马铃薯X病毒运动蛋白基因的原核表达、抗血清制备及应用

Expression of movement protein gene and antiserum preparation of Potato virus X

构建马铃薯X病毒运动蛋白(P25)原核表达载体,并在大肠杆菌BL21(DE3)pLysS中诱导表达融合蛋白,所表达的P25蛋白经SDS-PAGE电泳纯化后免疫小鼠,抗血清效价为1∶4000,抗血清与PVX有特异性反应(P/N>2),且具有较强的反应特异性,而与PVY、TMV均不发生反应。该方法制备的抗血清可用于ELISA检测PVX感病植株及Western blot检测转基因植株。

Recombinant plasmid pET-P25 was constructed by ligating movement protein （P25） gene of Potato virus X （PVX） into the expression vector pET-30a and then transferred into E. coli BL21 （DE3） pLysS. The results of SDS-PAGE showed that 29 kD specific fusion protein was produced after induction by IPTG. The fusion protein was injected into mice to prepare antiserum after being purified crudely. And the titer was determined to be 1 ： 4 000. The antiserum has special response with PVX （ P/N 〉 2 ） compared with no response with PVY and TMV, Using this antiserum, we can examine the infected leaves by PVX and PVX-P25 transgenic tobacco. And the result showed that P25 gene had been expressed and translated to protein in some transgenic plants.