The Research On Optimization Of Purification Process Of Membrane Protein R₉-EGFP And Its Distribution In Mice

Kinds of diseases have become a major cause of global morbidity and mor tality which was a serious threat to human health.Other biological macromole cules like proteins played a quite significant role in the occurrence and treatm ent numerous diseases,which has participated various forms of life activities.Because of its poor biofilm permeability and relative rapid degradation,peptid es and oligonucleotides were generally considered to have limited its therapeu tic value.Peptide(CPPs),also known as protein transduction domains,have th e ability to be displaced by cell membranes and efficiently transduce small mo lecules and drugs through the cell membrane.The discovery of small molecule cell penetrating peptide had extremely perfect application prospects in enhanci ng the absorption of drugs,drug transportation,clinical efficacy evaluation an d tumor targeting therapy.On account of its advantages such as a wide range o f histocompatibility,low toxicity,low immunogenicity,stability,specificity an d delivery characteristics,polymorphic arginine transmembrane with short pept ides might be more effectively applied to the cancer treatment.In my paper,the optimum conditions of the purification process of transm embrane protein R9-EGFP and the enrichment and distribution of R9-EGFP in mice were investigated.Firstly,based on the recombinant pET15bArg9EGFP pr okaryotic expression vector,the expression of OD,IPTG concentration,induct ion time and induction temperature were explored by orthogonal experiment.Meanwhile,the purification technology of AKTA purifier 100 system made a 1 arge amount of transmembrane protein with biological activity of bright green color have obtained.Next,Flow cytometry was used to analyze the efficiency o f cell migration in vitro.Finally,the distribution of R9-EGFP in mice was detec ted.The results showed that the optimal expression conditions of transmembra ne protein R9-EGFP were OD 0.6,IPTG concentration 0.2mM,temperature 30?,time 12h.And the protein expression level was 45%of the total bacterial protein.A549 cells used as target cells,the highest membranepenetrating effic iency was 53.07%relied on flowcytometry after 2 days.Interestingly,R9-EGF P with bright green color was mainly concentrated in lung,liver,kidney and st omach of mice.In this paper,the R9-EGFP of top purification conditions with protein activity have been examined successfully.What's more,the enrichment and distribution of the mice in vivo,proved that R9-EGFP has penetrating abil ity,which afforded a new strategy and direction for tumor drug targeting.