15 4 1999 8 V o l 15,N o 4 Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy A ug, 1999 3 IL -2gIFNΑ2b (, 200032), DNA IL 22 IFN Α, A FP galb, EA FP2PALB A, IL 22gIFN Α2b A FP IL 22 IFN IL 22gIFN Α, A FP,,, The Targeted Express ion of Human IL -2gIFNΑ2b Fused Gene in Hepa toma Cells H E P ing, TAN G Zhaoyou,L IU B inb in, YE Shenglong (L iver Cancer Institute, Z hong S han H osp ital, S hanghai M ed ical U niversity, S hanghai 200032) Abstract T he study exp lo red the u se of a tum o r2specific A FP enhancer and a liver2specific albu2 m in p rom o ter to ach ieve regu lated cytok ine IL 22gIFN Α2b fu sed gene exp ression fo r treatm en t of hepatocellu lar carcinom a (HCC) T he hum an A FP enhancer (EA FP) galbum in p rom o ter (PALB )w ere am p lified from hum an ch rom o sm e DNA by PCR m ethod A recom b inan t retroviru s including the selectab le m arker neo R gene and IL 22gIFN Α2b fu sed gene con tro led by the EA FP2PALB w as con2 structed T he liver2targeted exp ression pattern of the IL 22gIFN Α2b fu sed gene w as ob served w hen the p roduct w as tested in cu ltu re m edium of the infected cellṡ IL 22 activity w as 850 IU g10 6 cells, IFN Α activity w as 320 IU g10 6 cellṡ T he resu lts show ed that EA FP2PALB com b ind tran scrip tional regu lato ry sequences cou ld con tro l the targeted exp ression of cytok ine genes in A FP2po sitive hu2 m an hepatom a cells, and the exp ression level of IL 22gIFN Α2b fu sed gene w as po sitively co rrelated to the level of A FP exp ression in the infected cells T he exp ressed IL 22gIFN Α2b fu sed p ro tein had function s of bo th IL 22 and IFN Α T herefo re, th is study illu strates the superio rity of u sing tran2 scrip tionally targeted recom b inan t retroviru s vecto rs in cytok ine2based gene therapy Key words H um an IL 22gIFN Α2b fu sed gene, H um an A FP enhancer, H um an album in p rom o ter, T argeted exp ression, H epatocellu lar carcinom a, IFN,, N K CTL LA K, ;,, 3 (39400050),, [ 1 3 ] IL 22 T,, N K T :,, 1964 10,, T el: (021) 6404199022136 E2m ail: P inghell@usa net B : 1998207224, : 1998210208 1994-2006 China Academic Journal Electronic Publishing House All rights reserved http://wwwcnkinet
638 15 [ 4 ] GCT T 23 ), psv K32IL 22, PCR IL 22 cdna, 3 T GA, A GA, E cor g X ho g,, pcdna 23, pcdna (m IL 2, IL 22 IFN Α 2) DNA m IL 22 cdna,, 115 Po ly1 ( 5 2A T CTC 1 111-2 psv K32IL 22 A CC A GA TCC A CC GCC GGA TCC A CC GCC IL 22 cdna, A CC CTC GA G A T 23 ) PA GE, 459 bp, 153 112 JM 109 PU C19 (L inker) DNA DNA L XSN M D A ndson PU C ( IFN Α22b) 116, IFN Α22b cdna E cor g X ho g pcdna (m IL 22), 113 EAFP-PALB EA FP [ 5 ] 1 20 pm o l, PU C19, H ind g E cor g PALB 1 (5 2 T T G GA T CCC T TA A CT CT T TCA 23 ) 2 m IL 22 cdna PU C (Α22b) ( 5 2GGA A T T CCG CTA GGC TAA A T T A GC A CT AA 23 ), 5 PU C 19 ( IL 22gIFN Α22b) H ind g 3 B am H g E cor g DNA EA FP2PALB DNA 114 IL -2 cd NA P1 ( 5 2A GG AA T TCT T TA A TC A CT A CT CA C A 23 ), P2 ( 5 2AA C TCG A GA GT T A GT TA G T GT T GA GA T GA T GA G GGT GGC GGT GGA TCC GGC GGT GGA TCT GGT GGC GGC GGA TCT GAA T TC C 2 3 ), Po ly2 (5 2G GAA T TC A GA TCC GCC GCC pm o l, 95 5 m in,, E cor g X ho g, pcdna 23, pcdna m IL 22 cdna ( 508 bp ) ; pcdna (L inker), PA GE 57 bp, PU C ( IL 2L ) E cor g PU C ( IL 2L ), E cor g, E cor g PU H 23, EA FP2PALB ( 822 bp ) H ind g PU C 19 ( IL 22gIFN Α22b), E cor g, EA FP2PALB,, L XSN X ho g ( ), L (FL 2m IL 22gIFN 2Α2b) SN, F ig 1 F ig 1 T he construction of recom binant retrovirus L (FL 2IL 22gIFN Α2b) 117 > 10 5 ( 8 L ipofectin L (FL 2IL 22gIFN 2Α2b) Λggm l) A FP H epg2 PA 317, G418, A FP SMM C7721 N IH 3T 3 H el a (A FP ), G418, 10 5-80 118 119 1994-2006 China Academic Journal Electronic Publishing House All rights reserved http://wwwcnkinet
4 : IL 22gIFN Α2b 639 11911 PCR : DNA, N eor IL 22 IFN Α N eor 1: 5 2CAA GA T GGA T T G CA C GCA GG23, 2: 5 2CCC GCT CA G AA G AA C TCG TC23 ; IL 22 1: 5 2TCT GAA T TC A T G TA C A GG A T G CAA CTC G23 ; 2: 5 2AA C TCG A GA GT T A GT GT T GA G A T G A T G CT T 23 ; IFN Α 1: 5 2GA G GAA T TC A T G T GT GA T CT G CCT C23 ; 2: 5 2GAA 2 211 L (FL - IL -2gIFN-Α2b) SN 21111 EA FP PALB : EA FP [ 7 ] [ 8 ] H ind g B am H g ; PALB [ 8 ], B am H g E cor g GTC GA C TCA T TC CT T A CT TC23 21112 m IL 22 cdna : F ig 2, 11912 SD S2PA GE ( ) [ 6 ] : 469 bp, 5 E cor g, 3 X ho g, 24 h,, 8 Λl 2 T GA GA G, 95 5 m in, IL 22 cdna 11913 : M T SgPM S IL 22 ; F ig 2 T he DNA sequences of IL 22 cdna m utant [ 9 21113 IFN Α22b : ], 5 E cor g, 3 S a l g L XSN, 23, IFN Α22b 21114 L (FL 2IL 22gIFN 2Α2b) SN : F ig 3 L (FL 2IL 22gIFN ) SN : E cor g 526 bp, 7 126 bp ; B am H g 584 bp, 893 bp, 6 211 bp ; X ho g 592 bp, 7 096 bp 212 21412 SD S2PA GE ( ) : ( F ig 4 ), L L (FL 2IL 22gIFN 2Α2b) SN L XSN, 215 712 10 5 CFU gm l L (FL 2IL 22gIFN 2Α2b) SN, 214 21411 PCR :, L (FL 2IL 22gIFN Α2b) SN 2H ep G2 L (FL 2IL 22gIFN Α2b) SN 27721 N eor IL 22 IFN Α ; L XSN 2H epg2 L XSN 27721 N eor ( FL 2IL 22gIFN 2Α2b ) SN 2H epg2 L ( FL 2IL 22g IFN Α2b ) SN 2SMM C7721, 213 317 kd, 1994-2006 China Academic Journal Electronic Publishing House All rights reserved http://wwwcnkinet
640 15 T ab le 1, EA FP2 PALB ( A FP ) IL 22gIFN Α2b IL 22 IFN Α2b Table 1 T he h ighest activity of IL 22 and IFN Α in culture m edium of IL 22gIFN Α2infected cells ( IU g10 6 cells) L (FL 2IL 22 gifn Α2b) SN H epg2 SMM C7721 H el a IL 22 IFN Α IL 22 IFN Α IL 22 IFN Α 850 320 450 160 325 80 L XSN ND ND ND ND ND ND ND: T he activities of IL 22 and IFN Αw ere no t detected in culture m edium F ig 3 T he m ap of L (FL 2IL 22gIFN Α2b) SN cut by restric2 tion endonucleases 11PCR M arkers (bp) : 1 000, 750, 500, 300, 100, 50; 21L (FL 2IL 22gIFN Α2b) SN ge cor g (bp): 526, 7126; 31L (FL 2IL 22gIFN Α2b) SN gb am H g (bp): 584, 893, 6211; 41L (FL 2IL 22gIFN Α2b) SN gx ho g (bp) : 592, 7096; 51 ΚDNA gh ind g M arkers (bp ) : 564, 2 027, 2 322, 4 361, 6 557, 9 416, 23 130 F ig 4 T he SD S2PA GE of serum 2free m edium of infected H epg2 and SMM C7721 cells 11LXSN 2H epg2; 21L (FL 2IL 22gIFN Α2b) SN 2H epg2; 31LXSN 2SMM C7721; 41L (FL 2IL 22gIFN Α2b) SN 2SMM C7721; M Standard p ro tein mo lecular w eigh ts IL 22gIFN Α2b, IL 2 3 IL 22gIFN Α2b,,, : 11 ; 21, 19 [ (Gly2Gly2Gly2Ser) n ],,,, IL 22 IFN Α, :, ; ; ;,,,,, EA FP2PALB, IL 22gIFN Α : A FP H epg2 IL 22 IFN Α, A FP A FP SMM C7721 H el a, 2gIFN Α2b, 21413 : M T SgPM S EA FP2PALB IL 22, IFN, A FP 1994-2006 China Academic Journal Electronic Publishing House All rights reserved http://wwwcnkinet
4 : IL 22gIFN Α2b 641 IL 22gIFN Α,, IL 22gIFN Α References 1 Curtis B M, w illiam s D E, B roxm eyer H E, D unn J, Farrah T, Jeffery E, C levengerw, D eroo s P,M artin U, friend D, C raig V, Gayle R, P rice V, Co sm an D, M arch C J, Park L S Enhanced hem atopo ietic activity of a hum an granulocytegm acrophage co lony2stim ulating facto r2interleukin 3 fusion p ro tein P roc N atl A cad S ci U SA, 1991, 88: 5809 5813 2,,,,,,,,, (TN F) ΑA ( IFN ΑA ) (Zhi Gang, Hou Yunde, Zhang D ezhen, J in Q i, Zhou Yuan, L i Yuying, Zhang Zhiqing, H u Yuw en, Su Chengzhi, W ang Kew ei Construction and ex2 p ression of recom binant hum an tumo r necro sis facto r and inter2 feron ΑA fusion p ro tein Ch in B iochem J ), 1991, 7 (1) : 89 94 3 Klim ka A, Barth S, D rillich S,W els W, van Snick J, Renauld J C, T esch H, Boh len H, D ieh l V, Engert A A deletion m utant of P seudomonas exo toxin2a fused to recom binant hum an inter2 leuk in29 ( rh IL 292ETA ) show s specific cyto toxicity against IL 2 92recep to r2exp ressing cell lines, Cy tok ines M ol T her, 1996, 2 (3) : 137 146 4 Robert W, H atho rn M D, Cho2L ea T so, Kaboo R, Pang S, F iglin R, Saw yers C, dekernion J B, Belldegrun A In v itro modulation of the invasive and m etastatic po tentials of hum an renal cell car2 cinom a by interleukin23 andgo r interferon2alpha gene transfeṙ Cancer, 1994, 74 (7) : 1904 1911 5,,, (H e P ing, Ye Shenglong, H e B in, T nag Zhaoyou C loning of transcrip tional regulato ry sequences in hu2 m an Α2fetop ro tein gene Ch in J Cancer B iother), 1996, 3 (3) : 222 225 6 Sam brook J, F ritsch E F,M aniatis T M olecu lar C loning, A L ab2 oratory M anual, 2nd ed, N ew Yo rk: Co ld Sp ring H arbo r L abo ra2 to ry L abo rato ry, 1989 7 W atanabe K, saito A, T am aok i T Cell2specific enhancer activity in a far up stream region of the hum an Α2fetop ro tein gene, J B iol Chem, 1987, 262 (10) : 4812 4818 8 U rano Y,W atanabes K, SakaiM, T am aok i T T he hum an albu2 m in gene J B iol Chem, 1986, 261 (7) : 3244 3251 9 StreuliM, N agata S,W eissm ann C A t least th ree hum an type Α interferons: structure of Α2 S cience, 1980, 209: 1343 1347 1994-2006 China Academic Journal Electronic Publishing House All rights reserved http://wwwcnkinet