Development of a dual-expression vector facilitated with selection-free PCR recombination cloning strategy

Abstract

Abstract The conventional procedure for the construction of recombinant expression vector of a target gene includes PCR cloning and restriction enzyme mediated subcloning, which is time-consuming and sometimes troublesome because of the inefficiency of ligation. A variety of ligase-independent PCR cloning strategies have been developed, but they either involve complicated PCR procedures or need other DNA modifying enzymes. In this study, we report the design, and construction of an omnipotent expression vector pOmni, with which a target gene can be easily cloned through innovative selection-free PCR recombination cloning strategy with only one pair of primer and two times of PCR in one work day, without using any restriction enzymes, ligase and other DNA modifying enzymes. Furthermore, the target gene cloned in pOmni is ready to be high-efficiently expressed in either Escherichia coli cells or eukaryotic cells because of the elaborate design of compatible T7 promoter and CMV promoter expression elements in the vector. The cloning capability and reliability of selection-free PCR recombination cloning with pOmni were validated through cloning of 6 DNA fragments with length from 315 to 4557 bp, and the dual-expression function of the vector was verified through the cloning and expression of EGFP in E. coli BL21 and HeLa cells. pOmni developed in our study provides a powerful tool for gene cloning and expression, and is of special value for researches in which both prokaryotic and eukaryotic expression of a target gene are necessary.

Keywords

• PCR cloning
• Prokaryotic expression
• Eukaryotic expression
• Lysis gene E
• Positive selection