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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 Bxvap2 gene was synthesized by overlap extension PCR gene synthesis method according to the codons preferred by E. coli and ligated into prokaryotic expression vector pET-29b (+). The obtained recombinant was transformed into E. coli BL21 (DE3) and induced with IPTG. SDS-PAGE analysis showed that the target protein was highly expressed in soluble form, accounting for 33.5% of the total bacterial protein. The expressed product was purified by Ni-NTA affinity column after mass spectrometry, the results show that the purified protein is BXVAP2 protein. Polyclonal antibody was prepared by immunizing New Zealand White rabbits with the prepared BXVAP2 protein. The antibody sensitivity was 5.4 ng.mL-1 (titer 1: 1 360 000) by indirect ELISA. To further investigate the tissue localization and function of the protein, Gene in the pathogenesis of the mechanism of action laid the foundation.