Protein chip basic principles and technology research status
With the further development of molecular biology chip technology research work, DNA chip technology has been gradually applied to the detection and comparison of various known or unknown nucleic acid sequence expression in biological samples. However, a significant portion of the protein that is a functional component of chemical reactions in biological cells has no direct relationship with the mRNA expressed by the active gene. Therefore, the cDNA chip technology as a high-throughput gene expression analysis platform The application process is subject to certain restrictions. In addition, because various small chemical changes in protein structure and conformation can cause changes in activity or function, in order to further reveal the relationship between various metabolic processes and proteins in the cell and the molecular mechanism of certain diseases, it must be The function of the protein is studied in more depth. With the continuous maturity of DNA chip technology and the remarkable achievements made by genetic research institutes, it has further promoted the research of protein functions and the development of related technologies, and protein chip technology has emerged. This article intends to review the basic principles of protein chips, the overview of corresponding technology research and existing problems.
1 The basic principle of protein chip technology The basic principle of protein chip technology is to orderly fix various proteins on various carriers such as titration plates, filters and slides to become a chip for detection, and then, label specific fluorescence The protein or other components of the antibiotic body interact with the chip. After rinsing, the components that cannot be complementary to the protein on the chip are washed away, and then the fluorescence intensity of each point on the chip is measured using a fluorescence scanner or laser confocal scanning technology. Fluorescence intensity analyzes the relationship between protein and protein interaction, thereby achieving the purpose of measuring the function of various proteins. In order to achieve this goal, we must first fix the protein on a suitable carrier by a certain method, while maintaining the protein's natural conformation, that is, we must prevent its denaturation to maintain its original specific biological activity. In addition, due to the diversity of proteins and the complexity of functions in biological cells, the development and establishment of high-throughput protein core technology with parallel processing capabilities of multiple samples and rapid analysis will help simplify and accelerate the progress of protein function research.
2 Research status of protein chip technology In the research process of protein chip technology for many years, researchers have made unremitting explorations in order to find suitable substances as protein carriers. For example, Japanese scholar Velev uses liposomes containing cationic surfactant (HTAB) as a carrier to prepare a nano-level assembly by combining glutaraldehyde with a ferritin-coated component to assemble. The body can enter and fix ferritin molecules on the inner surface of the envelope to form a protein carrier under appropriate pH conditions. Adachi et al. Use some solid surfaces or films covered with electrolyte-containing films as carriers, which can transfer colloidal protein particles to the films to form protein chips. Uetz et al. Analyzed the interaction process of various proteins in the full-length reading frame of the Saccharomyces cerevisiae genome sequence, using plates with different pore numbers as carriers, and established a plate protein chip system consisting of about 6000 yeast transformants. Each well contains a yeast transformant, which can generate a protein according to the coding expression of the open reading frame of its active functional region. The system can be used for protein function detection and analysis. Arenkov et al. Used polyacrylamide gel plates as supports, placed protein samples on the gel, and then separated them by electrophoresis to make them protein arrays for further research. Recently, the Harvard University Protein Chip Research Center Gavin et al. Used the needle-like spotting tip of a high-precision manipulator that prepared DNA chips to prepare the first protein with a sample number of 10800 on a glass slide that is only half the size of a microscope slide. chip. This chip uses purified protein, G spotted 1799 liters per spot 10799 times, another time with FRB (FK-BR12-rapamycin binding domain of FKBP-rapamycin-associated protein) spotted. In order to ensure that spotted proteins of different molecular weights can be immobilized on the slide, they first coated BSA on the slide surface, and then activated the surface of the BSA with N, N'-disuccinimidyl carbonate Lysine, aspartic acid and glutamic acid residues become BSA-NHS slides. Their function is to promote the binding of BSA and spotted proteins so that the proteins are fixed on the slides. During the preparation of the chip, in order to ensure that the protein fixed on the carrier still maintains its natural conformation and biological activity, they added 40% glycerol to the protein-spotted phosphate buffer to prevent the evaporation of the body. Protein denaturation. After spotting, after 3 hours of warm bath and immersing the tablets in a buffer solution containing bovine serum albumin (BSA), the surface of the chip contains a layer of bovine serum albumin, which is used to block non-specific binding sites with other proteins And aldehyde groups that did not participate in the reaction on the surface. In order to detect the application of the chip, they labeled IgG and FKBP12 (12Kd FK506-binding protein) that can specifically bind to protein G and FRB with different fluorescent antibodies and acted on the protein chip to observe the interaction between these proteins and the protein chip. Clearly show that the protein G on the chip and the single FRB spot are marked with blue and red fluorescence, respectively. This experimental study establishes a micro-spotting technique for protein samples and allows the protein to retain the original conformation and biological activity when immobilized on the carrier. This will provide parallel research or rapid analysis of multiple protein samples in the future-for the preparation of Qualcomm The protein chip for quantitative function detection has laid the technical foundation.
3 Application research of protein chip At present, scholars are carrying out research on the application of protein chip technology. Holt and others use protein chip technology to screen specific antibody antigen components that can bind to each other. They use 12 kinds of antibodies that express strong but have not been exposed to any antigen. Fragment screening protein chip containing 27648 human fetal brain proteins, from which four sets of highly specific antigen (protein) -antibody complexes were identified, of which three antibodies bound protein function is unknown, but due to low expression levels , Indicating that this antigen-antibody combination technology is a screening method with high specificity and sensitivity, which can be used for high-throughput screening and separation of various antibody components, or to detect gene expression and protein molecules. Interaction, which will help to study the molecular mechanism of the pathogenesis of certain diseases (including tumors) and assist in the search for target molecules for disease diagnosis and treatment. According to the basic principle of protein chip technology, different antibodies can be fixed on a carrier to become an antibody protein chip for detecting proteins produced by different tissues.
In the study of protein interactions, Ge uses a universal protein chip system, which is sensitive and effective, has a wide range of applications, can be quantitatively determined and reused, and is easy to operate.
Gavin et al. Used protein chip technology to observe the interaction between related substances and enzymes in metabolic processes. They chose three pairs of kinase-actant systems, namely dependent on 3 ', 5'-phosphoprotein kinase-Kemptide; protein kinase II-protein phosphatase inhibitor 2 and p42 mitogen-activated protein (MAP) kinase (ErK2 ) -E1K1, first fix the action of each system on the three BSA-NHS slides in sequence, and bathe with different protein kinases in the environment with the isotope γ-³³P ATP, and then, the slides are treated with emulsion It can be observed under light microscope that various protein kinases catalyze phosphorylation reaction of specific targets. The results suggest that the protein chip technology can be applied to the study of the interaction between the acting substance and the enzyme and the analysis of the metabolic mechanism.
Gavin et al. Also used three small molecule traits with corresponding protein receptors, such as DIG, biotin and synthetic hexahydropyrrolidone (AP1497), in the research process of protein chip technology. Interaction. They first fixed the receptor proteins of the three small molecular substances on the same carrier in sequence and prepared them into the same four protein chips, labeled BSA with fluorescent substances (Alexa488, Cy3, or Cy5), and distributed them. DIC, biotin and AP1497 are combined into probes, and each of the probes with different fluorescent labels acts on the chip. As a result, only the receptor protein corresponding to the small molecule can be labeled with fluorescence. The above results indicate that there is no cross-excitation excitation and luminescence between the used fluorescent agents. Therefore, three different types of receptor proteins can be simultaneously labeled with different fluorescence by mixing the three labeled probes and acting on the protein chip. The research results suggest that the protein chip technology is very useful for the discovery of new drugs, because it is very convenient to find the target of the new drugs.
4 Existing problems and application prospects Although the first protein chip with high-throughput analysis of ginseng has been successfully produced, the spotted protein can maintain its natural conformation and biological activity through proper technical processing. However, there are still various problems in the study of protein function using protein chip technology. For example, most of the current cloning systems from cDNA libraries cannot encode proteins through the correct reading frame; or they cannot be correctly expressed to produce protein molecules with full amino acid sequences; Proteins expressed by bacteria cannot form a normal spatial conformation, etc., which will directly affect the study of protein functions. In addition, in order to facilitate the functional detection and analysis of a wide variety of proteins, proteins obtained through different ways or methods and used to make protein chips must first be purified. The difference between protein-protein interactions under normal and abnormal conditions requires further exploration.
In summary, the establishment of high-throughput analysis platform protein chip technology will provide fast, high-information and more direct research methods for protein function and related research. Compared with other molecular biological analysis methods, protein Chip technology has the advantage of being fast and parallel. The establishment and application of this method will help humans reveal the molecular mechanism of disease occurrence and find more reasonable and effective treatment methods and approaches. So far, this brand-new technology still has some defects, and needs to be further improved and developed.
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