Technology for producing monoclonal antibodies by Escherichia coli.



Antibody productionbyE.coli. Hybridoma, like most other animal cell cultures, grow relatively slowly, it does not reach the high density and require complex and expensive media. The resulting monoclonal antibodies are very expensive wich does not allow their extensive usage them in the clinic. To solve this problem, there have been attempts to create a sort of "bioreactors" from genetically modified bacteria, plants and animals. Frequently for the effective delivery and functioning of some immunotherapeutic agents one antigen binding region of an antibody (Fab-or Fv-fragment) is enough, ie, the presence of Fc-antibody fragment is optional. The essence of the method for production of functional antibodies using E. coli is the following: using mRNA extracted from the antibody-producing cells (B lymphocytes), cDNA is synthesized. Then separate PCR amplification of cDNAs encoding H-and L-chain is conducted. After that, the amplified cDNA was treated with specific endonucleases, and then inserted into a vector on the basis of bacteriophage l. cDNA of H-and L-chains have different endonuclease sites, which facilitates integration of each specific nucleotide sequence in its vector. At this stage, cloning of many different segments of H-and L-chains is occur. Next, common "combinatorial" vector is inserted into cDNA of one H and one L-chain, so that the bacteriophage synthesized both strands Ig and form a "full» Fv-fragment. Synthesis of H-and L-chains occurs during the lytic cycle of bacteriophage l, therefore it is possible to screen libraries of combinatorial phage clones to determine their antigen-binding activity. At the connection of cDNA H-and L-chains in a single vector a wide spectrum of different antibody genes is formed. Some of them encode unique binding sites and obtain them with conventional hybridoma technology would be impossible. Pool of mammals antibodies includes 106-108 different antibodies. Phage library contains about the same amount of clones, so we may expect that one combinatorial library will generate the same amount of different antibodies (Fv-molecules), just like any mammal. In addition, once created initial combinatorial library allow us to combine L-and H-chains and get Fv-fragments, which recognize unusual epitopes. Even greater diversity can be achieved using non-specific mutagenesis. Identification of Fv-fragments with the desired specificity takes from 7 to 14 days. For comparison, screening hundreds of hybridoma cell lines usually takes months. Vectors based on bacteriophage 1 is not very suitable for the production of large amounts of protein molecules. To solve this problem, such a vector was constructed in which DNA of H-and L-chains were integrated into site, flanked by plasmid DNA. Such plasmid containing the DNA of H-and L-chain can be cut out of the vector and transformed to E. coli. As part of plasmid DNA of Fv-fragments will be repeatedly replicated in E. coli with the formation of a large number of products which can be used for diagnostic and therapeutic purposes. Screening of combinatorial libraries of antibody fragments can be carried out by ELISA. The essence of the method is the following: samples (aliquots) from the library placed in wells of plates containing the target antigen. The wells is washed to remove unbound phage particles. Conjugate composed of an antibody that binds to the protein shell of the phage and enzyme is added in each well. The wells is washed to remove unbound conjugate and chromogenic substrate splitting by the enzyme associated with the phage is added to each of them. In this case those wells are stained which contain phage particles carrying antibodies to the target antigen. Selecting phage that synthesizes desired antibody it is possible to extract encoding DNA fragment and subclone it into the expression vector. Thus, the tools of biotechnology offer new promising perspectives in the creation of unique medicines and biological products for the needs of medicine and veterinary.

 


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