title: Recombinant expressed vector pET32a (+) S constructed by ligation independent cloning.
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.
authors.affiliation: Medicinal Chemistry and Pharmacology Institute, Inner Mongolia University for the Nationalities, Tongliao 028000, Inner Mongolia, China. shen348@126.com.
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Medicinal Chemistry and Pharmacology Institute, Inner Mongolia University for the Nationalities, Tongliao 028000, Inner Mongolia, China. gongguohua0211@163.com.
authors.forename: Guo-Hua
authors.initials: GH
authors.lastname: Gong
authors.affiliation: Medicinal Chemistry and Pharmacology Institute, Inner Mongolia University for the Nationalities, Tongliao 028000, Inner Mongolia, China. weichengxi1224@163.com.
authors.forename: Cheng-Xi
authors.initials: CX
authors.lastname: Wei
authors.affiliation: State Key Laboratory of Medical Microbiology and Biosafety, Academy of Military Medical Sciences, Biotechnology Institute of Beijing, Beijing 100071, China. ll@bmi.ac.cn.
authors.forename: Long
authors.initials: L
authors.lastname: Liang
authors.affiliation: Medicinal Chemistry and Pharmacology Institute, Inner Mongolia University for the Nationalities, Tongliao 028000, Inner Mongolia, China. bzh9911@163.com.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhang
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins