Cloning And Prokaryotic Expression Of Chicken Interferon-α Gene

Objective:In order to Construct prokaryotic expression system for chicken IFN-a (ChIFN-α) gene and to optimize the expression conditions.Methods:1. Find the chicken interferon-a (ChIFN-α) gene and obtain their gene sequence and protein sequence from the GenBank, specific primers for PCR were designed by using the software DNAman.2. Chicken interferon a mature protein gene was cloned and amplified by reverse transcription-polymerase chain reaction (RT-PCR) from the total mRNA in the lymphocyte of chicken blood stimulated with ConA for 8～10 hours. PCR production reclaimed and purified in low-melting point agarose and connected with plasmid PMD18-T, named pMD18-T-Ckα.It was transformed into E.coli DH5a competent cells and screeing positive colony in ampicillin plate and evaluated in enzyme-cutting by restriction endonucleases Nde I and EcoR I and PCR, in the same time to sequence.3. pMD18-T-Cka was enzyme-cutted and purified,the product connected with PET30a vector, named PET30a-Cka. It was transformed into E.coli BL21 (DE3) competent cells and screening positive colony in knar plate, evaluated by restriction enzymes Nde I, EcoR I and Xho I and PCR.The recombination vector was expressing with IPTG.4. The recombination vector expression level was detected by SDS-PAGE and was expressed high performance in the E.coli BL21(DE3).Through induction expression in temperature and time and inducer, we determined a optimization expression system.5. The inclusion body containing target protein ChIFN-αwas separated and washed.The preliminary purified ChIFN-αwas dissolved in different concentration urea and renatured by gradient dilution.Results:1. Total RNA was extracted successfully, chicken interferon-a (ChIFN-α) gene was amplified by PCR successfully.2. The recombinant plasmid PET30a-Cka was construted successful and the target protein ChIFN-αcan express as inclusion body in E.coli BL21(DE3). SDS-PAGE showed the expressed ChIFN-αprotein was mainly inclusion bodies and had a molecular weight of 22 KD.3. The ChIFN-αexpression level of protein was highly in E.coli BL21(DE3) and the most optimization expression condition is that 37℃cultivates OD600 with 0.7 and lmmol/L IPTG induces to expresse 3h and the ChIFN-αprotein accounted for 30% of whole bacteria protein.4. Further purification expression, using the 0.1 mol/L Tris.cl (PH 8.5) containing 2 mol/L urea to wash the inclusion bodies,the expression deal could achieve 40%. wash the best, accounting for bacterial protein expression of about 40%. ChIFN-αprotein using nickel affinity chromatography column with 200 mmol/L imidazole elution can obtain purified protein.Conclusion:Through of the gene engineering, we had successed to screen the ChIFN-αgene and constructed the recombinant plasmid PET30a-Cka;Through the grading-up,we had obtain the optimization strain E.coli BL21(DE3)and the optimization expreeion condition,which can express the recombinant ChIFN-αprotein highly;The purity ChIFN-αprotein had been attained in using the urea washing and purify; The ChIFN-αprotein has the well application prospect.The results of our study have provided the reliable experimental methods for expression and purification in large scale as well as the foundation for further study of its anti-viral function and may pave the way for future anti-viral application.