A brief talk on the phage panning

  • Gene cloning is the basis of gene engineering and molecular biology research. With the rapid development of biotechnology in the 21st century, gene cloning technology is more and more widely used. Since Hofstetter et al. first successfully constructed the cDNA library by hybridizing plasmids in 1976, the construction and screening of the cDNA library has become more and more mature and popular. It has become the basic technology of gene cloning and functional genomics research, and is one of the most popular and classical methods of cloning full-length gene sequences. The traditional sieve library method is based on labeled nucleic acid probe technology, which has the advantages of high accuracy and sensitivity. The disadvantage is that the radiolabeled probe is easy to cause pollution and the half-life of isotopes is short and unstable. The non-radiolabeled probe is based on biotin and digoxin, which is expensive and requires a lot of work. CR-based methods for screening cDNA libraries are fast and sensitive, and are favored by many researchers. For example, Yang Xiaoming and other researchers use CR-mediated array of DNA libraries to screen hybrid genomic libraries, Qu Wenquan to establish SSS (subsscreening) methods for screening DNA libraries, Wang Zhicheng and other methods based on 96 holes. Screening of cDNA libraries by plate CR method, and so on. In this study, we attempted to construct a CR-based method combined with limited dilution method for screening cDNA libraries, which can quickly and effectively isolate target genes and reduce the workload of screening the cDNA libraries.

    Phage display technology is a high throughput in vitro screening technology that extracts the required polypeptide/protein from a large number of variant colonies. Because of its high efficiency, practicability and convenience, it has been widely used in many fields of drug research and development, and has also been more and more widely used in target proteins of active ingredients of traditional Chinese medicine. Target proteins are binding sites of drug molecules in vivo, and good targets are the basis for obtaining good drugs. This paper reviews the research progress of phage display technology and its application in screening target proteins of active molecules of traditional Chinese medicine. Phage display technology (PDT) has attracted much attention in recent years as an application technology for rapid screening of drug molecular target proteins. It was first established in 1985. It has been fully developed in various fields of drug research due to its simple operation, controllable process, reliable results and powerful screening function. It provides a new and convenient tool for discovering target proteins bound by small scfv antibody library screening in vivo. It also provides new ideas and methods for studying the mechanism of action of active molecules in traditional Chinese medicine.

    Phage display technology was initially established by Smith Equivalent of the University of Missouri in 1985 when they linked foreign gene fragments to the gene III (g3) of the bacteriophage fd-tet. It was found that the polypeptide encoded by the foreign gene fragment could be displayed on the surface of the bacteriophage in the form of fusion with the coding protein of the bacteriophage gene. Later, it was confirmed by experiments that the adhesion, invasion and integration function of phage particles themselves would not be affected by the insertion of exogenous DNA fragments, and the expression of exogenous fragments in the phage shell maintained its original three-dimensional conformation. Since then, bacteriophage display technology has been gradually promoted because of its simple, effective, easy to control characteristics and the advantages of genotype and phenotype unification, and its application has become more and more extensive and in-depth. In turn, the continuous expansion of this application field has also made the bacteriophage display more carefully revised and supplemented at the technical level. It is maturing day by day. At present, llama antibodies technology has been brought into full play in many fields, such as epitope analysis, molecular interaction, preparation of monoclonal antibodies, drug screening, vaccine development, pathogenesis of diseases and functional genomics, and its application is still being explored and broadened, such as some people recently. A commercialized sequencing technology platform has been developed using phage display technology for the development of targeted drugs and disease diagnosis. Some people have linked phage display technology with nano-transmitter system to prepare monodisperse emulsions to promote macromolecular drugs to penetrate the blood-brain barrier and improve their bioavailability.

    The screening principle of phage display technology, also known as biological elution, is based on the specific affinity between molecule and corresponding ligand. Firstly, the target molecule to be studied is immobilized and added into the phage library to make full interaction for binding selection. In this process, phage clones containing specific binding ligands of target molecule are incorporated into the immobilized molecule, and the unconjugated phage clones are washed out directly. Then the bound phage clones were eluted with buffer or free molecules, and the eluted phage clones were collected to infect the host cells for amplification, and the obtained phages entered the next round of screening. After 3-5 rounds of screening by "adsorption-elution-amplification" cycle, the final eluted phage clones were identified by preliminary binding tests, and then single clones were selected for sequence analysis. According to the sequencing results, the common conserved fragments were analyzed. The basic amino acid sequence corresponding to the sequence was the target protein for testing. Sequence.

    Only "possible" target proteins were screened from phage display libraries, and further validation was needed according to the different types of target molecules. As usual, ELISA was used to detect the specific binding of the protein sequence to the target molecule. The monoclonal interaction between the target molecule and the enriched bacteriophage was used to detect whether the monoclonal phage binds to the target molecule by the color reaction of the enzyme-labeled antibody synthesis. In addition, it is necessary to design experiments according to the biological functions of target molecules and target proteins, so as to verify their structure and function.