| Now the most serious problem for drugs used to treat cancer is lacking of specificity for tumor cells. These anticancer drugs currently used in the clinical often cause lethal damage while killing tumor cells, so the development of novel drugs targeting cancer cells becomes a highlight in field of cancer research. In the past decades, people have developed monoclonal antibodies, only binding to the specific cell surface epitomes but not penetrating into the cells. Because the cell membrane has a bilayer lipid structure, which has different permission for dieerernt kinds of molecules. Now micro-injection, lipofection, the virus infection, and other physical and chemical methods are generally used in transporting molecules into cells, but they all have varying degrees of defects, such as low efficiency, cell damage and even death, as well as no targeting characteristic internal cells. It greatly restricts their promotion and application.The emerge of penetrating peptides resolves the difficulty that macromolecular material penetrates through the membrane into cells. Penetrating peptides can effectively transport polypeptides, hydrophilic protein molecules and macromolecules such as DNA fragments into a variety of mammalian cells. They can guarantee the normal activity of the cargo molecules, and also in a certain range will not cause cell damage. Now with their special internalization activity, penetrating peptides have been deeply applied for gene therapy, drug transport, clinical efficacy evaluation, cellular immunology and other areas of research. They have attractive prospects and huge contribution for the life science research. As an effective tool for intracellular transport in cellbiology There are three different ways to combine cell penetrating peptide and cargo molecule together:①Connecting the cargo molecule which is the cDNA of the target gene to the N-terminal of the penetrating peptide gene, transfecting the fusion gene expression vector into eukaryotic cells, using their own protein expression system to express transfected fusion protein. And then these proteins are secreted into the extracellular and penetrating into the surrounding cells, but the transduction efficiency is markedly low.②Polypeptide synthesis, connecting the penetrating peptide and the proteins and other molecules in the form of covalent bonding in vitro, that we can obtain a high purity of fusion protein, but the cost is relatively large.③Inserting the cDNA of the target gene and penetrating peptide into the prokaryotic expression vector, getting expressed and purified fusion protein. This method is avilable to operate, with low expense, and high efficiency of transduction.In addition to application in research and development, we have focused on the internalization mechanism of penetrating peptides in recent years. As we know polar molecules crossing cell membrane is mediated by the transferrin, but the proteins, nucleic acids, polysaccharides and other macromolecules, and some particulate subjects is not permitted by the channels into cells. Biological macromolecules and particles are transported through the cell membrane, during the course the subjects are surrounded by a membrane to form vesicles to carry them out, so they are called vesicular transport, also known as cell internalization. It’s a complex process of cell kinetics, involving the participation of multiple signaling pathways, including a wide range of protein-protein and protein-lipid, protein-carbohydrate interactions. Until now the internalization of most cell-penetrating peptides is mediated by the way of forming bubble membrane, but because of limitation of technical means, experimental methods, the concrete process and the internalization mechanism is still unclear. In this research we received a large number of Hela cell penetrating peptides by biopanning, and analyzed these sequences by bioinformatics. It’s expected to study penetrating peptides from a proteomic perspective, to comprehend their peptide sequence features and the way of internalization, to further clarify the penetrating peptide internalization mechanism.Phage display technique is a biotechnology for screening functional peptides. Its principle is the DNA which encodes polypeptide fragments and the phage capsid protein encoding genes reorganized, expressed in the phage surface in form of a fusion protein. Based on the affinity of biological molecules and drug molecules (antibodies, receptors, antigen, enzyme substrate, etc.), phage display technology can execute rapid biopanning from the peptide libraries. Through adsorption-elution amplification process repeatly, we will get contain phage specifically binding to target protein from the phage library expressing exogenous proteins. After screened out, then enrichment, amplification and gene sequence determination, we will deduce the amino acid composition about exogenous proteins. The technology makes a connection between molecular binding activity and amplified ability. It is a highly efficient screening technology. The advantages of phage display system:①The genotype and phenotype of the exogenous proteins or peptides are uniformed in the same phage particle, so through the phenotype panning we can get their encoding gene.②It can be used in high-throughput screening, has low expression deviation, convenient to operate. It has been successfully used in epitome analysis, monoclonal antibody screening, and analog peptides or antagonist peptides according to protein function. Meanwhile it plays an important role in drug discovery, vaccine design, etc. The research choose the Ph.D.-C7C phage display peptide library as the screening tool, it randomly arranges seven fusion peptide gene sequences inserted into the phage pⅢgene which is a capsid protein coding genes, allowing the expression of random 7-peptide connecting to the expression of terminal phage capsid protein in the form of fusion protein, which build up a combinatorial library. At both ends of each of random seven peptides there is one cytokine, they can spontaneously form a disulfide bond, so that display peptide stylization.Based on the above backgrounds, the design of the subject and experimental results are as follows:One purpose of this experiment was to screen penetrating peptide for Hela cells using phage display technology. The results showed that after 4 rounds of screen, phages with cell penetrating peptide sequences were significantly enriched, recovery rate increased round by round, second, third and forth round respectively enriched 4.63,10.35, and 5.53 folds higher level.The second purpose was to establish penetrating peptide librariy for Hela cell, and get lots of exact penetrating peptide sequences, then analyse them by bioinformatics. In order to ensure the diversity of penetrating peptides, we extracted the gene products of the third round of phage biopanning, constructed them into the pET14b-MCS-EGFP vector, and then screened penetrating peptide library to get definitive peptides. Totally we screened 255 sequences, received 135 with penetrating ability, and 107 with different sequences.The third purpose was to study the internalization mechanism of penetrating peptides for Hela cells. Based on prior studies on penetrating peptides, we selected the highest peak DHYHPFS, and TDAKKLR with the opposite charge, different PH value as a control to invest the mechanism of internalization. For the facility of observation, we constructed and purified DHYHPFS, TDAKKLR fusion protein with the EGFP tag. Pathway of internalization is mainly related to the time-dependent, energy-dependent; heparan sulfate proteoglycan(HS) which combines the protein on the cell surface; caveolin and/or clathrin which form vesicles; microtube and microfilaments which transport vesicular to lysosome or other cell organelle to be degraded. We choosed internalization pathway related inhibitors, there were low temperature (energy), heparin (heparan sulfate proteoglycan), MβCD (caveolin), sucrose (clathrin), CytoD (microtubules), nocodazole (microfilaments). The results showed, the two protein sequence DHYHPFS and TDAKKLR at the concentration of 20μmol/L and 5μmol/L appeared varying degrees of differences in the penetrating process. In addition, they had significant different ways of internalization on different concentrations.The forth purpose of experiment was to combine a functional molecule with penetrating peptide. Then we detect the internalization and effectiveness of the functional molecules. Chicken anemia virus (VP3) can induce apoptosis in transformed cells and tumor cells, but has no inducing apoptosis effect in normal human diploid cells. We constructed the VP3 into the penetrating peptide fusion expression vector and studied whether penetrating peptide play a role in internalization and indirectly inducing tumor cell apoptosis. We selected the non cell specific penetrating peptide TAT as control, and choosed different types of cells Hela cells, HEK293 cells, NIH3T3 cells, PC-3 cells for research. The results showed, His-DHYHPFS-EGFP-VP3 fusion protein showed significant specificity for Hela cells, His-VP3-TAT-EGFP fusion protein showed no cell selectivity. His-DHYHPFS-EGFP-VP3 can effectively induced apoptosis, and the control His-DHYHPFS-EGFP didn’t. The difference was statistically significant (t=-42.017, P=0.000)In conclusion, we explored a high efficiency way for research that was using molecular biology techniques to build a library and making use of bioinformatics research methods for data analysis, to have a systemic understanding for groups of protein with similar function about their molecular structure and characteristic. We selected the specific sequence to study their internalization mechanism, biological function and application. This thesis gained hundred of penetrating peptides different from the previous reported ones. Their internalization mechanism differs from the typical penetrating peptides; and they can effectively carry molecule of Apoptin into Hela cells and play a role in the promotion of apoptosis and showed a certain degree of cell specificity. |
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