松材线虫类毒过敏原蛋白的原核表达及多克隆抗体的制备

Prokaryotic expression of venom allergen-like protein of Bursaphelenchus xylophilus and preparation of its polyclonal antibody

利用重叠延伸PCR基因合成法,按大肠杆菌偏爱密码子,合成松材线虫类毒过敏原蛋白基因(Bxvap2),将其连接到原核表达载体pET-29b(+)上。获得的重组子转化大肠杆菌BL21(DE3)后,用IPTG进行诱导表达。SDS-PAGE分析表明,目的蛋白以可溶形式高效表达,占细菌总蛋白的33.5%。将表达产物经Ni-NTA亲和柱纯化后进行质谱分析,结果显示纯化的蛋白为BXVAP2蛋白。用制备的BXVAP2蛋白免疫新西兰白兔制备多克隆抗体,间接ELISA测得抗体灵敏度5.4 ng.mL-1(效价为1∶1 360 000),为进一步研究该蛋白的组织定位及功能,明确该基因在致病过程中的作用机理奠定了基础。

The venom allergen-like protein gene of Bursaphelenchus xylophilus （Bxvap2） was optimized using the overlap extension PCR technique for expression in Escherichia coli. It was subsequently cloned into pET-29b （ ＋ ） vector and the fusion protein was highly expressed in E. coli BL21 （DE3） in soluble form which covered 33.5% of total cellular proteins. After purification through affinity Ni-NTA column, the fusion protein was fur- ther identified as BXVAP2 by mass spectrometry （MS）. White rabbits were immunized with BXVAP2 protein. The sensitivity of polyclonal antibody was 5.4 ng·mL-1（ the titer was l： 1 360 000） tested by indirect ELISA. This result will be an important basis for tissue localization and functional study of BXVAP2 protein of B. xylo- philus.