Method for amplifying mouse monoclonal antibody heavy light chain gene sequence, primers thereof and method for screening primers

Abstract

The invention discloses a method for amplifying a mouse monoclonal antibody heavy light chain gene sequence, primers thereof and a method for screening the primers. Forward primers used by all composite primers are all compositions, the composite primers are obtained by screening primer pairs capable of amplifying non-functional gene sequences in advance, and moreover, the primers for amplifying aSp2 / 0 cell non-functional light chain gene are removed from each group of the forward primers, PCR amplification is performed by using the composite primers, the obtained primers do not require TA cloning, the purity requirement of sequencing can be met, further, the number of the amplification tubes can be reduced by using the composite primers, the amplification operation can be simplified, thedetection sensitivity can be improved, and the PCR amplification operation is simpler and more convenient.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for amplifying heavy and light chain gene sequences of monoclonal antibodies in mouse hybridoma cells, primers thereof, and a method for screening the primers. Background technique [0002] Monoclonal antibodies are widely used in the field of biomedicine, and play an important role in fields such as in vitro diagnosis (In Vitro Diagnosis, IVD). Obtaining antibody heavy and light chain sequences from hybridoma cells through gene sequencing technology is the first step for permanent preservation of monoclonal antibody resources, construction of recombinant antibodies, and in vitro affinity maturation of antibodies. [0003] Sp2 / 0 cells used for hybridoma cell fusion contain endogenous non-functional genes, and these sequences cannot be efficiently translated into proteins. Therefore, when the antibody light chain gene sequence is amplified from hybridoma cells, th...

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Problems solved by technology

[0004] At present, a variety of strategies are used to amplify light chain antibody genes from hybridoma cells, the most commonly used method is to use restriction endonuclease method to remove endogenous non-functional genes, but this method is complicated and inefficient; Another method is to construct TA clones from the amplified products, and then sequence and identify multiple clones obtained after TA cloning and purification to exclude endogenous non-functional genes. However, this method is also time-consuming and laborious.

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