CAJ | 학술논문

以鲢(Hypophthalmichthys molitrix)肌肉cDNA为模板,利用小清蛋白特异性引物进行PCR扩增,克隆得到β小清蛋白两种不同亚型,即Ⅰ型、Ⅱ型编码区基因。将目的基因片段连接到 pET28a (+)表达载体,并在大肠杆菌[E.coli BL21(DE3)]中诱导表达。结果表明,经诱导的小清蛋白重组质粒菌株有特异的蛋白表达。SDS-PAGE分析显示,目的蛋白的分子量约为13 kD,与预期大小一致。菌体超声破碎后发现2种亚型的小清蛋白均为可溶表达。利用Ni2+亲和层析柱对重组蛋白进行纯化,得到高纯度的重组小清蛋白PVⅠ和PVⅡ。经Western Blot 鉴定,重组小清蛋白 PVⅠ和 PVⅡ均能与抗鲢小清蛋白单克隆抗体反应。本研究为进一步分析小清蛋白的结构与致敏性的关系提供了重要的基础。
이련(Hypophthalmichthys molitrix)기육cDNA위모판,이용소청단백특이성인물진행PCR확증,극륭득도β소청단백량충불동아형,즉Ⅰ형、Ⅱ형편마구기인。장목적기인편단련접도 pET28a (+)표체재체,병재대장간균[E.coli BL21(DE3)]중유도표체。결과표명,경유도적소청단백중조질립균주유특이적단백표체。SDS-PAGE분석현시,목적단백적분자량약위13 kD,여예기대소일치。균체초성파쇄후발현2충아형적소청단백균위가용표체。이용Ni2+친화층석주대중조단백진행순화,득도고순도적중조소청단백PVⅠ화PVⅡ。경Western Blot 감정,중조소청단백 PVⅠ화 PVⅡ균능여항련소청단백단극륭항체반응。본연구위진일보분석소청단백적결구여치민성적관계제공료중요적기출。
Parvalbumin (PV) is a major fish allergen that is involved in IgE-mediated food hypersensitivity. Sensitized individuals can develop some clinical symptoms including urticaria, angioedema, asthma, and even fatal anaphylaxis after ingestion of trace quantitiesof fish. As the largest producer and consumer of fresh water fish in the world, a high number of Chinese people suffer from allergies associated with consumption of fresh water fish. Despite this, little is known about the allergens in freshwater fish products that are available in China. We extracted total RNA from silver carp (Hypophthalmichthys molitrix) skeletal muscle, and synthesized first-strand cDNA by reverse transcriptase with an oligo (dT)18 primer. Some specific primers were designed based on the sequences of silver carp PV mRNA (GenBank nos. FJ216937 and FJ216938). Using these primers and the synthesized cDNA, two PV isoform genes (PVI and PVII) were cloned. The full-length coding region of both PVs was 330 bp, which encoded a protein of 109 amino acid resi-dues. The PCR products were cloned into a pMD18-T vector for sequencing. Both the positive plasmid and the plasmid pET28a were digested by Nde I and BamH I.The target genes were subcloned into pET28a for expression in [E.coli BL21 (DE3)] by 1 mmol/LIPTG induction at 37℃ for 4 h. The two target protein bands were~13 kD, which was con-sistent with the predicted size. SDS-PAGE analysis indicated that the recombinant PVI and PVII both existed in the soluble fraction of the proteins. The recombinant PVI and PVII were further purified by Ni-NTA agarose affinity chromatography, and the target proteins were eluted by 100 mmol/L imidazol. Both purified proteins yielded a single band on SDS-PAGE. Similar to the native PV, the recombinant proteins reacted strongly with anti-silver carp PV monoclonal antibody in the western blot analysis, suggesting that the recombinant PVI and PVII have strong IgG binding activity. Thus, we obtained two isoforms of purified and biological active recombinant PV. The interaction force of intermolecularcross linkage may have a close relationship with the stability and allergenicity of PV. However, there are few reports concerning the relationship between the structure and allergenicity of PV. Therefore, further research will be carried out to determine the relationship between the structure and allergenicity of freshwater fish PV and the impact of thermal processing on the stability and allergenicity of different PVs.