Penetration Efficiency Research Of Cell-Penetrating Peptides In Artemia Sinica

Cell penetrating peptides (CPPs), a class of short peptides which could translocate into cells and mediate the exogenous biological molecules transduction, has been reported in recent years. Its length is often less than30amino acids. Artemia sinica, a kind of crustacean animals, is a good model experiment animal for its eggs are easy to keep and incubate.As for the research, first, we pan out the CPPs of Artemia sinica using phage display library of12random peptides, and we would got the base sequence of the CPPs we pan out. Then, we constructed prokaryotic expression vectors of CPP-EGFP fusion proteins. After the fusion proteins expressed and purified, we check out the CPPs’penetration efficiency of through the cell membrance of Artemia sinica. It would lay a foundation for the further study on the function of cell-penetrating peptides in crustacean animals.The results were as follow:1. The special CPPs screened in Artemia sinicaThe CPPs of Artemia sinica were pan out by phage display library of12random peptides for four cycles. Then, we detected the recovery of phage every cycle. When it reached to10"5, we concluded the phages were succeeded in concentrating. And, if the recovery of single phages we pick out was more than it in last screening cycle, it would show that we succeed in screening. In the experiment, after four screen cycles, we succeeded got the sequences of CPPs in Artemia sinica.2. The expression purification and penetration efficiency of CPP-EGFPThe recombinant expression plasmids pGEX-2T-EGFP-CPP were constructed and expressed in E.coli BL21(DE3). In the control of0.1mM IPTG and16℃, we got the soluble protein successfully. One of fusion proteins purified could penetrate the gastrointestinal cells and began to enrich in Artemia sinica3h after the treatment by watched through fluorescence microscope. The penetration efficiency maximized6h atter the treatment. We conclude the way of concentration was spread to all the intestinal canal gradually and the longer the time was, the higher the penetration efficiency was.3. the reaserch method establishment of CPPs in Crustacea The two parts of the research was a full methods to screen and test especial CPPs in rustacea. It lay a foundation for the further study on the function of cell-penetrating peptides in other crustacean animals...