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

Construction of Eukaryotic Expression Plasmid pSecTag2B-msBlyS Expressing Mouse Soluble B Lymphocyte Stimulator

目的 克隆BALB c小鼠可溶性B淋巴细胞刺激因子 (msBlyS)的cDNA片段并测序分析 ,为研究msBlyS诱导的抗肿瘤作用提供目的基因。方法 采用反转录聚合酶联反应 (RT_PCR)法从BALB c小鼠脾脏组织总RNA中获得 5 6 6bp的msBlyS基因cDNA。通过TA克隆测序无误 ,再将其定向连接到pSecTag2B真核表达载体上 ,转化E .coliXL1_blue。限制性内切酶筛选出阳性克隆pSecTag2B_msBlyS ,末端终止法完成msBlyScDNA的序列测定。结果 测得的msBlyScDNA序列与基因文库中报道序列完全相同 ,插入相位正确 ,未改变目的基因的阅读框架。结论 成功克隆了小鼠可溶性B淋巴细胞刺激因子基因 ,为研究其诱导的抗肿瘤作用奠定了基础。

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 EcoRⅠ. 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 sucessfully been cloned. This will provide us an opportunity to do further research work on BlyS.