[Construction of middle fragment deletion mutant with improved gene splicing by overlap extension].

OBJECTIVE

To construct a phosphatidylinositol 4-kinase beta (PI4K-beta) mutant with the 325th to 373rd amino acid codons deleted, and try to develop a simple method for constructing middle fragment deletion mutant.

METHODS

In line with the mechanism of gene splicing by overlap extension(SOE), an additional PCR was used to get the PI4K-beta mutant in which the 325th to 373rd amino acid codons were deleted. Then the mutated gene was cloned into pCMV-Tag4A mammalian expression vector.

RESULTS

A mutant with the 325th to 373rd amino acid codons deleted was constructed successfully.

CONCLUSION

The improved SOE is a very effective and reliable method to construct middle fragment deletion mutant. It is worthy to be popularized.