中国水产科学Issue(4)：669-675,7.DOI:10.3724/SP.J.1118.2014.00669
鲢小清蛋白的cDNA克隆及在大肠杆菌中的原核表达
Cloning and prokaryotic expression of parvalbumin from silver carp (Hypophthalmichthy molitrix) skeletal muscle
摘要
Abstract
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.关键词
鲢/小清蛋白/cDNA克隆/原核表达Key words
silver carp/parvalbumin/cDNA cloning/prokaryotic expression分类
农业科技引用本文复制引用
王慈,曹敏杰,郑晓江,詹春兰,刘光明,蔡秋凤..鲢小清蛋白的cDNA克隆及在大肠杆菌中的原核表达[J].中国水产科学,2014,(4):669-675,7.基金项目
国家自然科学基金项目(31301440) （31301440）
福建省科技重大专项(2011NZ0002-1) （2011NZ0002-1）
福建省自然科学基金项目(2011J01227) （2011J01227）
