Design and production of recombinant interferon beta construct with specific mutations in Kozak sequence due to promote translation

Interferon beta is one of the most important members of group I interferons and is the main drug for multiple sclerosis treatment. Interferon beta has short half life and this compels patients to make frequent use of medicine. According to its clinical usage there is broad effort to improve translation level and protein production. There are several important factors which effect protein production that Kozak sequence is one of the most important one. Specific primers were designed due to induce site directed mutations by SOEing PCR (Splicing by overlap extension / Splicing by overhang extension (SOE) PCR) method. Three PCR reactions needed to amplify recombinant interferon beta gene. The final recombinant gene and pSVM plasmid were digested by EcoRI and then ligated by DNA ligase enzyme. After transformation plasmid extraction was done and the structure of the recombinant plasmid confirmed by digestion and PCR. Finally, approved recombinant plasmid was transfected to CHO cell line by using Lipofectamine kit. Genetic engineering methods were used to produce recombinant interferon beta gene. Production of recombinant construct including specific mutations in Kozak sequence was done by using SOEing PCR method. The white colonies were detected after transformation. Plasmid extraction was done and the structure of recombinant plasmid was confirmed by digestion and PCR. Expected length of fragment was observed after digestion. The CHO cells were cultured and recombinant plasmids were transfected to CHO cell line. These mutations will enhance and improve interferon beta protein production. The influence of these mutations in protein production will survey by using ELISA method in next phase of research.