Absorption of cholecystokinin ( CCK) yolk antibody from the duodenum of the rat 3

50 (3) :375-379 2004 A cta Zoologica S inica 3 33 210095 CCK SDS2PA GE EL ISA Western blot CCK SD CCK 14 SD 150 mg/ ml CCK (2 ml/ ) 2 3 4 h SDS2PA GE CCK 200 kd EL ISA 2 3 h CCK 1 128 Western blot 2 3 h CCK SD [ 50 (3) : 375-379 2004 ] CCK Absorption of cholecystokinin ( CCK) yolk antibody from the duodenum of the rat 3 ZHAN G Xiao2Ping HUAN G Hao2Long ZHU Zu2Kang CHEN Jie ZHAO Ru2Qian 33 Key Laboratory of Animal Physiology and Biochemistry Ministry of Agriculture Nanjing Agricultural University Nanjing 210095 China Abstract To clarify whether CCK yolk antibody can be absorbed from duodenum in rats SDS2PA GE electrophoresis EL ISA and Western blot analysis were employed to detect the immunoreactivity and the chemical form of CCK yolk anti2 body in duodenal venous blood after gastric infusion of CCK yolk antibody. Fourteen adult male SD rats were divided at random into treatment and control groups. Two ml of CCK yolk antibody (150 mg/ ml) or physiological solution (019 % NaCl) was administered intragastrically to rats in treatment and control groups respectively. Duodenal venous blood was collected from duodenal venous catheters at the 2nd 3rd and 4th hour after infusion. SDS2PA GE electrophoresis showed that the molecular weight of CCK yolk antibody was 200 kd. CCK yolk antibody was detected in the duodenal venous plasma of animals in the treatment group with EL ISA at the 2nd and 3rd hour after administration with the titer of 1 128. Western blot analysis confirmed the finding of EL ISA that intact CCK yolk antibody existed in duodenal venous plas2 ma of treatment group. These results suggest that CCK yolk antibody can be absorbed or partly absorbed from the duode2 num and play its role in systematic circulation [ Acta Zoologica Sinica 50 (3) : 375-379 2004 ]. Key words Rat CCK Yolk antibody Absorption Ig Y ( Shin et al. 2002) ; ( Enterotoxin2 (Yolk immunoglobulin Ig Y) Ig Y genic E. coli ETEC) Ig Y ETEC ( Yokoyama and Hashi 1997) ; Ig Y 200 mg/ kg ( Pseudomonas 3 aeruginosa) Ig Y ( Carlande et al. 2002) ; ( Helicobacter pylori Hp ) (Stadelman 1999) ( Cholecystokinin CCK) 2003211224 2004201218 3 [ This research was funded by Hongkong Walcom Bio2Chemicals Industrial Limited] 33 (Corresponding author). ν 2004 Acta Zoologica Sinica E2mail : lapb @njau1edu1cn

376 EL ISA Western blot SD CCK CCK ( 2001) Ig Y CCK (Danny 1998) CCK CCK 111 CCK CCK CCK EL ISA CCK 1 12 800 112 (Cook et al. Ξ ) 70 96 ( Costar) 550 mg/ kg CCK ( B IO2RAD) ( Eppen2 2 % (Adalsteinsson ΞΞ ) dorf) D YY28B D YCZ223A Ig Y D YCP240C ( ) Ig G2HRP ( Sout hern Biotech2nology Associates Ig Y Inc. ) ( Hybond) High Pro2 tein Mark ( ) Ig Y CCK 113 SD? Ig Y SD ( ) 4 350 5 g 14? 150 mg/ ml CCK (2 ml/ ) 12 h 1 h 20 mg/ ml 2 ml/ kg ( Pacha 2002) Na + ( 100 mg/ ml) PE2100 ( 2 3 4 h SD Peptide t ransporters PEPT) 018 ml ( Shen et al. 2001) - 20 114 IgY 6 h Akita and Nakai (1993) Ig Y ( Pacha 2002) 12 CCK p H IGF21 ( Philipps et al. 2002) 512 10 4 6 h Ig Y? 4 000 r/ min 20 min 50 % Ig Y 4 2 h 4 000 r/ min 20 min 1 ml PBS (p H 714 SD SDS2PA GE 1 50 012 mol/ L) 30 % Ξ ΞΞ Cook Mark E Miller Cheryl C Pimentel J ulio L 1998. CCK antibodies used to improve feed efficiency. United States Patent 5 827 517. Adalsteinsson O Fitzpatrick ME Sandra G Hunchar J G Jeffery G 2000. Method of increasing muscle protein and reducing fat in animals. United States Patent 6 086 878.

3 : 377 3 PBS 24 h - 20 Bradford Ig Y Ig Y SDS2PA GE 115 SDS2PAGE SDS2PA GE Ig Y 3 % 8 % 100 V 3 h Ig Y 116 EL ISA CCK2 (p H 916 0101 mol/ L ) 1 100 96 100 l/ 4 ( p H 714 0101 mol/ L PBS2Tween20) 3 1 % 100 l/ 37 1 h 3 Ig Y 0101 mol/ L PBS 3 1 IgY SDS2PAGE 100 l 37 1 h 3 Ig G2HRP 0101 mol/ L PBS (1 4 000) 100 l / 37 1 h 3 (2 mg/ ml 011 % H 2 O 2 ) 100 l/ 15 min 2 mol/ L H 2 SO 4 OD 490 117 Western blot 10 20 5 % SDS2PA GE Ig Y 1 h TBS 3 10 ml (016 mg/ ml 3 3 2 l EL ISA SD 2 3 h CCK 011 % H 2 O 2 ) SDS2PA GE Ig Y Western blot 118 SD 2 3 h SPSS 1110 for windows Ig Y (Mean S E) 3 h Kodak 1D 2 h 4 h ( 3 ) SD 2 3 h 2 211 IgY Bradford Ig Y 221624 mg/ ml 212 SDS2PAGE CCK Ig Y 200 kd ( 1) Ig Y 8318 % Mr : 1 : 2 : 3 : Fig11 SDS2PAGE patterns for IgY purif ied from egg yolk antibody Mr : Protein molecular weight marker. 1 : IgY obtained after salting out. 2 : Water2soluble fraction. 3 : Egg yolk. 213 EL ISA 1 128 100 V 150 min 28 ma 3 h 2 h 4 h 3 h 5 % CCK ( 2) 1 h Ig G2HRP TBS (p H 715 150 SD 2 3 h mmol/ L NaCl 50 mmol/ L Tris) 1 6 000 CCK 214 Western blot 3 ( ) SD Ig Y 3 Ig Y

78 3 50 2 EL ISA SD CCK Fig12 Titer of CCK yolk antibody in duodenal venous plasma of SD rat detected with EL ISA after gastric administration Fig. 3 SDS2PAGE patterns and Western blot analysis for duodenal venous plasma of SD rat after gastric administra2 tion Mr : Protein molecular weight marker. 1 : Ig Y. 2 : Control plasma. 3 : 4th h plasma of treatment. 4 : 3rd h plasma of treatment. 5 : 2nd h plasma of treatment. Left : SDS2PAGE. blot. Right : Western Ig G Ig Y Ig Y Ig G Fc A ( RF) ( Tini et al. 2002) Ig Y Ig Y (63 15 min) ; 37 p H 310 1 h ; 37 p H 719 215 h ( 2001) Ig Y (p H 410) 10 h 63 % ; 8 h 39 % Ig Y 3 SDS2PAGE Western blot SD ( Hatta et al. 1993) Ig Y Mr : 1 : 2 : 3 : 4 h 4 : 3 h 5 : 2 h : SDS2PAGE : Western blot Ig Y ( Shin et al. 2002) Ig Y PEP T PEPT PEPT 1 ( ) PEPT 2 ( ) PEPT 1

3 : 379 PEPT 1 mrna Western blot CCK 4 d 28 d PEPT1 mrna ( Shen et al. 2001) Ig Y ( Shin et al. 2002) CCK CCK (Danny 1998 ; Cook et al. 1998) CCK CCK CCK SD EL ISA Western blot SD 2 3 h Ig Y 3 h Ig Y 2 h 4 h Ig Y SD 2 h Ig Y 3 h CCK Ig Y CCK SD CCK : (1) Ig Y ; (2) (Shen et al. 2001) EL ISA SD ( References) Akita EM Nakai S 1993. Comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immu2 nized with an enterotoxigenic E. coli strain. J. Immunol. Meth2 ods 160 : 207-214. Carlander D Kollberg H Larsson A 2002. Retention of specific yolk IgY in the human oral cavity. Bio. Drugs 16 (6) : 433-437. Chang S Zhang YP Xia XG 20011Stability and activities of im2 munoglobulin Y against Candida albicans IgY in vit ro. Acta A2 cad. Med. Militaris Tertlae 23 ( 12) : 1 390-1 392 ( In Chi2 nese). Danny MH 1998. Studies show benefits of cholecystokinin antibodies. Feedstuffs 2 : 12-15. Hatta H Tsuda K Akachi S Kim M Yamamoto T Ebina T 1993. Oral passive immunization effect of anti2human rotavirus IgY and its behavior against proteolytic enzymes. Biosci. Biotechnol. Biochem. 57 : 1 077-1 081. Pacha J 2002. Development of intestinal transport function in mam2 mals. Physiol. Rev. 80 : 1 633-1 667. Philipps AF Kling PJ Grilli J G Dvorak B 2002. Intestinal transport of insulin2like growth factor21 in the suckling rat. J. Pediatr. Gas2 troenterol. Nutr. 35 (4) : 539-544. Shen H Smith DE Brosius FC 2001. Developmental expression of PEPT1 and PEPT2 in rat small intestine colon and kidney. Pedi2 atr. Res. 49 (6) : 789-795. Shin J H Yang M Nam SW Kim J T Myung NH Bang WG Roe IH 2002. Use of egg yolk2derived immunoglobulin as an alterna2 tive to antibiotic treatment for control of Helicobacter pylori infec2 tion. Clin. Diagn. Lab. Immunol. 9 (5) : 1 061-1 066. Stadelman WJ 1999. The incredibly functional egg. Poult. Sci. 78 (6) : 807-811. Tang CW Han J 2001. The physiological and pathologic physiological effect of CCK regulated human pancreas. Chin. J. Dig. 21 (1) : 43-45 ( In Chinese). Tini M Jewell UR Camenisch G Chilov D Gassmann M 2002. Generation and application of chicken egg2yolk antibodies. Comp Biochem. Physiol. A. Mol. Integr. Physiol. 131 ( 3) : 569-574. Yokoyama H Hashi T 1997. Effect of oral egg antibody in experimen2 tal F18 + E. coil infection in weaned pigs. J. Vet. Med. Sci. 59 (10) : 917-921. 2001. Ig Y. 23 (12) : 1 390-1 392. 2001.. 21 : 43-45.