Phage demonstration technology

phage
demonstration technology is to insert the
DNA
sequence of an exogenetic protein or
peptide
into the appropriate position of the phage shell protein structure
gene
so that the exogenetic gene is expressed with the expression of the shell protein, and the exogenetic protein is shown to the phage surface biotechnology with the reassembly of the phage. So far, people have developed a single-stranded silk phage display system, phage display system, T4 phage display system and several other phage display system. This paper mainly summarizes the basic principles of phage display technology, phage display system research and technical characteristics, and tracks the latest research progress and development prospects in this field.
keywords: phage display; assembly; fusion protein
In 1985, Smith G P1 first inserted the exogenetic gene into gene III of the silk phage f1, so that the gene-encoded peptides in the form of fusion proteins on the phage surface, thus creating a phage display technology. The main feature of this technique is to unify the genotypes and ideotypes of a particular molecule within the same virus particle, i.e., to display a specific
protein
on the surface of the phage, and to contain the structural genes of the protein in the core DNA of the phage.
, the technique combines gene expression products with affinity screening to select the target protein or peptide using the appropriate target protein. In recent years, with the improvement of phage display technology, the impact of this technology in many basic and applied research fields has become increasingly obvious.
, the principle of phage display technologyPhage display technology is to clone peptides or protein coding genes or the intended gene fragments into the appropriate position of the phage shell protein structure gene, in the reading box correctly and does not affect the normal function of other shell proteins, so that the exogenetic peptides or proteins and shell protein fusion expression, fusion proteins with the reassembly of the child phage and display on the phage surface. The polypeptides or proteins shown can maintain relatively independent spatial structures and biological activity to benefit the identification and binding of target molecules. After incubation of the target protein molecules on the peptide reservoir and solid phase for a certain period of time, the unbinded free phage is washed away, and then the phages that are adsorbed in combination with the target molecule are washed away by competing subjects or acidic escapes, and the phage infection is washed away. After the host cells are multiplied and amplified, the next round of enchantment, after 3 to 5 rounds of "absorption-enchantment-amplification", the phages specifically combined with the target molecules are highly rich. The resulting phage system agent can be used to further atrophy target phages with desired binding properties.
, phage display system2.1 Single-stranded silk phage display system
(1) PIII. display system.
is a single-stranded DNA virus, PIII. is a secondary shell protein of the virus, located at the end of the virus particles, is necessary for phage infection with E. coli. Each virus particle has 3 to 5 copies of PIII.protein, which can be structurally divided into N1, N2 and CT 3 functional regions, which are connected by two glycine-rich connection peptides G1 and G2. Among them, N1 and N2 are related to phage adsorption of E. coli and penetration of cell membranes, while CT forms part of the protein structure of the phage shell and anchors the C-side domain of the entire PIII.protein to one end of the phage. PIII. There are 2 bits available for exogenetic sequence insertion, and when exogenetic peptides or proteins are fused between PIII.protein signal peptides (SgIII.) and N1, the system retains the complete PIII.protein, phage It is still infectious, but if the exogenetic peptide or protein is directly connected to the CT domain of the PIII.protein, the phage loses its infectiousity, at which point the infectiousness of the recombinative phage is provided by the complete PIII.protein expressed by the auxiliary phage. PIII. Proteins are easily hydrolyzed by protein hydrolysis enzymes, so when there is an auxiliary phage super-infection, each phage can show an average of less than one fusion protein, the so-called "unit price" phage.
(2) PVIII. and other display systems.
PVIII. is the main shell protein of silk phage, located on the outer side of the phage, C end and DNA binding, N end protruding outside the phage, each virus particle has about 2,700 PVIII.copy. The N-end of PVIII. can fuse five peptides, but not longer peptide chains, because larger peptides or proteins can cause spatial barriers that affect phage assembly. However, when assisted phages are involved, wild PVIII.proteins can be provided to reduce the price, at which point peptides and even antibodies can be fused
antibody
fragments. In addition, there are still research reports on silky phage PVI. display systems. The C-end of the PVI.protein is exposed to the phage surface and can be used as a fusion point of the exogenetic protein, which can be used to study the regional function of the C-end structure of the exogenetic protein. From the literature, the system is mainly used
cDNA
surface display library construction, and achieved good screening results.
2.2 bacteragus display system
(1) PV display system.
PV protein of the pyrophage forms the tulle part of its tail, which consists of 32 disc-like structures, each of which consists of 6 PV sub-base. PV has two folding areas, and the folding domain (non-functional area) on the C end can be inserted or replaced by an external sequence. At present, the active large molecular protein β-semi-lactose glycosidease (465 ku) and plant exogenetic coagulant BPA (120 ku) have been successfully demonstrated with the PV system. The assembly of phages is carried out in cells, so it can show peptides or proteins that are difficult to secrete. The system shows an average of 1 molecular/phage copy of an exogenetic protein, indicating that an exogenetic protein or peptide may interfere with the tail assembly of the phage.
(2) D protein display system.
the molecular mass of the D protein is 11 ku and is involved in the assembly of the head of the wild phage. The analysis of cryogenic electroscopy showed that the D protein protruded on the surface of the shell grain in the form of a tripolymer. When the mutant phage genome is less than 82% of the wild genome, it can be assembled without the D protein, so the D protein can be used as a carrier of exogenetic sequence fusion, and the exogenetic peptides shown are spatially accessible. The assembly of viral particles can be in vivo or in vitro, in vitro assembly is to bind D fusion protein to the surface of the D-phage, and in vivo assembly is to contain D fusion gene particles into the P. E. coli Essia species containing D-soluble source, thus compensating for the D protein missing from the lysobacteria, by thermal induction and assembly. The system has a good feature that the ratio of fusion proteins to D proteins on phages can be controlled by the host's inhibition of tRNA activity, which is particularly useful for demonstrating proteins that can cause damage to phage assembly.
2.3 T4 phage display system
The T4 phage display system is a new display system established in the mid-1990s. It is characterized by the ability to combine two completely different properties of exogenetic peptides or proteins with shell protein SOCs (9 ku) and HOC (40 ku) on the surface of the T4 shell to be displayed directly on the surface of the T4 phage, so that the proteins it expresses do not require complex protein purification, avoiding protein denaturation and loss due to purification. T4 phages are assembled within the host cell and do not need to be secreted, so peptides or proteins of all sizes can be displayed with little restriction. Wu Jianmin and others successfully displayed the size of about 215 aa SOC/m E2 fusion protein on the surface of the T4 phage shell. It is interesting to note that the presence or failure of SOC and HOC proteins does not affect the survival and reproduction of T4. SOC and HOC can be assembled better than DNA packaging on the surface of the shell when the phage is assembled, in fact, when the DNA packaging is suppressed, T4 is the only two-stranded DNA phage in the body can produce empty shell phage (SOC and HOC are also assembled at the same time). Therefore, when using recombinant T4 as a vaccine, it can show the purpose
antogen
on the surface of the empty shell, which lacks DNA and has a very bright future in biosecurity.
3. Limitations of phage display technology
(1) In the process of phage display must go through bacterial transformation, phage packaging, some display systems also go through the cross-membrane secretion process, which greatly limits the capacity and molecular diversity of the built library. At present, the number of molecules containing different sequences in the commonly used phage display library is generally limited to 109.
(2) Not all sequences can be well expressed in phages, as some protein functions need to be folded, transported, membrane inserted and complexed, resulting in the need for selection pressure when screening the body. For example, in a phage presentation library test, since some unfolded proteins are easily degraded in bacteria, careful control conditions must be taken to ensure that the library displayed on the bacteragus surface is not degraded. In addition, the mouse-sourced antibody has poor expression in the phage and is an example of the pressure of choice in the body. The poor expression of endocrine proteins in bacteria is due to the difference between their protein synthesis and folding mechanisms.
(3) phage display library is built, it is difficult to carry out effective in-body mutation and recombination, which in turn limits the diversity of molecular genetics in the library.
(4) Because the phage display system relies on the expression of genes in cells, some molecules that are toxic to cells, such as biotoxin molecules, are difficult to be effectively expressed and demonstrated.
4. Conclusion
Phage display technology, after nearly 20 years of development and improvement, has become an important technology in the field of life sciences, widely used in the establishment of antigen antibody bank, drug design, vaccine research, pathogen detection, gene therapy, antigen prototyping research and cell signal transduct ion research. The phage display system simulates the natural immune system, making it possible to simulate antibody production in the body and build a high affinity antibody library. Because phage display technology realizes the effective transformation of genotype and ideosis, the researchers realize the in-body control of protein composition on the basis of gene molecular cloning, thus providing a powerful means to obtain expression products with good biological activity. In addition, phage display technology has become a new way to obtain specific human-specific antibodies without immunity, providing an important means for obtaining monoclonal antibodies of diagnostic and therapeutic value for human and animal diseases.