大豆主要过敏原Gly m Bd 28K基因的克隆表达、纯化及多抗血清的制备

CLONING, EXPRESSION AND PURIFICATION OF MAJOR SOYBEAN ALLERGEN Gly m Bd 28K GENE AND PREPARATION OF ITS POLYCLONAL ANTIBODIES

RT-PCR克隆大豆主要过敏原Gly m Bd 28K基因,根据序列设计引物,扩增目的基因的开放阅读框,与MBP载体连接,测序鉴定,转化至大肠杆菌BL21(DE3)中,并用IPTG诱导表达.亲和层析法纯化重组蛋白,SDS-PAGE验证蛋白的纯度,免疫新西兰大白兔,收集多抗血清,用ELISA法测效价.扩增出含Gly m Bd 28K目的基因片段,诱导并获得高浓度和预期相符的表达产物,经层析纯化出Gly m Bd 28K蛋白,免疫新西兰大白兔,获得效价为1∶1.6×106多抗血清.

A major soybean allergen, Gly m Bd 28K gene, was cloned by RT-PCR. Primers were designed according to the sequence; the ORF （open reading frame ） of a target gene was amplified and inserted into vector MBP; and the recombinants was transformed into E. coli BL21 （DE3） after sequencing and induced by IPTG. We purified the recombinant protein through affinity chromatography and verified its purity by SDS-PAGE. New Zealand white rabbits were immunized with recombinant protein and the sera were collected. Titers of polyclonal antibodies were determined by ELISA. The results indicated that an expected fragment of the target gene was amplified and the purified product with high concentration was obtained through affinity chromatography from the recombinant host bacteria containing Gly m Bd 28K gene. The titer of polyclonal antibodies obtained through immunization of New Zealand white rabbits with Gly m Bd 28K was 1∶1.6×10^6. The present study might provide a foundation for the further studies on preparation of monoclonal antibody of Gly m Bd 28K and localization of antigenic binding epitopes.