克隆、表达及测序一体化的原核表达载体pLCM182的构建及在细胞因子表达中的应用

Objective　To simplify the steps of cloning, sequencing andexpressing of heterogeneous genes,a new integral plasmid pLCM182 has been designed andconstructed. Methods　This new plasmid is characterized with the pL promoter andRBS of phage T7 gene 10 for high expression of the inserted gene,the universal sequencingprimer sites for directly sequencing the inserted gene,and a multiple cloning sitespecified for fitting the optimizing of translation.Thus once cloning of a PCR productinto this plasmid,the gene expression and sequencing can be done without the need ofsubcloning, and much more time can be saved. Results　By use of this plasmid,several genes obtained byPCR,including human IL-17,human IL-4,and human IL-1RA have been cloned,sequenced,andhighly expressed. Conclusion　The integrative plasmid is applicable and convenient forcytokines expression.

Construction of an integrative plasmid pLCM182 for heterogeneous gene cloning, sequencing, expression and its application in cytokine expression

Objective To simplify the steps of cloning, sequencing and expressing of heterogeneous genes,a new integral plasmid pLCM182 has been designed and constructed. Methods This new plasmid is characterized with the pL promoter and RBS of phage T7 gene 10 for high expression of the inserted gene,the universal sequencing primer sites for directly sequencing the inserted gene,and a multiple cloning site specified for fitting the optimizing of translation.Thus once cloning of a PCR product into this plasmid,the gene expression and sequencing can be done without the need of subcloning, and much more time can be saved. Results By use of this plasmid,several genes obtained by PCR,including human IL 17,human IL 4,and human IL 1RA have been cloned,sequenced,and highly expressed. Conclusion The integrative plasmid is applicable and convenient for cytokines expression.