The Targeted Express ion of Human IL -2gIFNΑ2b Fused Gene in Hepa toma Cells
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1 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, IL -2gIFNΑ2b (, ), 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 ) 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 ( ),, [ 1 3 ] IL 22 T,, N K T :,, ,, T el: (021) E2m ail: P inghell@usa net B : , : China Academic Journal Electronic Publishing House All rights reserved
2 [ 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 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, 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, China Academic Journal Electronic Publishing House All rights reserved
3 4 : IL 22gIFN Α2b 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 L (FL - IL -2gIFN-Α2b) SN 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 TC m IL 22 cdna : F ig 2, 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 : M T SgPM S IL 22 ; F ig 2 T he DNA sequences of IL 22 cdna m utant [ IFN Α22b : ], 5 E cor g, 3 S a l g L XSN, 23, IFN Α22b L (FL 2IL 22gIFN 2Α2b) SN : F ig 3 L (FL 2IL 22gIFN ) SN : E cor g 526 bp, bp ; B am H g 584 bp, 893 bp, bp ; X ho g 592 bp, bp SD S2PA GE ( ) : ( F ig 4 ), L L (FL 2IL 22gIFN 2Α2b) SN L XSN, CFU gm l L (FL 2IL 22gIFN 2Α2b) SN, PCR :, L (FL 2IL 22gIFN Α2b) SN 2H ep G2 L (FL 2IL 22gIFN Α2b) SN N eor IL 22 IFN Α ; L XSN 2H epg2 L XSN N eor ( FL 2IL 22gIFN 2Α2b ) SN 2H epg2 L ( FL 2IL 22g IFN Α2b ) SN 2SMM C7721, kd, China Academic Journal Electronic Publishing House All rights reserved
4 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 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, 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, : M T SgPM S EA FP2PALB IL 22, IFN, A FP China Academic Journal Electronic Publishing House All rights reserved
5 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: ,,,,,,,,, (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) : 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) : 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) : ,,, (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) : 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, 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) : 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) : 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: China Academic Journal Electronic Publishing House All rights reserved
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Supplemental file 3. All 306 mapped IDs collected by IPA program. Supplemental file 6. The functions and main focused genes in each network.
LIST OF SUPPLEMENTAL FILES Supplemental file 1. Primer sets used for qrt-pcr. Supplemental file 2. All 1305 differentially expressed genes. Supplemental file 3. All 306 mapped IDs collected by IPA program.
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Supplementary Table 1. Primers used for RT-qPCR analysis of striatal and nigral tissue.
Supplementary Table 1. Primers used for RT-qPCR analysis of striatal and nigral tissue. Gene Forward Primer (5-3 ) Reverse Primer (5-3 ) Dopaminergic Markers TH CTG GCC ATT GAT GTA CTG GA ACA CAC ATG GGA
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