, ) ( , ) ( , 011 mmol/ L IPTG. , 16 kd. , Western2blot %, (leptin) kd, ( Zhang et al., 111

Transcription

1 47 (5) : , 2001 A cta Zoologica S inica 3 33 (, ) (, ) (, ) PCR cdna, 5 B am H,3 EcoR, 5 CCC CCG, 459 bp, p GEX22 T B am H EcoR,, 011 mmol/ L IPTG 42 kd, 26 kd p GEX22 T, 16 kd,, Western2blot (ob),, 4911 %, (leptin) 167, 1 16 kd, ( Zhang et al., ) 1994 Rockefeller J. M. Friedman,, p GEX22 T Amer2, leptin sham Pharmacia, B am H EcoR ( Pelleymount et al., T 4 DNA BioLab, 1995 ; Halaas, 1995 ; Campfied et al., 1995) PE, ( E. coli ) DH5 BL21 J M83 J M107 J M109 C600, (Considine et al., 1995 ; Carsson et 112 al., 1997),, leptin Y (Dai et al., 2000), CCC, CCG: 1 5 GGGA TCCGTGCC2 (Ogawa et al., 1995 ; Masuzaki et al., 1995 ) GA TCTGGA GA GTCCA GGA TG GGAA TTCTTCAA G2 cdna (Dai et al., 2000), GCTTCA GCA GCCA GGG3 (Xi et al., 2000), PCR GeneAmp 9600 PCR s, s, s 30, 72 7 min, 4, 110 % , (No ) 33 E2mail : bta. net,, 33,, :

2 PCR PCR Triton2100 STE, r/ min p GEX22 T B am H EcoR DH5, mol/ L p GEMX2POB, PCR (p H 818) BL21 10 mmol/ L ( p H 813 ), 4 2 h., 2 % p GEMX2POB r/ min, 10 mmol/ L Tris Cl (p H BL21, LB, OD ), 3, IPTG, 011 mmol/ L 215 h ( = 3 1 Western2blotting ) p GEMX2POB DH5 211 cd NA BL21 J M83 J M107 J M109 C600, OD IPTG 1 2 PCR 215 h POB BL21 OD , IPTG 215 h,, 8915 % 8419 % ; cdna mmol/ L 8710 % 8219 % p GEMX2OB 212 pgemx2pob BL21 BL21 OD , IPTG, , DNase 37 1 h, 1 %, 3 5,,, r/ min,, Western2Blot, 2 cdna, IPTG p GEMX2 cdna, 1 cdna BL21,, p GEMX2POB BL h (OD 600 : ) ,, SDS2PA GE 2 1 cdna Fig. 1 The comparison of ob cdna sequence encoding mature protein in porcine, human and mouse

3 5 : pgemx2pob BL21 Table 1 Expression of pgemx2pob fusion protein from E. coli BL21 transformant OD 600 (Different OD 600 ) ( %) ( Expression level) pgemx2pob Table 2 Expression of pgemx2pob fusion protein from different E. coli transformant (Different E. coli) DH5 C600 BL21 J M83 J M107 J M109 2 GST2POB BL21 SDS2PAGE Fig. 2 SDS2PAGE analysis for expression of GST2OB fusion protein from transformant 1 8 : p GEMX2POB BL2 LB ( OD ) (Bacterial protein from BL21 transformed with p GEMX2 POB and induced with 011 mmol/ L IPTG in different OD 600 ) 9 : p GEX22 T BL2 (Bacterial proteins from BL21 transformed with p GEX22 T and induced with 011 mmol/ L IPTG) 10 : p GEX22 T BL2 (Bacterial proteins from Bl21 transformed with p GEX22 T and induced without IPTG) 11 : BL21 (Bacterial proteins from BL21 in2 duced with 011 mmol/ L IPTG) 12 : BL21 (Bacterial proteins from BL21 in2 duced without IPTG) M : 213 pgemx2pob p GEMX2POB 2, 3 p GEMX2POB ( %) ( Expression level) BL21 J M83, IPTG) M : DH pgemx2pob SDS2PAGE Fig. 3 SDS2PAGE analysis for expression of pgemx2pob fusion protein from different E. coli transformant 1 6 : p GEMX2POB DH5 C600 BL21 J M83 J M107 J M109 (Bacterial proteins from different E. coli DH5 C600 BL21 J M83 J M107 and J M109 transformed with p GEMX2POB and induced with 011 mmol/ L IPTG at OD ) 7 : p GEX22 T BL21 ( Bacterial proteins from BL21 transformed with p GEX22 T and induced with IPTG) 8 : p GEX22 T BL21 (Bacterial protein from BL21 transformed with p GEX22 T and induced without 3 IPTG pgemx2pob BL21 Table 3 Expression of pgemx2pob fusion protein from E. coli BL21 transformant in different concentration of IPTG IPTG (mmol/ L) (Different concentration of IPTG) ( Expression level %)

4 IPTG pgemx2pob BL21 SDS2PAGE Fig. 4 SDS2PAGE analysis for expression of pgemx2pob fusion protein from E. coli BL21 transformant in different concentration of IPTG 1 10 : IPTG p GEMX2POB BL21 (Bacterial proteins from E. coli BL21 transform with PGEMX2POB and induced with IPTG from 0101 to 10 mmol/ L) 11 : p GEX22 T BL21 (Bacterial proteins from BL21 transformed with p GEX22 T and induced with 011 mmol/ L IPTG) 12 : p GEX22 T BL21 (Bacterial proteins from BL21 transformed with p GEX22 T and induced without IPTG) 13 : BL21 (Bacterial proteins from BL21 induced with 011 mmol/ L IPTG) 14 : BL2 (Bacterial proteins from BL21 in2 duced without IPTG) M : 5 pgemx2pob BL21 Fig. 5 SDS2PAGE analysis for expression of pgemx2pob fusion protein from E. coli BL21 transformant in different inducement time 1 6 : p GEMX2POB BL21 (Bacterial proteins from E. coli BL21 transformed with p GEMX2POB and induced in different time) ck1 : p GEX22 T BL21 (Bacterial proteins from BL21 transformed with p GEX22 T and induced with 011 mmol/ L IPTG) ck2 : p GEX22 T BL21 (Bacterial pro2 teins from BL21 transformed with p GEX22 T and induced without IPTG) M : 6 SDS2PAGE Fig. 6 SDS2PAGE analysis for purif ication protein 1 : p GEMX2POB BL21 (Bacterial proteins from E. coli BL21 transformed with p GEMX2POB and induced with 011 mmol/ L IPTG) 2 3 : ( The includbody of bacterial lysate) 4 : ( The supernate of lysated from expression of p GEMX2POB) 5 : ( Purification of recombinant protein) M : 7 Western2blot Fig. 7 Western2blotting analyzing the expression of pgemx2pob gene in E. coli 1 : p GEX22 T BL21 (Bacterial pro2 teins from BL21 transformed with p GEX22 T and induced with2 out IPTG) 2 : p GEMX2POB BL21 (Bacterial proteins from BL21 transformed with p GEMX2POB and induced without IPTG) 3 4 : p GEMX2POB BL21 (Bacterial pro2 teins from E. coli BL21 transformed with p GEMX2POB and in2 duced with 011 mmol/ L IPTG) MW : 214 IPTG pgemx2pob 215 pgemx2pob BL21 BL21 IPTG p GEMX2POB p GEMX2POB BL21 BL21 3, 4 4, 5,

5 5 : 551 OD , 011 mmol/ L ITGP, h, 2 h 4 pgemx2pob BL21 Table 4 Expression of pgemx2pob fusion protein from E. coli BL21 transformant in different induce time ( Induce time : hr) ( Expression level %) , 7 3 cdna, p GEMX2POB,, p GEMX2POB IPTG,, p GEMX2POB,,,, IPTG 0101 mmol/ L, 216, OD p GEMX2POB IPTG 2710 %, STE,, 4210 %, 5 p GEMX2POB : 4812 %, 9116 % BL21, OD , 1/ 3 IPTG, IPTG 217 Western2Blot 011 mmol/ L, 215 h, 4911 % ( References) Campfield, L. A., F. J. Smith, Y. Guisez, R. Devos and P. Burn 1995 Reconbinant mouse ob protein : evidence for a peripheral signal linking adiposity and central neural network. Science 269 : Carlsson, B., K. Lindell, B, Gabrielsson, C. Karlsson, R. Bjarnason, O. Westphal, U. Karlsson, L. Sjostrom and L. M. Carlsson 1997 Obese (ob) gene defects are rare in human obesity. Obes. Res. 5 : Considine, R. V., E. L. Considine, C. J. Williams, M. R. Nyce, S. A. Magosin, T. L. Bauer, E. L. Rosato, J. Colberg and J. F. Caro 1995 Evidence against either a premature stop codon or the absence of obese gene mrna in human obesity. J. Cli n. Invest. 95 (6) : Dai, R. J., N. Li and C. X. Wu 2000 Clonins and analysis of pig obese cdna gene. J. Genetics 27 (4) : [,, 2000 cdna. 27 (4) : ] Halaas, J. L., K. S. Gajiwala, M. Maffei, S. L. Cohen, S. K. Burley and J. M. Friedman 1995 Weight2reducing effects of the plasma protein encoded by the obese gene. Science 269 : Ogawa, Y., H. Masuzaki, N. Isse, T. Okazaki, K. Mori, M. Shigemoto, N. Satoh, N. Tamura, K. Hosoda and Y. Yoshimasa 1995 Molec2 ular cloning of rat obese cdna and augmented gene expression in genetically obese zucker fatty (fa/ fa) rat. J. Cli n. Invest. 96 (3) : Masuzaki, H., Y. Ogawa, K. Hosoda, T. Kawada, T. Fushiki and K. Nakao 1995 Augmented expression of the obese gene in the adipose tissue from rat fed high2fat diet. Biochem. Biophy. Res. Com m un (1) : Pelleymounter, M. A., M. J. Cullen, M. B. Baker, R. Hecht, D. Winters, T. Boone and F. on body weight regulation in ob/ ob mice. Science 269 : Collins 1995 Effects of the obese gene product Xi, F. G, R. J. Dai, N. Li, G. S. Cao, Y. F. Zhao and C. X. Wu 2000 RFL P polymorphic analysis on obese gene locus in swin. J. Biotech. 8 (2) : [,,,,, 2000 RFL P. 8 (2) : ] Zhang, Y., R. Proenca, M. Maffei, M. Barone, L. Leopold and J. M. homologue. N at ure 372 (6 505) : A gr. Friedman 1994 Positional cloning of the mouse obese gene and its human

6 ( Abstract) HIGH EXPRESSION OF PORCINE OBESE GENE IN E. COL I 3 XI Fen2Gong L I Ning 33 DAI Ru2J uan L IU Zhao2Liang WU Chang2Xin ( N ational L aboratory of A gribiotechnology, Chi na A gricult ural U niversity, Beiji ng , Chi na) ( Instit ute of Microbiology, Academy of Xi njiang A gricult ural Science, U rumqi , Chi na) ( College of A ni mal Science and Technology, Chi na A gricult ural U niversity, Beiji ng , Chi na) A 459 bp fragment of porcine obese cdna, which encodes ob mature protein without signal peptide, was amplified by PCR technique. The DNA fragment has been modified as follow : an EcoR site was included in the 5 end and the codon CCC of it was changed to CCG, which was more frequently used in E. coli, and a B am H site was designed in the 3 end. Then the fragment was cloned into expression vector p GEX22 T and the correct const ruction was confirmed by sequencing. 011 mmol/ L IP T G can induce high level expression of GST2 OB fused protein under the control of P tac promote, which consisted of 26 kd GST and 16 kd leptin. The yield of recombinant protein was over 4911 % of total cellular and expressed as inclusion bodies. The recombinant pro2 tein was purified with the different p H. Western2blotting testing of the porcine expression of p GEMX2POB in E. coli BL21 was positive. Key words Porcine, Obese gene, E. coli, Expression 3 This work was supported by the National Natural Science Foundation of China (No ) 33 Corresponding author