusion expressed in E.coli. and successfully purified with the molecular mass of about 36.7 Ku and more than 95% pure on SDS-PAGE, which indicated the correct chIFN-α/chIL-2 fusion protein had been obtained. By using SOE-PCR method, we have successfully constructed the mosaic gene of ChIFN-α-linker-ChIL-2 and its recombination-expression carrier pQE-30/ChIFN-α-linker-ChIL-2, the sequencing result shows that the length of the ChIFN-α-linker-ChIL-2 mosaic gene, which is inserted by recombined and expressed plastids, is 909bp and encoded 300 amino acid, it has been correctly inserted and there was no variation of the sequence of nucleotide acid. The molecular weight of the expressed rChIFN-α-linker-ChIL-2 protein is about 35.9kD and corresponds with our expectation; after purifying, the pure degree of protein is above 96%. The study of activity shows as follows, rChIFN-α-linker-ChIL-2 protein has obvious antivirus of ND activity in cells, its active units ( 9.36×107IU/mg ) in CEF cell is higher than the single rChIFN-α( 4.09×105IU/mg ) .With the increase of dosage, the multiplicative activity of rChIFN-α-linker-ChIL-2 protein over chicken T-lymphocyte is increasing; rChIFN-α-linker-ChIL-2 protein can develop specific immunity with ChIL-2, the relative content of ChIL-2 protein in rChIFN-α-linker-ChIL-2 protein is about 8.12×10-7mg/mL, this indicates that ChIL-2 protein in rChIFN-α-linker-ChIL-2 has immune bioactivity. rChIFN-α-linker-ChIL-2 protein can decrease the bleeding of chicken embryo caused by NDV virus and AIV H9N2 virus, and also prolong the life of chicken embryo remarkably, this indicates that rChIFN-α-linker-ChIL-2 protein has remarkable antiviral activity in chicken embryo. rChIFN-α-linker-ChIL-2 protein has obvious immune strengthen effect on NDV inactivated vaccine, but it has no such apparent effect on NDV live vaccine, this indicates that rChIFN-α-linker-ChIL-2 protein has not only antiviral activity in chicken, but also immune strengthen effect on IL-2. The above results indicate that rChIFN-α-linker-ChIL-2 protein has the dual bioactivity of ChIFN-αand ChIL-2 both in and out of the chicken body.This study has successfully constructed the mosaic gene of ChIFN-α-linker-ChIL-2, and expressed and purified it, too. The purified ChIFN-α-linker-ChIL-2 protein has the dual bioactivity of ChIFN-αand ChIL-2 both in and out of the chicken body, it also has obvious antiviral activity in chicken embryo. This study settles a solid foundation for the development of genetic engineering antiviral preparation for chicken and the study of the control and treatment of chicken virosis.