Construction Of Fusion Plasmid HPV16E7-IL28B And IL7-IL15 And Expression In Pichia Pastoris System

Objective This experiment was performed by constructing pPIC9k-his-HPV16E7-IL-28B(Linker)and pPIC9k-IL-7-IL-15(Linker)secreted recombinant expression plasmid(hereinafter abbreviated as pPIC9k-E7-IL28 B,pPIC9k-IL7-IL15),Pichia pastoris transformed to induce expression,which provides a new reference and basis for the study of its immunotherapeutic effect on cervical cancer.Methods 1.According to the Pichia pastoris codon usage frequency table,the E7-IL28 B gene was codon-optimized by OptimumGene software;2.The recombinant expression plasmids pPIC9k-E7-IL28 B and pPIC9k-IL7-IL15 were constructed by molecular cloning technique;the recombinant expression vectors pPIC9k-E7-IL28 B and pPIC9k-IL7-IL15 were electrotransformed into P.pastoris GS115 and multi-copy rotors were performed.filter;3.The expression of fusion protein pPIC9k-E7-IL28 B and pPIC9k-IL7-IL15 under different conditions such as methanol concentration,induction temperature,induction time and pH value of the medium were explored.These two fusion proteins were analyzed by SDS-PAGE.Result The recombinant expression plasmid vectors pPIC9k-E7-IL28 B and pPIC9kIL7-IL15 were constructed successfully,and high copy transformants were screened by geneticin(G418);The selected high-copy transformants were subjected to PCR verification,and the PCR-corrected and correctly sequenced strains were selected for preliminary expression of the fusion protein.After several experiments,After several experiments,we found the suitable expression conditions for the fusion proteins E7-IL28 B and IL7-IL15,that is to say the optimized fusion protein E7-IL28 B was expre-ssed at a methanol concentration of 1%,29?,230 rpm,a pH of 6.0,and induction of 96 h;While without genetic optimizationing,the fusion protein pPIC9k-HPV16E7-IL-28 B did not see the target band;The fusion protein IL7-IL15 was obtained at a metha-nol concentration of 1%,29?,230 rpm,pH 6.0,and induction for 120 h and the amount of expression is high.Conclusion 1.We have constructed Pichia pastoris expression vectors pPIC9kHPV16E7-IL-28 B and pPIC9k-IL7-IL15 successfully,and integrated their gene sequences into the genome of Pichia pastoris;2.Codon optimization strategy promotes the fusion protein E7-IL28 B express in Pichia pastoris;3.When the other induction conditions were the same,the expression level of the fusion protein IL7-IL15 was the highest at a methanol concentration of 1%.