嵌合体SBR-CT^(ΔA1)植物表达质粒的构建

Construction of Plant Expression Plasmid of Chimera SBR-CT^(ΔA1)

目的 构建含编码嵌合体SBR_CTΔA1 基因的植物表达质粒。方法 用PCR扩增的含编码嵌合体SBR_CTΔA1 基因的P2片段 (34 98～ 5 378bp) ,经TA克隆与pGEM_easy载体结合得到pTSC质粒 ,pTSC质粒经BamHⅠ和KpnⅠ双酶切后得到P2片段 ,P2片段与BamHⅠ和KpnⅠ双酶切后的植物高效表达质粒pPOKⅡ定向克隆 ,构建pROSC表达质粒 ;且将此质粒与bar基因 (抗除草剂基因 )连接 ,构建重组质粒pROSB ,并经酶切电泳及DNA序列测定鉴定。结果 含编码嵌合体SBR_CTΔA1 基因的P2片段整合到pPOKⅡ的适当部位 ,插入相位正确 ,未改变目的基因的阅读框架 ;从pBARGUS分离出的bar基因整合到pROSC ,构成重组质粒pROSB。结论 本实验成功构建了携带编码嵌合体SBR_CTΔA1 基因的植物表达质粒pROSC和pROSB。

Objective The purpose of this study is to construct plant expression plasmid containing the gene encoding chimera SBR-CT+{ΔA1}.Methods The target gene fragment P2, including the gene-encoded chimera SBR-CT+{ΔA1} (3 498～5 378 bp), was obtained by standard PCR amplification. The PCR products were ligated with pGEM-easy vector through TA clone to form plasmid pTSC. The plasmid pTSC and plasmid pPOKII were digested by restricted endonuclease BamHI and KpnI, and the digested products were extracted and purified for recombination. Then the purified P2 and plasmid pPOKII were recombined by T4 DNA ligase to form recombinant plasmid pROSC; inserting bar gene into the plasmid and form pROSB plasmid. The recombined plasmids were isolated and identified by restricted endonuclease cutting and Sanger dideoxy DNA sequencing.Results P2 gene was linked to pPOKII plasmid and formed recombinant plasmid pROSC. The DNA sequence and orientation were corrected. And bar gene was inserted into pPOSC and form recombinant plasmid pROSB.Conclusion Plant expression vector pROSC and pROSB containing the gene encoding chimera SBR-CT+{ΔA1}, which may provide useful experiment foundation for further study on edible vaccine against caries have been successfully constructed.