Reconstruction Of The Cre-LoxP Recombination Enzyme System Based On The Cell Penetrating Peptides

In 1988, Green Loewenstein and Frankel Pabo of American first reported and confirmed that I human immunodeficiency virus trans-activator protein Tat polypeptide can pass across alone a range of cells, then opened the prelude to the research about cell penetrating peptide CPPs (cell penetrating peptide, CPP); In 1994, FaweH reported that Tat can mediate exogenous protein into cells, and determine the shortest sequence of the protein internalization of Tat protein containing 11 amino acids from 47 to 57 in amino acid sequence, which is called "Tat protein transduction structure" (peptide transduction domain, PTD), the matter is an important milestone in the CPPs’developing history; In 1997, the first CPP designed by research group of Heitz and Divita which can convey nucleic acid into the cell by non-covalent way opened the gate of artificial design and synthesis of CPP; Another major breakthrough in the field of CPP is that the Dowdy group’s research using CPP in vivo transportal experiments for the first time, they used Tat 47-57 in sequnce to connect with high molecular weight protein P-gal glucosidase (β-galactosidase,β-Gal) by gene engineering method, after intraperitoneal injection into mice, the staining results showed that, Tat 47-57 in his whole sequence can carry portable beta-Gal into multiple tissues including brain, body organs. From then on, the study upon CPP expands from vitro to vivo. In the meanwhile, more and more CPPs were found by the researchers, expecially large numble of nature proteins such as Drosophila transcription factor Antennapedia(Antp), VP22 protein of human cyclin, herpes simplex virus (human period 1), signal transduction protein Syn B1, fibroblast growth factor FGF-4, fusion protein gp41, exotoxin A, G enzyme and prion, which were found to have the protein polypeptide sequence to penetrate cell membrane.A large number of studies have shown that cell penetrating peptide is a kind of substance that can carry exogenous substance amino, whose acid length is often less than 30 polypeptides, and is also called membrane penetrating peptide (membrane penetrating peptide, MPP), PTD or membrane translocation sequence. It can not only penetrate cell membrane, but also can be used as carrier for exogenous substance into the types of different kinds of extensive cell. There are more than 300 kinds of exogenous substance transported by CPP. Exogenous substances are mainly divided into three categories:protein, nucleic acid and a small amount of small molecule substance. The proteins exogenous chemicals includ enzymes, apoptosis, protein hormones, molecular chaperones and intracellular signal transduction molecules such as proteins, superoxide dismutase, apoptosis regulating factor p53, neurotrophic factor, heat shock protein 27, Rho protein. Nucleic acids exogenous chemicals always are composed of plasmid, double-stranded DNA, antisense oligonucleotides and small interfering RNA (small interfering RNA, siRNA); other substances are dye (medical imaging),200nm liposome, phage and superparamagnetic particles. But compared with previous carrier, it has advantages of simple operation, rapid, high transduction efficiency, relatively safe, less damage to cells. Therefore, in recent years, CPPs has received widespread attention at home and abroad, research mainly focuses on two aspects of application and internalization mechanism of membrane. Nowadays the methods of CPP delivery of material in vivo and in vitro gradually become mature. In the fundamental research, it has become a useful tool for interaction of intracellular molecular; in application field, some drugs linking CPP have entered preclinical, clinical stage I and II test, especially shows obvious advantages in the treatment of tumor, diseases of the central nervous system and cardiovascular disease. Because of all sorts of characteristics and advantages of cell penetrating peptides based on CPPs, the researchers said in the pharmaceutical, medical diagnosis and treatment, research CPPs will shows more and more important significance.The process of carrying cargo molecules into cells by these CPPs is complex, all the function of CPP is similar, but the structure, physicochemical properties and mechanisms of endocytosis are not the same. At present, point view of the researchers about CPPs’penetrating into the cell mainly has three reasonable mechanisms: endocytosis, directly through the membrane and depending on the specific membrane structure.The specific mechanism of endocytosis of a CPP molecule depends on several related parameters, such as molecular size, temperature, cell types, cell internal and cell external environmental factors. For example, short CPPs with lots of Arg mainly depend on endocytosis of cell uptake, Tunnemann reported that Tat peptide linked cargo into cells is a fast, non endocytosis process directly into cell, and with the big goods after fusion through the endocytic pathway of endocytosis. Duchardt recently reported when the concentration of Antp is over 40μmol/L, its internalization mechanism may be involved in multiple cellular pathways including depending on the specific membrane structure.Gene knockout technology is a kind of genetic engineering technology, according to a known sequence but unknown function sequence, it can cause genetic change of gene function, making specific loss function, so that part function will become barrier, and further impact will be occured on the organism, and then researcher can infer biological function of the gene. It is an experiment method to study gene function which is the most commonly used, mainly through the mutant gene related construction, in-depth study of gene function. Up to now, gene knockout technology is composing of Cre-LoxP system, FLPI system and other systems. But the Cre-LoxP system is widely used for conditional knockout gene method in recent years, Cre-LoxP recombination system belongs to one of the site-specific recombination system, is currently on a site-specific recombination system widely used at home and abroad. The system is from phage P1, containing two important parts:one is the LoxP locus; another is Cre recombinase which can connect with LoxP recognition site. As early as 1981 the system has been found for the first time. In 1993 by the Gu researchers formally as a gene manipulation methods used since, has been widely used in gene cloning, artificial antibody library construction, antibody rearrangement, conversion animal model and chimeric genome research. Furthermore, it shows great advantages, and the system has been successfully used in a variety of eukaryotic and bacterial genes knockout field.In recent years, it is reported that targeted genes can be silenced through transfecting in use of liposomes combined with Cre recombinase, but the operation is complex, and it is not only high cost, but the characteristics of silencing effect is aslo not apparent. Nevertheless, the process of cargo molecules into cell mediated by penetrating peptides is fast, direct, few cellular toxicity. At the same time, we sceen peptides for cells through phage display technology in the preliminary sceening stage, then get lots of CPPs for HeLa cell, and then analisised sequence by bioinformatics. Now we want to explore use of CPPs screened out and Cre sequence of exogenous combined expression of fusion protein to internalize cells to target gene knockdown effect in the cell level.Nuclear localization sequence (Nuclear localization signal, NLS) is a domain of protein, also signal peptide as another form, being generally typically 4-8 amino acid.The protein can be transported into the nucleus with the nuclear carrier. The first NLS is SV40 (Simian vacuolating virus 40) T antigen (92kDa), NLS is the most mature research, features of NLS:(1) the core NLS containing 4 lysine or arginine peptides composed of six amino acids; (2) not containing acidic amino acids and high molecule; (3) the side core NLS is proline or glycine; (4) existing in the middle of flanking sequence of hydrophobic amino acids to ensure the molecules on the surface of NLS located in the protein. With the help of this special signal peptide, effiency can be increased for CPPs in the nucleus translocation of Cre recombinant enzyme.The purpose of my research is to do further study on 152 CPPs polypeptide on the basis of previous screened:(1) To compare the peptide internalization of MEF cell efficiency to screening peptide which have strong internalization ability in MEF cells; (2) To establish the evaluation method of polypeptide internalization ability by fluorescence intensity quantitative way; (3) To choose higher internalization capacity peptide of MEF cells to connect Cre-NLS to construct prokaryotic fusion protein expression vector; (4) To use fusion protein His-SBP-Tat-NLS-Cre-EGFP to knockout Cdc42 gene whose two sides connect LoxP site in C57 mouse peritoneal macrophages, and to evaluate the system knockout gene effect at the protein level.Based on the above research background, In the research work, I choose to purify protein by using green fluorescent protein tags to screene 152 peptides; to use M5 to campare internalization effiency of 5 peptides; to analyse relation of physicochemical property and internalization capability of two screening polypeptide with the method of bioinformatics; to construct recombinant plasmid containing NLS and Cre; to induced to isolate peritoneal macrophages of mice with C57 with the method of PBS as exogenous stimulus; and to use fusion protein His-SBP-Tat-NLS-Cre-EGFP to internalize peritoneal macrophages of mice to detect gene knock effency at the protein level.Through the study, we get the following results:(1) Completed the screening of 152 CPP for MEF cell line, observation of protein internalization in living cell through Zeiss macroscope:17 strong internalization ability peptides,12 common internalization ability peptides,123 few internalization ability peptides;(2) Completed 5 peptides internalization effiency quantitative comparision for MEF cells by M5;(3) Completed Analysis of polypeptide molecular weight, charge, hydrophilic and hydrophobic properties of amino acids, multi-angle analysis characters:After internalization, all charge characteristic for granular both strongly internalized polypeptide and weak internalization peptides:positive charge or a zero charge; water coefficient of strongly internalized polypeptide:-2.286-2.7; isoelectric point distribution of strongly internalized polypeptide:3.49-12.00; strong internalization ability polypeptide generally contain basic amino acids, and the nature of partial hydrophilic;(4) Successful construction of recombinant plasmid such as: pET14b-SBP-Tat-NLS-Cre-EGFP,pET14b-ARE-NLS-Cre-EGFP, pET 14b-HDG-NLS-Cre-EGFP, pET 14b-HPT-NLS-Cre-EGFP, and pET 14b-TVL-NLS-Cre-EGFP;(5) Successful separation of peritoneal macrophage in C57 mice with LoxP site;(6) Successful Cdc42 gene knockout of fusion protein His-SBP-Tat-NLS-Cre-EGFP in mouse macrophages.In summary, the research verify firstly the internalization ability of 152 CPPs for MEF cell, then analysis the relation of the polypeptide physicochemical properties and internalization ability with the method of bio informatics, and do the comparision of internalization effiency for MEF cell line among five strong internalization ability polypeptides. At the same time, do the construction of Tat-NLS-Cre containing good classical recombinant prokaryotic plasmid, then purify exogenous protein to use it as a carrier, finally ultilise it to knock out gene. The above work has made beneficial exploration in animal and tissue level for gene knockout, also wides application prospect of the CPPs in gene manipulation and treatment fields in the future.