Abstract
The aim of this work was to develop a new method for constructing vectors, named ligation-independent cloning (LIC) method. We constructed the S label expression vector and recombinant pET32a (+) S-phoN2 by LIC. The recombinant proteins were expressed in E. coli at a high level, and then the specificity of the recombinant proteins was identified by western blot. The target band was detected by S monoclonal antibody and Apyrase polyclonal antibodies but not Trx monoclonal antibody and HIS monoclonal antibody. Finally, we obtained protein Apyrase in E. coli (BL21), with a protein-only expression S tag. Collectively, our results demonstrated that LIC is effective for the construction of new vectors and recombinant plasmids. Free from the limitations of restriction enzyme sites and with a higher positive rate, LIC processes should find broad applications in molecular biology research.
Highlights
The purpose of DNA molecular cloning is the insertion of a particular fragment into a vector to construct a recombinant plasmid
Traditional cloning methods are widely used in the construction of vectors and recombinant plasmids [1,2]
Traditional cloning techniques use restriction enzymes and ligation of DNA in vitro, which can be hampered by a lack of appropriate restriction-sites and inefficient enzymatic steps
Summary
The purpose of DNA molecular cloning is the insertion of a particular fragment into a vector to construct a recombinant plasmid. It is widely used in the medicine and biology field. Traditional cloning methods are widely used in the construction of vectors and recombinant plasmids [1,2]. Because of the properties of the original restriction sites of a particular fragment, the experiment has to use some rare restriction This greatly increases the difficulty and cost of the experiment [7]. Using the LIC method to construct vectors and recombinant plasmids shortens the experiments, and reduces the cost of experiments. We report the development of an effective, inexpensive method named the LIC method to construct the expression vector pET32a (+) S and pET32a (+) S-phoN2, which can generate controllable overhangs
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