Expression of pro-urokinase cDNA gene in chinese hamster ovary cell line

Vol. 63, No. 2

ABSTRACTS;

CHINESE-JAPANESE

EXPRESSION OF PRO-UROKINASE cDNA CELL LINE Fana. X&&n Li. WewYu.

GENE

SYMP. 1990

IN CHINESE I

271

HAMSTER

OVARY

I

I01. Suwen. Honadi R-ochena Hu. mi Guan, Honsa. Cuifen Huang. Institute of Biotechnology, Academy of Military Medical Sciences, Beijing, China A full length pro-UK cDNA gene coding for 411 Aa residues of structural gene and 20 Aa residues of signal peptide were obtained and identified by reverse transcription, restriction mapping and DNA sequencing analysis. pSV2-pro-UK and pSVL-pro-UK recombinant plasmids containing SV40 early and late promotor respectively were transfected into CHO-dhfr- and COS-7 cells using calcium phosphate The transfected cells precipitation and DEAE-dextran techniques. pSV2-pro-UK and pSV2could synthesize and secret active pro-UK. dhfr or MMTV-dhfr expression vectors were cotransfected into CHOTwo of dhfr- cells by calcium phosphate precipitation techniques. the colonies, named CLE-14 and DLF-8, exhibited high expression level of U-PA activity (0.16 pg/106 cells/48h and 0.085 pg/lOc cells/48 h). The U-PA secreted by stable transformed cells can be completely inhibited by anti-UK serum but not by anti-t-PA serum and normal rabbit serum.

CLONING AND EXPRESSION OF HUMAN TISSUE-TYPE PLASMINOGEN ACTIVATOR cDNA IN CHINESE HAMSTER OVARY CELLS . J-0. Jun.Institute of Biotechnology, Academy of Military Medical Sciences, Beijing, 100850 China Poly(A) mRNA was isolated from the Bowes Melanoma cells and single and double-stranded cDNA by standard used to prepare residues, procedures. The cDNA was extended with deoxy(C) PstI-cleaved pBR322 and used to annealed to deoxy(G)-tailed transform E coli Cc00 strain. One positive clone, named E. coli The results of the restriction enzyme CSOO(PMG6oo) I was obtained. analysis have identified that plasmid pMG600 contained about 2.0kb t-PA segment. The sequencing results have shown that the sequence of cloned t-PA cDNA fragment was the same as that of t-PA BgIII reported by Pennica et al, but the signal peptide fragment sequence was absent. The signal peptide sequence was chemically The fullsynthesized and joined to the cloned cDNA sequence. length cDNA was cloned into vector pSV2-dfr, and then introduced into the CHO dfr- cells. The clones expressed t-PA were detected fibrin-agar plate, and the expressed products were by characterized by the neutralization assay. The results have indicated that the expressed products are identical to natural tPA from Bowes Melanoma cells.