小麦蓝矮植原体免疫膜蛋白(Imp)的跨膜区影响其在大肠杆菌中的表达

Influence of the transmembrane region of immunodominant membrane protein (Imp) from wheat blue dwarf phytoplasma on Imp expression in Escherichia coli

根据跨膜区预测结果,设计ImpF1/ImpR1和ImpF2/ImpR2两对特异性引物。以ImpF1/ImpR1,ImpF1/ImpR2,Imp-F2/ImpR1和ImpF2/ImpR2共4个组合经PCR扩增得到4个目标片段,即:免疫膜蛋白Imp基因的全长(Imp-B);切除C-端跨膜区的Imp基因的基因序列(Imp-N);切除N-端跨膜区的Imp基因的基因序列(Imp-C);切除N-和C-端跨膜区的Imp基因的基因序列(Imp-S)。经酶切连接到原核表达载体Pmal-c2x,并转入E.coliBL21(DE3)PlysS菌株中。SDS-PAGE电泳验证,只有切除N-、C-端跨膜区的Imp基因的基因序列(Imp-S)得到大量表达,结果表明:小麦蓝矮植原体免疫膜蛋白的跨膜区影响其在大肠杆菌中的表达。根据跨膜区预测结果,设计ImpF1/ImpR1和ImpF2/ImpR2两对特异性引物。以ImpF1/ImpR1,ImpF1/ImpR2,Imp-F2/ImpR1和ImpF2/ImpR2共4个组合经PCR扩增得到4个目标片段,即:免疫膜蛋白Imp基因的全长(Imp-B);切除C-端跨膜区的Imp基因的基因序列(Imp-N);切除N-端跨膜区的Imp基因的基因序列(Imp-C);切除N-和C-端跨膜区的Imp基因的基因序列(Imp-S)。经酶切连接到原核表达载体Pmal-c2x,并转入E.coliBL21(DE3)PlysS菌株中。SDS-PAGE电泳验证,只有切除N-、C-端跨膜区的Imp基因的基因序列(Imp-S)得到大量表达,结果表明:小麦蓝矮植原体免疫膜蛋白的跨膜区影响其在大肠杆菌中的表达。

Two pairs of specific primers,ImpF1/ImpR1 and ImpF2/ImpR2,were designed according to the prediction result of possible transmembrane helices for immunodominant membrane protein（Imp） of wheat blue dwarf（WBD） phytoplasma.Four sequences,Imp-B,Imp-N,Imp-C and Imp-S were respectively obtained by using combined primers ImpF1/ImpR1,ImpF1/ImpR2,ImpF2/ImpR1 and ImpF2/ImpR2.The overall length of Imp was named Imp-B;the Imp sequence excision of the C-transmembrane region was Imp-N;the Imp excision of the N-transmembrane region was Imp-C and the Imp excision of the C-and N-transmembrane regions was Imp-S.After double enzyme digestion,these four sequences were respectively sub-cloned into the expression vector Pmal-c2x and then transformed into E.coli strain BL21（DE3） PlysS.SDS-PAGE analysis showed that only Imp-S with cleaved N-terminal and C-terminal transmembrane regions could be expressed.The results demonstrated that the transmembrane region can influence the Imp expression of WBD phytoplasma in E.coli.