山东寿光地区番茄黄化曲叶病毒外壳蛋白基因克隆及其在大肠杆菌中的表达

Cloning of Coat Protein Gene of Tomato Yellow Leaf Curl Virus Strain from Shouguang Region of Shandong Province and Its Expression in E. coli BL21 (DE3)

利用双生病毒简并引物PA/PB,对山东寿光地区保护地疑似感染番茄黄化曲叶病毒(TYLCV)的番茄植株进行PCR检测,结果证明番茄病叶由TYLCV侵染所致。以提取的病叶总DNA为模板,扩增得到长约770bp的TYLCV-CP基因片段,克隆至pEASY-T1Simple载体,然后用XhoI和EcoRI将其切下并插入到pET-32a表达载体中,构建了寿光地区TYLCV分离物的外壳蛋白基因原核表达载体,转入大肠杆菌BL21(DE3),经IPTG(异丙基-β-D-硫代吡喃半乳糖苷)诱导获得了50kD重组蛋白,Ni2+-NTA亲和层析纯化和Western印迹分析证明目的蛋白为TYLCV外壳蛋白,且具有良好的抗原活性。

The PCR test was carried out on tomato(Lycopersicon esculentum Mill.)leaves which were infected by TYLCV in the greenhouse in Shouguang region of Shandong Province using the prime PA/PB.The results showed the yellow leaf curl disease found in Shouguang was caused by TYLCV.A DNA fragment(770 bp in length)encoding TYLCV coat protein(TYLCV-CP)was amplified and inserted into pEASY-T1 Simple vector.After double digestion with Xho I and EcoR I,the fragment was inserted into vector pET-32a.A 50 kD recombinant protein of TYLCV-CP from isolate of Shouguang highly expressed by IPTG in E.coli BL21(DE3)showed a high antigenic activity.