摘要
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。
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
出处
《中国水产科学》
CAS
CSCD
北大核心
2014年第4期669-675,共7页
Journal of Fishery Sciences of China
基金
国家自然科学基金项目(31301440)
福建省科技重大专项(2011NZ0002-1)
福建省自然科学基金项目(2011J01227)
关键词
鲢
小清蛋白
CDNA克隆
原核表达
silver carp
parvalbumin
cDNA cloning
prokaryotic expression
