Construction of eukaryotic expression plasmid pSecTag2B-msBlyS expressing mouse soluble B lymphocyte stimulator / 华西口腔医学杂志

ABSTRACT

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>The sequencing data indicated that inserted msBlyS gene had correct DNA sequence and orientation.</p><p><b>CONCLUSION</b>Eukaryotic expression vector pSecTag2B. Expressing mouse BlyS have successfully been cloned. This will provide us an opportunity to do further research work on BlyS.</p>