ISSN 100727626 CN 1123870gQ 16 3 Ch in. J. B iochem. M o l. B io l. 2000, V o l. 16, N o. 3, 306 311 2000 6 T IM P-1 cd NA 3 CO S-7 3 3 (,,, 100853) GenBank T IM P21, R T 2PCR T IM P21 cdna T 2A pcr R 2. 1, DNA T IM P21 cdna pcdna 3, pcdna 3gT IM P21, DO TA P CO S2 7,N o rthern CO S27 T IM P21, T IM P21 CO S27, T IM P21 T IM P21,, CO S27 Q 78 Clon ing of Human T IM P-1 cd NA and its Expression in COS-7 Cells L IN Hong2li, CH EN X iang2m ei 3 3, YU Zh i2heng, L IW en2ge, FU Bo (D ep artm ent of N ep h rology, General H osp ital of Ch inese PL A,M ilitary K id ney Center, B eij ing 100853, Ch ina) Abstract T IM P21 fu ll length cdna con tain ing the signal pep tide w as am p lified by reverse tran2 scrip tion and po lym erase chain reaction (PCR ) from to tal RNA of hum an no rm al renal tissueṡ Confirm ed by the DNA sequence analysis, the T IM P21 cdna fragm en ts w ere then in serted to m amm alian exp ression p lasm id pcdna 3 respectively. T hu s pcdna 3gT IM P21 recom b inan t eu2 karyo tic exp ression vecto r w as con structed. M o reover, th is recom b inan t p lasm id w as tran sfected in to CO S27 cells w ith lipofectin DO TA P. N o rthern b lo t analysis and in situ hyb ridization dem on2 strated that no T IM P21 m RNA w as detected in no rm al CO S27 cells, w h ile T IM P21 m RNA w as detected in pcdna 3gT IM P21 tran sfected CO S27 cells. Fu rther study indicated that the endoge2 nou s T IM P21 overexp ression cou ld induce the p ro liferation of CO S27 cells. Key words T IM P21, C lon ing, T ran sfection, CO S27 cells, : T IM P2 [ 1 1 ],, 21 (tissue in2, h ib ito r of m etallop ro teinase21, T IM P21) T IM P21,, T IM P21, (m atrix m etallop ro teinases,mm P s) MM P s 3 (962056) 3 3 T el: (010) 68280985, Fax: (010) 68223803 E2m ail: xm chen@public. bta. neṫ cn T IM P21 T IM P21,, 1966 11,, : 1999212210, : 2000202203
3 : T IM P21 cdna CO S27 307 T IM P21 cdna, pcdna 3g T IM P21, 55, 30 s, 72, 2 m in, 72 (CO S27), N o rthern T aq DNA 15 m in 1% CO S27 T IM P21, T IM P2 1 T IM P21 116 TIM P-1 cd NA T-A CO S27 1 111 TRL ZO 1 T EL 2T EST Inc,DNA T 4 P rom ega 117 pcd NA3-TIM P-1 T 2A clon ing Invitrogen PCR L ipofectin T IM P21 A vag T IM P21 SP6gT 7 SuperScrip t TM RN ase H - G418 118 GibcoBRL DNA Q IN 2 CO S27 18 h 1 GEN 112 pcdna 3 DH 5Α CO S27, 400 ggl G418, 10 d 113 RNA 2 mm 2 mm 3 mm, TRL ZO 1,, 70%, D FPC 260 nm T GCA CGCA GG23 ; 3 : 5 2CCCGCTCA 2 RNA 114 GenBank T IM P21 1 m in, 25, 72 7 m in PCR T IM P21 cdna : T IM P21 cdna, 5 5 2T T GAA T TCCCA CCA T GGCCCC CT T T GA GCC23 ; 3 5 2CT GAA T TCGCA GGA T TCA GGCTA TCT G23 5 72 7 m in N, 1110 Northern TIM P-1 COS-7 E cor g 3 C, 15 g CO S27 RNA, [ 3 ] E cor g pcdna 3 neo r, neo r, 5 5 2CAA 2 GA T GGA T T GCA CGCA GG23 ; 5 2CC2 1111 TIM P-1 COS-7 CGCTCA GAA GAA CTCGTC23 115 RT-PCR TIM P-1 cd NA SuperScrip t TM RN ase H - pcdna 3gT IM P21, T IM P21 (T 7) cdna R T V en t DNA (Sp 6)RNA CO S27 PCR PCR PCR (con) pcdna 3 (vec) pcdna 3g : DNA 94 3 m in, 10 T IM P21 (PT ) CO S27 3 cm 94, 15 s, 55, 30 s, 72, 1 m in, 15 94, 15 s, PCR Geneclean T 2A clon ing, T IM P21 PCR pcr R 2. 1, pcr R 2. 1 T 7 M 13 [ 3 ] E cor g 50 DO TA P pcdna 3 pcdna 3gT IM P21 CO S27 119 COS-7 neo r CO S27 DNA, PCR, 5 : 5 2CAA GA T GGA T GAA GAA CTCGTC23, 790 bp : 94, 30 s, 60, 30 s, 72, 652 bp : 94, 30 s, 58, 30 s, 72, 1 m in, 30, N o rthern, - 70 1 h D IG RNA L abelling K it (Sp 6gT 7),, 40, RNA 016 ggm l,
308 16 40 15 h, NBT gbc IP 1112 con vec PT CO S27 pcr R 2. 1, E cor g N cog 10 000 gm l 24, pcr R 2. 1gT IM P21, F ig. 1, 6, 3, 5 d, 212 pcd NA3gTIM P-1 3 E cor g T IM P21 cdna pcr R 2. 12 2 RNA, R T 2PCR T IM P21 cdna, 652 bp T 2A T IM P21 cdna T IM P21, pcdna 3 ( F ig. 2 ), E cor g T IM P21 211 TIM P-1 cd NA cdna, 514 kp 652 bp ; F ig. 1 Partial sequence analysis of hum an T IM P21 cdna F ig. 2 Construction m ap of pcdna 3gT IM P21 recom binant p lasm id
3 : T IM P21 cdna CO S27 309 A vag T IM P21 cdna, 4 880 bp 1 119 bp 99 bp 3 (F ig. 3) 213 PCR neo r TIM P-1 COS-7 PCR pcdna 3 neo r, CO S27 pcdna 3gT IM P21 CO S27 790 bp CO S27 neo r, PCR CO S27 T IM P21cDNA, CO S27, pcdna 3gT IM P21 CO S27 652 bp, T IM P21cDNA CO S27, T IM P21 T IM P21 (F ig. 4) F ig. 3 Restriction m ap of recom binant p lasm id pcdna 3g T IM P21 L ane 1: 1 kb mo lecular w eigh t m arker; L ane 2: Recom binant p lasm id pcdna 3gT IM P21; L ane 3: pcdna 3gT IM P21 digested by E cor g ; L ane 4: pcdna 3gT IM P21 digested by A vag F ig. 4 Amp lification of neo r and T IM P21 cdna gene by PCR L ane 1: 1 kb mo lecular w eigh t m arker; L ane 2: CO S27 cells; L ane 3: CO S27 cells transfected by pcdna 3; L ane 4: CO S27 cells transfected by pcdna 3gT IM P21 214 COS-7 21412 T IM P21 m RNA CO S2 21411 N o rthern b lo ting T IM P21 m RNA 7 : T IM P21 CO S27 : 15 g RNA (T 7) T IM P21 RNA (Sp 6) pcdna 3gT IM P21 CO S27 pcdna 3gT IM P21 RNA, 32 P CO S27, T IM P21, 1 h, CO S27 Sp 6 T 7, pcdna 3g CO S27 T IM P21 T IM P21 CO S27 (F ig. 5) m RNA ; CO S27
310 16 F ig. 5 N o rthern blo tting of hum an T IM P21 mrna in CO S27 cells 1: CO S27 cells; 2: CO S27 cells transfected by pcdna 3; 3: CO S27 cells transfected by pcdna 3gT IM P21 CO S27 ( ), pcdna 3gT IM P21 CO S27 pcdna 3gT IM P21 CO S27 T 7 T IM P21 (F ig. 6), Sp 6 F ig. 6 In situ hybridization of hum an T IM P21 mrna in CO S27 cells A, B: no rm al CO S27 cell; C, D: CO S27 cells transfected by pcdna 3gT IM P21. A, C: In situ hybridyzation w ith sense T IM P21 RNA p robe (T 7) ; B, D: In situ hybridyzation w ith antisense T IM P21 RNA p robe (Sp6) 215 TIM P-1 COS-7 24 h, F ig. 7 : T IM P21 (PT ) (con) (vec),
3 : T IM P21 cdna CO S27 311 (P < 0. 01), T IM P21 T IM P21, CO S27 F ig. 7 T he effects of T IM P21 transfection on the p ro lifera2 tion of CO S27 cells 3 3 P < 0. 01 (PT vs con) ; P < 0. 01 (PT vs vec) 3 T IM P21 cdna 782 bp, 63 bp 5, 623 bp 99 bp 3 B lood, 1998, 92: 1342 1349 T IM P21 cdna, 652 bp V en t DNA R T 2 PCR m RNA, T aq DNA 3 25, PCR, T IM P21, Docherty [ 2 ] T IM P21, V en t 3 2 5 PCR 3 A, V en t PCR T 2A, PCR T aq DNA 72 PCR 15 m in, 3 A,, pcr R 2. 1 T 7 M 13, [ 4 ], T IM P21, T IM P21 T IM P21 pcdna 3, CO S27 N o rthern, pcdna 3gT IM P21 CO S27 1 h T IM P21, : T IM P21 CO S27, T IM P21, T IM P21 H ayakaw a [ 5 ] T IM P21, T IM P21 pcdna 3gT IM P21 CO S27, T IM P21 CO S27, T IM P21 CO S27 (References) 1 Guedez L, Courtem anch L, Stetler2Stevenson M. T issue in2 hibito r of m etallop ro teinases21 ( T IM P21) induce differentia2 tion and an antiapop to tic pheno type in germ inal center B cellṡ 2 Docherty A J P, L yons A, Sm ith B J,W righ t E M, Stephens P E, H arris H J R. Sequence of hum an tissue inh ibito r of m etallo2 p ro teinases and its identity to erythiod2po tentiating activity. N atu re, 1985, 318: 66 69 3 Sam brook J, F ritsch E F, M aniatis T. M olecu lar C loning : A L aboratory M anual, 2nd ed, N ew Yo rk: Co ld Sp ring H arbo r L abo rato ry P ress, 1989 4 Yo sh iji H, H arris S R, Raso E, Gom ez D E, L indsay C K, Sh ibuya M, Sinha C C, T ho rgeirsson U P. M amm ary cacinom a cells over2exp ressing tissue inhibito r of m etallop ro teinases21 show s enhanced vescular endo thelial grow th facto r exp ression. Int J Cancer, 1998, 75: 81 87 5 H ayakaw a T, Yam ash ita K, T anzaw a K, U ch ijim a E, Iw ata K. Grow th2p romo ting activity of tissue inhibito r of m etallop ro2 teinases21 (T IM P21) fo r a w ide range of cells. F EB S, 1992, 298 (1) : 29 32