- (SDS-PAGE) (Western blot), TQ93

Transcription

1 42 6 Vol.42, No OCEANOLOGIA ET LIMNOLOGIA SINICA Nov., 2011 * ( ) - (SDS-PAGE) (Western blot), (Tropomyosin, TM) (Parvalbumin, PV), / ph (SGF), TM, TM, / 10/1, ; ph 5, TM PV (0.33U / g, ph 3.5) SGF, TM, PV ;,,,,, SDS-PAGE, Western blot TQ93, (, 2009; Lopata et al, 2009),,,, (tropomyosin, TM) (Leung et al, 1998) (parvalbumin, PV) (Lopata et al, 2009) (Aas et al, 1967) (Lindstrøm et al, 1996) (Bugajska et al, 2000) (Hamada et al, 2003) PV IgE (Wickham et al, 2009),,, ph,, (Scylla serrata) (Eriocheir sinensis) (Penaeus monodon) (Penaeus vannamei) (Carassius auratus cuvieri) (Hypophthalmichthys molitrix) SDS-PAGE GE Fermentas, Western blot New England BioLab, DEAE-Sepharose Pharmacia, IgG-HRP Pierce, Sigma,, TM PV (Liang et al, 2008), *, , ;, 2010J06012 ;, NCETFJ-2007 ;, 2010A005,,, gmliu@jmu.edu.cn : , :

2 (Kinematica, ), (Beckman, ), (Vilber Lourmat, ), (Bio-Rad, ), (Memmert, ), (ND-1000, ) Cao (2004) TM TM Liang (2008), 0 4 : 20g, 10 A ( 50mmol/L KCl 2mmol/L NaHCO 3 ), 5000g 5min, 10 A,,, 4 10,, 10 B (20mmol/L Tris-HCl, ph 7.5, 1mol/L KCl 10mmol/L - ), 20000g 20min,, 3mol/L HCl ph 4.5, 20000g 20min, B 1mol/L NaOH ph % 60% B, ph 4.5,, 20mmol/L Tris-HCl (ph 7.5), min, 20000g 20min, TM, PV 10 (V/W) 20mmol/L (ph 7.0) ( 30s, 1min), 8000g 10min, (Lindstrøm et al, 1996) PV 1.2.3, min;, 15000g 15min, 70% 100%, 20mmol/L Tris-HCl (ph 7.5);, 20mmol/L Tris-HCl (ph 7.5) 24h, 3 ; 10000g 10min, DEAE- Sepharose, ( 1ml/min), 0, 0 0.5mol/L NaCl, 3ml/, A 280 A 220 (, 2009) (Simulated gastric fluid, SGF) SGF (US Pharmacopoeia, 1995), 8185U/mg, 1L NaCl 2g, HCl ph 1.2 SGF Thomas (2004), SGF, 37 15min,, 1ml, PV 1 : 50 (W/W), min 100 l 1.5ml, 30 l 200mmol/L Na 2 CO 3, 0min SGF Na 2 CO 3, ; / SGF, 100 l, TM 60 g/ml, 1/20 1/10 1/5 1/1 5/1 10/1 (W/W), 1.5ml : SGF 37,,, 37 30min ( PV 2min), 30 l 200mmol/L Na 2 CO 3,, ph SGF ph SGF, 100 l, TM 60 g/ml, 1/23 (W/W), 1.5ml : ph ( ) 37,,, 37 15min 30 l 200mmol/L Na 2 CO 3, SDS-PAGE Western blot SDS- PAGE (, 1999), R-250 ; Western blot Towbin (1979), (3,3 -diaminobenzidine, DAB)

3 6 : ph, 3 TM ph ,, TM 2.1 / SGF TM, ph, SGF / ph 5.0,, TM TM, 1, ( 3A) TM /TM TM ph , ph, ph 5.0, TM / 1/10, 30min ( 3B) TM, 34kDa 20kDa TM 15min, / 5/1,, ph 3.5 TM SDS-PAGE, 38kDa, (pepsin)( pep, ) TM 34kDa 20kDa ( 1A) TM, TM TM / 1/5, 30min, 1/1, TM, 10/1,, TM 34 38kDa 20kDa ( 1B) TM TM / 1/1, TM, 34kDa 22kDa, 1 / TM SGF, Fig.1 SGF digestion at different ratios of pepsin / crustaceans TM, TM (20kDa M: ; A: ; B: ; C: ; D: 18kDa) / 10/1 1 6 pepsin/tm, 1 1/20; 2 1/10; 3 1/5; 4 ( 1C 1D) 1/1; 5 5/1; 6 10/1; / SGF PV PV,, 2min 2A PV / 1/20, PV, 4kDa 2kDa, 1/1 SDS-PAGE PV, / 1/20, 2 / PV SGF ( 2B) Fig.2 SGF digestion at different ratios of pepsin / fish PV 2.3 ph SGF TM M: ; A: ; B: 1 6 pepsin/tm, 1 1/20; SGF ph (ph ), 2 1/10; 3 1/5; 4 1/1; 5 5/1; 6 10/1; 7

4 ph TM SGF Fig.3 SGF digestion of crustacean TM at different ph M: ; A: ; B: ; C: ; D: 1: ph 1.2; 2: ph 1.5; 3: ph 2.0; 4: ph 2.5; 5: ph 3.0; 6: ph 3.5; 7: ph 4.0; 8: ph 4.5; 9: ph 5.0 PV ph SDS-PAGE, ph 5.0, PV ( 4B) 2.5 SGF SGF, (0.33U / g, ph 3.5) SGF, IgE 5A, TM 34kDa 37kDa TM IgE, 28kDa 22kDa IgE, IgE,, TM( 5B) TM( 5C) TM, TM,, ( 5B, Lane 3) TM, 36kDa 34kDa, 22kDa IgE ( 5D), IgE 4 ph PV SGF Fig.4 SGF digestion of fish PV at different ph M: ; A: ; B: 1: ph 1.2; 2: ph 1.5; 3: ph 2.0; 4: ph 2.5; 5: ph 3.0; 6: ph 3.5; 7: ph 4.0; 8: ph 4.5; 9: ph 5.0 ( 3C 3D) 1h, ph 3.5,, ph, TM, ph 5.0, ( ) 2.4 ph SGF PV PV ph 4, PV ph , ph 4.5 PV,, ph 5.0, PV ( 4A) 3,,, (Fu et al, 2002) Wickham (2009),, SGF, /,,, TM PV,,, TM PV TM / 5/1, TM 10/1,,

5 6 : SGF TM Fig.5 Detection of the products of TM digested by SGF with the serum from crustacean-allergic humans by IgE-Immunoblotting M: ; A: ; B: ; C: ; D: 1: ; 2: ; 3: ; 4: 60min, ph, ph,, ph (Prichard et al, 1985) ph SGF, ph (ph 5),,, ph, Untersmayr (2005) ph SGF, ph , ph>2.75, PV, ph ;, ph, ( ), ph,,,, /, /,,, Moreno(2007),, 1 : 7500 (W/W),,, (0.33U / g, ph 3.5),, (, 2009),,,, , 30(3): ,,, (Scylla serrata)., 40(1): 62 67,,, , 9(4): F M, R, R E, 1999.,, ( ). :, Aas K, Jebsen J W, Studies of hypersensitivity to fish: Partial purification and crystallization of a major allergenic component of cod. Int Arch Allergy Appl Immunol, 32(1): 1 20 Bugajska S A, Grote M, Vangelista L et al, Purification, biochemical, and immunological characterisation of a major food allergen: different immunoglobulin E recognition of the apo- and calcium-bound forms of carp parvalbumin. Gut, 46(5): Cao M J, Shao W, Li Y et al, Identification of a myofibril-bound serine proteinase in the skeletal muscle of silver carp. J Food Biochem, 28: Fu T J, Abbott U R, Hatzos C, Digestibility of food allergens and nonallergenic proteins in simulated gastric fluid and simulated intestinal fluid-a comparative study. J Agr

6 Food Chem, 50(24): Hamada Y, Tanaka H, Ishizaki S et al, Purification, reactivity with IgE and cdna cloning of parvalbumin as the major allergen of mackerels. Food Chem Toxicol, 41(8): Leung P S, Chen Y C, Gershwin M E et al, Identification and molecular characterization of Charybdis feriatus tropomyosin, the major crab allergen. J Allergy Clin Immunol, 102(5): Liang Y L, Cao M J, Su W J et al, Identification and characterization of the major allergen of Chinese mitten crab (Eriocheir sinensis). Food Chem, 111(4): Lindstrøm C D, van Dô T, Hordvik I et al, Cloning of two distinct cdnas encoding parvalbumin, the major allergen of Atlantic salmon (Salmo salar). Scand J Immunol, 44(4): Lopata A L, Lehrer S B, New insights into seafood allergy. Curr Opin Allergy Clin Immunol, 9(3): Moreno F J, Gastrointestinal digestion of food allergens: effect on their allergenicity. Biomed Pharmacother, 61(1): Prichard P J, Yeomans N D, Mihaly G W et al, Omeprazole: a study of its inhibition of gastric ph and oral pharmacokinetics after morning or evening dosage. Gastroenterology, 88: Thomas K, Aalbers M, Bannon G A et al, A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins. Regul Toxicol Pharm: RTP, 39(2): Towbin H, Staehelin T, Gordon J, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. P Natl Acad Sci USA, 76(9): Untersmayr E, Poulsen L K, Platzer M H et al, The effects of gastric digestion on codfish allergenicity. J Allergy Clin Immunol, 115(2): US Pharmacopoeia, The National Formulary. Simulated Gastric Fluid and Simulated Intestinal Fluid, TS. In: The United States Pharmacopeia 23, The National Formulary 18, The United States Pharmacopeial Convention, Inc.: Rockville, MD, Wickham M, Faulks R, Mills C, In vitro digestion methods for assessing the effect of food structure on allergen breakdown. Mol Nutr Food Res, 53(8): OPTIMIZATION OF SIMULATED GASTRIC FLUID DIGESTION OF MAJOR ALLERGENS FROM AQUATIC PRODUCTS LIU Guang-Ming, HUANG Yuan-Yuan, CAI Qiu-Feng, WENG Ling, SU Wen-Jin, CAO Min-Jie (College of Biological Engineering, Key Laboratory of Science and Technology for Aquaculture and Food Safety in Fujian Province, Jimei University, Xiamen, ) Abstract Stability in simulated gastric fluid (SGF) is regarded as an important parameter in estimation of food allergenicity. The digestive conditions of crustacean allergens (tropomyosin, TM), fish allergens (parvalbumin, PV) and nonallergenic proteins from muscles of mud crab (Scylla serrata), Chinese mitten crab (Eriocheir sinensis), grass prawn (Penaeus monodon), whiteleg shrimp (Penaeus vannamei), Crucian carp (Carassius auratus cuvieri) and Silver carp (Hypophthalmichthys molitrix) in SGF were compared. Porcine pepsin was used to simulate digestive proteinases from human. Stabilities of allergens were studied with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blot at different pepsin/protein ratios and ph. It was found that the apparent susceptibility of allergens to proteolysis was dependent on the ratio of pepsin to allergen. TM was of relatively higher stability; even at pepsin/allergen = 10/1, some stable degradation fragments still existed. Under optimized conditions (0.33U pepsin/ g protein, ph 3.5), crustacean allergen TM was relatively resistant to pepsin, but fish allergen PV was rapidly degraded within a short period of time. Further study by Western-blot using sera from crustacean-allergic patients indicated that allergenicity of TM was partially decreased, but the products of TM digested by pepsin still had a little IgE-binding capacity. Key words Aquatic product allergens, Simulated gastric fluid digestion, Tropomyosin, Parvalbumin, SDS-PAGE, Western-blot