鸡PLA2基因的克隆与原核表达及PLA2蛋白卵黄抗体的制备

Cloning and expression of chicken PLA2 gene and preparation of hen egg yolk immunoglobulin against chicken PLA2

【目的】克隆并原核表达鸡PLA2基因,小量制备鸡PLA2卵黄抗体,为大规模制备PLA2卵黄抗体作为饲料添加剂提供参考。【方法】提取鸡胰脏总RNA,利用RT-PCR方法获得鸡PLA2基因,将其克隆至pGEX-4T-1原核表达载体中,构建表达载体pGEX-4T-1-PLA2,转入大肠杆菌BL21(DE3),IPTG诱导表达GST-PLA2融合蛋白。将GST-PLA2融合蛋白免疫青年蛋鸡,每次0.3mg/只,共免疫4次,收集鸡蛋,提取卵黄抗体,采用West-ern Blotting检测抗体的特异性。【结果】成功克隆出了鸡PLA2基因,构建了pGEX-4T-1-PLA2原核表达载体,并诱导表达了分子质量为44.59ku的GST-PLA2融合蛋白。GST-PLA2融合蛋白免疫蛋鸡后获得了卵黄抗体,经West-ern Blotting检查,制备的卵黄抗体具有很好的特异性。【结论】构建了表达鸡PLA2基因的表达系统,并成功的制备了抗鸡PLA2的卵黄抗体。

【Objective】 The research prepared the high titer and specific hen egg yolk immunoglobulin（IgY） against chicken PLA2.【Method】 Gallus gallus phospholipase A2 gene was amplified by RT-PCR,inserted into vector pGEX-4T-1,and expressed in recombinant E.coli strain BL21（DE3）.The GST-PLA2 fusion protein was isolated from the bacteria cells and injected into hens at 300 μg dose four times.Western blotting was performed to detect antibodies in egg yolk.【Result】 The chicken PLA2 gene was cloned and inserted into expression vector pGEX-4T-1.The fusion protein GST-PLA2 was expressed in E.coli,and relative molecular mass of fusion protein was 44.59 ku.After injecting fusion protein into hen,we collected eggs and extracted IgY.Western blotting was used to show that the IgY was specific.【Conclusion】 The expression system pGEX-4T-1-PLA2 could express recombinant protein GST-PLA2,and the prepared IgY had a good immunized specificity.