Arginine Kinase From Scylla Serrata: Purification, Cloning, Expression And Allergenic Analysis

Food allergen is a common immunity disease. With the development of society and globalization, the rate of food allergen was higher and higher, and became a global attention food safety problem. Food allergen was mainly caused by proteins in food, the symptom contained asthma, urticaria, and even threaten life. Seafood is a kind of serious allergy food, some seafood allergen such as tropomyosin and Parvalbumins has been widely studied by researchers from home and abroad. For arginine kinase is a new allergen or not, its physical feature and allergenicity need futher research.Mud crab (Scylla serrata) with rich nutrition tastes good and sells very well to asia countries, it is an important economic crab globally. Our research focus on purification, antibody preparation, clone, expression and allergenicity of mud crab(Scylla serrata). For our research, arginine kinase was confirmed as a kind of crab first time and the in vitro recombinant arginine kinase provided a technology basis for standardization production of seafood allergen.Purification of native arginine kinase includes two simple steps as ammonium sulfate from 70 % to 90 % saturation and HiTrapQ Sepharose. SDS-PAGE and 2D-PAGE suggested the molecular mass of native arginine kinase is 40kDa and the pI is 6.5, which is similar to Pen m 2 from shrimp.Arginine kinase gene was amplified with sequence analysis showed that cloned DNA fragments had open reading frames of 1021 bp, predicted to encode proteins with 356 amino acid residues. The obtained gene was deposited in GenBank under accession number of GQ: 851626. Sequence alignment revealed that Mud crab arginine kinase share high homology to other crustacean species.Mud crab arginine kinase gene was further recombined with the vector of pGEX-4T-3 and expressed in Escherichia coli BL 21. The expressed protein revealed a band of about 65 kDa on SDS–PAGE, and pI after digestion was 6.3 on 2D-PAGE, which is similar to its native form, suggesting the successful expression of glutathione S-transferase–arginine kinase (GST–AK) fusion protein.The native and recombinant arginine kinase were further confirmed by MALDI-TOF MS. Immunoblotting analysis and GICA using sera from subjects with crustacean allergy confirmed that the native and expressed fusion protein reacted positively with these sera, indicating arginine kinase is a minor allergen of Mud crab. Both native and expressed arginine kinase could be used in seafood allergen diagnosis.