马铃薯卷叶病毒CP基因的突变及其原核表达

Mutation and prokaryotic expression of potato leafroll virus coat protein gene

通过人工合成DNA的方法,对马铃薯卷叶病毒外壳蛋白（CP）基因第52～177核苷酸（126bp）这段序列进行了突变,将其中12个AGA、AGG、CGA等精氨酸稀有密码子变成了原核高效表达的同义密码子CGT与CGC,将另外两个AGA精氨酸密码子变成了错义密码。构建了突变基因的原核表达载体pBAD-LRCP2,Bsp1407Ⅰ与MssⅠ酶切及DNA测序结果表明,基因突变符合要求,表达载体的构建正确。在37℃,大肠杆菌工程株TOP10（pBAD-LRCP2）用0.2%L-阿拉伯糖诱导培养4h,SDS-PAGE显示蛋白图谱上有一条36ku的诱导表达的融合蛋白条带,结果表明突变基因在PBAD启动子驱动下在大肠杆菌中实现了表达。

The 52-177 nucleotides （126 bp） ofpotato leafrollvirus coatprotein （CP） gene were mutated by artificial synthesis of DNA. Twelve rare arginine codons of AGA, AGG, and CGA were replaced by synonymous codons of CGT and CGC with high-level prokaryotic expression, and othertwo arginine codons of AGA were changed into missense codons. The prokaryotic expression vector of the mutant gene named pBAD-LRCP2 was constructed. Digestion of Bsp1407 Ⅰ and Mss Ⅰand DNA sequencing confirmed the accuracies of gene mutation and expression vector construction. The engineered strain TOP10 （pBAD- LRCP2） of Escherichia coli was induced with 0.2%L-arabinose at 37 ℃ for4 h, a 36 ku induction expression band of fusion protein was found in the SDS-PAGE pattern. The result indicated that prokaryotic expression ofthe mutantgene was realized underthe drive ofPBADpromoter.