大豆球蛋白G5A3亚基加工破坏表位的初步定位

Preliminary Localization of Processing-destroyed Epitope of G5A3 Subunit of Glycinin

大豆球蛋白是重要的大豆过敏原,为探究大豆球蛋白G5亚基中A3酸性多肽链的破坏表位,对A3亚基的基因进行重叠分段并设计合成引物、构建克隆载体后进行双酶切,与T7 vector arms连接构建重组噬菌体并表达目的蛋白;制作热处理及超高压联合热处理破坏抗原表位的特异性抗体,用间接竞争ELISA法检测不同肽段的抗原性高低及加工破坏表位。结果表明,A3链与其3个分段基因的长度分别为:960、420、450、450 bp,与预期片段大小相符;经间接竞争ELISA法检测后,片段1抗原性最高,通过阴性对照,超高压联合热处理对该亚基的破坏效果优于单独热处理试验,其中对片段1的破坏效果最好。因此,超高压联合热处理的方法对蛋白抗原性破坏效果显著,且对片段1破坏最强。

Glycinin is an important soybean allergen.In order to explore the destructive epitopes of the A3 acidic polypeptide chain in the G5 subunit of glycinin,the gene of A3 subunit was overlapped and segmented,then primers were designed and synthesized,and the cloning vector was constructed for double digests.At the same time,T7 vector arms were reconnected to construct recombinant phage and express the target protein.Heat treatment and ultra-high pressure combined heat treatment(UHPCH)were used to destroy the specific antibody of the epitope.The indirect competitive ELISA(icELISA)method was used to detect the antigenicity of different peptides and the processing and destruction of epitopes.The results indicated that the lengths of the A3 subunit and its three segmented genes were:960,420,450,450 bp,consistent with the expected fragment size,after detection by indirect competitive ELISA,fragment I had the highest antigenicity,through the negative control,the UHPCT treatment had better destructive effects on the sensitized subunits than the heat treatment alone,among them,the destruction effect on segment I was the best.In summary,the method of UHPCT treatment had remarkable effect on protein antigenic destruction,and it had the strongest damage to fragment one.