小鼠可溶性B淋巴细胞刺激因子真核表达载体pSecTag2B-msBlyS的构建

Objective The purpose of this studywas 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 inthe 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 byHindⅢandEcoRⅠ. The di- gested 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 Eu- karyotic expression vector pSecTag2B. Expressingmouse BlyS have sucessfully been cloned. Thiswill provide us an opportunity to do further research work on BlyS.