LfcinB. Expression and Iden tif ica tion of Lfc inb Gene in P ich ia pastoris

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Ῥαχήλ Χλόη Βασιλειάδης
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1 265 ( LfcinB) 3 ( ) : ( Pichia pastors) ( bovine lactoferricin, Lf2 cinb), LfcinB pp IC9K, pp IC9K2LfcinB Sal, SMD1168 G418,, PCR LfcinB LfcinB, : (LfcinB),,, : Q782, Q784, Q786 : A : (2007) Expression and Iden tif ica tion of Lfc inb Gene in P ich ia pastoris YI Jun2Bo HUANG De2Xin L IL ing2yun L IN Feng (N ational Engineering Center forb iotechnology, Shenzhen ) Abstract: In present study, bovine Lactoferricin was first secretly exp ressed in Pichia pastors yeast expression system1the synthesized Lf2 cinb gene fragmentwas cloned into expression vector ppic9k, and then obtained recombinant p lasm id, designated as ppic9k2lfcinb, was linearized and transformed into Pichia pastors strains SMD1168 by electroporation1the transformantswere screened with Geneticin and multi2 p ly2copy colonies were harvested, in which LfcinB gene was verified to inserted into yeast chromosome stably1the positive recombinant Pichia strains were induced with methanol to exp resslfcinb in culture supernatant1 Itπs exp ressive products has high activity of killing bacteri2 a1w e concluded that LfcinB gene was cloned and integrated into yeast chromosomes, and obtained exp ression pep tide was tested to have high antibacterial activity1 Key words: Bovine lactoferricin, Gene, Expression vector, Pichia pastors yeast expression system ( antim icrobial pep tides),,,,, 1975 Boman (Hyatophoracecrop ia), cecrop in,, 80, ; 90,,,, [ 1 ], Groves,,,,, [ 2 ],, LF, Horoa2 ki [ 3 ] : LF ph,, LF,,,, LF LfcinB [ 4 ], 25, ( lactoferrin, LF ) 1960 : F K C R R W Q W R M K K L G A P 3 Tel: : , :

2 (2) S I T C V R R A F [ 5 ], LfcinB W, [ 6 ] LfcinB 400 [ 7 ],,,,,, LfcinB ( G + ) ( G - ), [ 8, 9 ],,, LfcinB [ 10 ],,,,,, : SMD1168 ( his4, pep4) DH5 pp IC9K S1au reus [ CMCC26003 ] : OXO ID Tryp tone Yeast extract Mycological pep tone,, T4 DNA, ( dntp), Ex Taq DNA, EcoR N ot, DNA DNA Q iagen, DNA DL2000, ( SDS2Na), Tris, B iorad, G418 Invitrogen, CENTRE MasterPure TM EP I2 Yeast DNA Purification Kit, PCR : YEPD BMGY BMMY MD MM Invitrogen Pichia exp res2 sion Kit PCR : [ 6 ], [ 11 ] LfcinB : FKCW RWQWRW KKLGAPSITCVRRAF : P1 ( EcoR I) 5 2GGC GAA TTCTTCAAATGTTGGAGAT23 P2 3 ( N ot I) 1 5 2GGCGCGGCCGC TTAAAAGGCTCTTCTA LfcinB PCR :, P1 P2, PCR, PCR 94 4 m in : s, s, s, 30 ; 72 5 m in 115%, PCR, PCR : EcoR N ot LfcinB PCR,, EcoR N ot pp IC9K, DH5, PCR, ( 1) : 100mL 10mL YEPD, SMD1168, 30, 250 r/m in ; , OD 600 = , 3000 r/m in 5m in, 100mL 50mL 2, 25mL 1mol/L 1,, 200 L 1mol/L, pp IC9k2LfcinB S al, g DNA 80ul, 012cm, 5m in, 10500V 25 F 200, 1mL 1mol/L,, MD, d, (pp IC9K2LfcinB /SMD1168)

3 267, 3mL LB, 37, 300 r/m in OD 560 = L 6mL LB, 2 mm, 5 L, 0101 mg/ml, pp IC9K ( pp IC9K/SMD1168 ), 37,, SDS2PAGE : ( TCA ) 10 : 1mL 100 L 100%, 30m in, g 5m in,, 10% [ 12 ], 1615% SDS2PAGE 1 pp IC9K2LfcinB G418 : ( pp IC9K2LfcinB / SMD1168) G418 G mg/ml YEDP, MD MM, d, PCR LfcinB EP ICENTRE MasterPure TM Yeast DNA Purification Kit DNA, Invitrogen PCR LfcinB, pp IC9K ( pp IC9K/SMD1168 ), pp IC9K2Lf2 cinb DNA : G418 PCR 25mL BMGY, 30, r/m in OD 600 = , 4000r/m in 5m in,, mL BMMY, OD 600 = 110, 30, r/m in, h, 1mL; 10000r/m in 2m in, - 80, 24h, 1% : L fc inb PCR PCR 115%, :, 100bp, (98bp) ( ) ( 2), LfcinB 2 LfcinB PCR 1, 2 PCR, M DNA marker 212 EcoR N ot 80bp,, 80bp LfcinB pp IC9K, pp IC9K2LfcinB ( 3) 213 G418 pp IC9K2LfcinB

4 (2) SMD1168, G418, G418 2 mg/ml YDP 9 G418, (Mut + ) 214 PCR 9 (Mut + ) PCR, 9 LfcinB SMD1168 ( 4) 3 pp IC9K2LfcinB 1 pp IC9K2LfcinB EcoR + N ot 80bp, M DNA marker 4 PCR 1 ( pp IC9K2LfcinB DNA PCR ), 2 10 pp IC9K2LfcinB PCR, 11 ( PCR, M DNA marker (Mut + ),,, 2,, pp IC9K ( pp IC9K/SMD1168 ) ( 5) 5 0 (pp IC9K ), , 36, 48, 60, 72, 84 h, 7 (0101mg/mL ) 216 SD S2PAGE 2 10 SDS2PAGE, 316kD 1, pp IC9K ( 6), 2 3, pp IC9K, ( p1, p2 ), 5 3 EcoR N ot, LfcinB,, S al DNA, SMD1168, G418, (Mut + ), DNA PCR, LfcinB,,,, 10 SDS2PAGE,,, Reichhart [ 13 ] defensin A, ml 215 g; Andersons [ 14 ] cecrop ina

5 269 6 SDS2PAGE 1 ( pp IC9K ), , 36, 48, 60, 72, 84h, 8 (314kD), M marker ; [ 15 ],,,,,,, [ 1 ] Robert E W H1The Lancet infectious D iseases, 2001, 1: [ 2 ] B rock J1 Immunology Today, 1995, 16 (9) : [ 3 ] Horoaki A1J Dairy Sci, 1991, 74 (1) : [ 4 ] BellamyW1B iocem B iophys Acta, 1992, 1121 (2) : [ 5 ],, 1, 2003, 39 ( 3 ) : [ 6 ] Teraghcui S. B iochem Cell B iol, 2002, 80: [ 7 ] 1, 2004, 13 (4) : [ 8 ] Elizabeth J D1D igestive D iseases and Sciences11998, 43 ( 12 ) : [ 9 ] Pyong W P, Gerald B P, M ichael T H, et al1nature, 2000, 411: [ 10 ], 1, 2003, 7: [ 11 ],, 1, 2000, 6 ( 3 ) : [ 12 ] Hames B D1Gel Electrophoresis of Proteins1A Practical Ap2 p roach1 In: Hames B D, R ickwood D, eds1london: IRL Press L im ited, 1981, 2901 [ 13 ] Reichhart J M, Petit I, Legrain M, et al1 Invert Peprod Dev, 1992, 21: [ 14 ] Andersons D, Engstrm A, Josephson S, et al1b iochem J, 1991, 280: [ 15 ],,, 1 ( ), 2002, 30 (2) : , gx, X, M ean s, SD s - x, S E t t F F 2 r n P

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