Procaryotic cell in vivo antibody library construction screening and optimizing of antibody and use
An antibody library, prokaryotic cell technology, applied in the direction of antibodies, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problems of difficult operation and slow growth of yeast, and achieve the effect of high affinity and high specificity
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Embodiment 1
[0040] Embodiment 1: Embodiment 1 is to further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention (the same applies to the following embodiments). The method for constructing an antibody library in a prokaryotic cell comprises the following steps: (1) taking immune-related tissues and cells such as peripheral blood, lymph nodes, spleen, bone marrow, and liver of organisms; (2) isolating RNA from the taken tissues and cells, and Reverse transcription to obtain cDNA; (3) Design DNA primers for the variable region of the heavy chain of the immunoglobulin, the variable region of the light chain and the third constant region respectively, and amplify the variable region of the heavy chain of the immunoglobulin, the variable region of the heavy chain and the third constant region of the obtained cDNA. light chain variable region and the third...
Embodiment 2
[0041]Embodiment 2: the method for screening specific antibody in prokaryotic cells, comprising the following steps:
[0042] (1) The full-length or partial open reading frame (ORF) of the antigen gene is inserted into the DNA sequence that can be used as a bait plasmid in bacterial two-hybrid. Bait plasmids include all plasmids that can be used in prokaryotic cells, that is, bacterial two-hybrids. In addition to the common protein-coding genes of general plasmids, this plasmid also contains partial sequences of special protein-coding genes such as DNA-binding proteins or adenylyl cyclase. . The inserted antigen gene can form a fusion gene with the DNA-binding protein sequence or the partial sequence of adenylyl cyclase. (2) The plasmid containing the antigen obtained from step (1) is mixed with the plasmid containing the antibody library obtained from the library construction step (4), and the plasmid containing the reporter gene (such as antibiotic resistance gene and β-gal...
Embodiment 3
[0043] Example 3: A method for constructing a secondary antibody library in prokaryotic cells, comprising the following steps: (1) Using the original antibody light chain and heavy chain variable region as a template, the following method is used to construct a derived secondary antibody library, but Not limited to the following 4 methods. The light chain and heavy chain variable regions were amplified by random mutagenesis of polymerase chain reaction (PCR), inserted into the above-mentioned plasmids used to construct antibody libraries, and combined with specific protein-coding genes in the plasmids such as RNA polymerase α subunit or The partial sequence of adenylate cyclase forms a fusion gene to form a secondary antibody library; (2) After inserting the original antibody light chain and heavy chain variable regions into the above-mentioned plasmids used to construct the antibody library, use special host bacteria Cause random mutations, extract plasmids, and form a second...
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