[Construction of eukaryotic expression plasmid pSecTag2B-msBlyS expressing mouse soluble B lymphocyte stimulator].

OBJECTIVE

The purpose of this study was to clone the soluble form of the mouse BlyS (msBlyS) and insert it into a eukaryotic expression vector pSecTag2B in order to further elucidat the antitumor activity induced by msBlyS expressed by the recombined plasmid pSecTag2B-msBlyS.

METHODS

Full length cDNA of mouse soluble BlyS (msBlyS) was amplified by reverse transcription-PCR from total RNA of mouse spleen. The PCR product was ligated directly with linearized vector pCR2.1 supplied in the TA cloning kit. The recombined plasmid pCR2.1-msBlyS which was selected and identified using blue-white screening method and restriction map analysis and the purified original plasmid pSecTag2B were both cut by HindIII and EcoR I. The digested fragments were extracted and purified from low-melting temperature agarose and ligated by T4DNA ligase. The recombined plasmid pSecTag2B-msBlyS were isolated and identified by restricted endonuclease cutting and Sanger dideoxy DNA sequencing.

RESULTS

The sequencing data indicated that inserted msBlyS gene had correct DNA sequence and orientation.

CONCLUSION

Eukaryotic expression vector pSecTag2B. Expressing mouse BlyS have successfully been cloned. This will provide us an opportunity to do further research work on BlyS.