Molecular Acquirement And Performance Analysis Of Interferon Alpha-2b Mutant

IntroductionAs a type of important cytokines interferon a have a broad spectrum of biological effects,including antiviral effects,antiproliferative and immuno-modulatory actions.It is used widely in clinic for the treatment of virosis and tumor.As a protein medicine, the mainly shortcoming of IFN are short circulating half-life and unstable activity.The former need frequent injection and the latter lead to greater side-effect caused by using IFN in large dose and long time.The prospect of research and development of IFN will be increased bioactivity and improved stability.By changing the target residue of proteases,the performance of IFN will be improved.In this study,the target residues of a-chymotrypsin and endoproteinase Glu-C were choosen as the mutant site,which are not binding site of IFNAR but locate on the surface of spatial structure rather than conserved sequence.Glu at position 58, Tyr at position 89 and Glu at position 159 were substituted by His via site-directed mutagenesis.After substituted by His,these three residues were physically and functionally similar to the original residues and would not bring about new target residue of proteases through the analysis of percent accepted mutation matrix.Rebuild the structure in order to obtain a new mutant of IFNα-2b with higher potency.One of the aims of this study is to modify IFNα-2b gene to abtain two mutant molecules: IFNα-2bHis⁵⁸and IFNα-2bHis⁸⁹/His¹⁵⁹.Clone the IFNα-2b mutant gene and original IFNα-2b gene in order to compare them in bioactivity and stability.The other aim of this study is to discuss whether it can retain the necessary bioactivity of IFN as well as improve the circulating stability. Methods and results1.Gene cloning and construction of IFNα-2b and its two mutantsIn this study,Glu at position 58 was substituted by His and IFNa-2b mutantl was obtained.Tyr at position 89 and Glu at position 159 were substituted by His via site-directed mutagenesis and IFNα-2b mutant2 was obtained.The original IFNα-2b gene was amplified in order to compare these three genes which were cloned into expression vector pET23b.The recombinant plasmids were transformed into E.coli BL21(DE3).Analysis of PCR,restrictive enzyme digestion and DNA sequence indicated that these genes have been cloned correctly and the recombinant plasmids have been constructed successfully.2.Expression and purification of IFNα-2b and its two mutant proteinsThose three proteins were expressed in engineering bacteria.When the culture was finished,the bacteria was centrifuged and washed and the inclusion bodies were obtained by sonication.The inclusion bodies were denatured by 7mol/L guanidine hydrochloride,and renatured by the borate buffer.The protein was purified by anion-exchange and cation-exchange chromatography.The expressed products were analyzed by 15%reducing SDS-PAGE,the result of analysis revealed the yield of protein was up to 40%and the protein mainly existed in the inclusion body and the molecule weight was about 20Kda which was similar to its theoretical molecular weight.The purity of IFNα-2b and its mutants was determined by 15%non-reducing SDS-PAGE,which was more than 95%.3.Quality determination of IFNα-2b and its two mutant proteinsThe molecular weight of IFNα-2b mutant was determined by matrix assisted laser desorption ionisation time-of-flight mass spectrometry.The concentration of IFNα-2b and its two mutants were determined by Lowry method.The bioactiveities of them were determined by the cytopathic-effect inhibition assay and their specific activities were calculated. The molecular weight of IFNα-2bHis⁵⁸is 19239.98,which was completely in agreement with its theoretical molecular weight.The specific activity of IFNα-2b was above 1.68×l0⁸IU/mg and IFNα-2bHis⁵⁸above 3.46×10⁸IU/mg, IFNα-2bHis⁸⁹/His¹⁵⁹above 2.80×10⁸IU/mg.The results indicated that IFNα-2bHis^58,s activity was increased over one time and IFNα-2bHis⁸⁹/His^159,s activity was higher than unmodified one.4.Pharmacokinetic examination of IFNα-2b and its two mutant proteinsEighteen SD rats were divided into three groups and administered the three proteins in dose of 2.5×10⁷ IU/kg subcutaneously.Blood was obtained from caudal vein at the corresponding points.Serum was obtained for detection of the residual IFNαactivity via cytopathic-effect inhibition assay.Draw activity-time curve and analysis pharmacokinetic parameters by DAS software.The half-life of IFNa-2b was 1.72h and the half-life of IFNα-2bHis⁵⁸and IFNα-2bHis⁸⁹/His¹⁵⁹was respectively 2.30h and 2.34h,which was improved slightly in rats.Conclusion1.Two IFNα-2b mutant molecules were designed through method of molecular biology, bio-informatics and computer assist in this study.Recombinant plasmids and engineering bacteria have been constructed successfully.2.Conformed the cultivation condition of IFNα-2b mutant.The yield of protein was above 40%.3.The procedure of purification of IFNα-2b mutant was got in the study,which is simple and in higher yield as well as purity.It may give a simple strategy to generate the recombinant IFNα-2b.4.The results of CPE conformed that the bioactivity of IFNα-2b mutants was higher than the unmodified one so we obtained new molecules in higher performance.5.The stability in vivo of IFNα-2b mutants has been improved which certified that substitution of target residue of proteases could gain the goal of long effect.The study is expected the theoretical and experimental basis of further research in the important protease cleavage site.