CMV Promoter Controlled Expression And The Establishment Of A Semi-quantitative Assay Of Tissue-type Plasminogen Activator

Human tissue-type plasminogen activator(ht-PA), composed by 527 amino-acid residues, is a kind of serine proteinase that lyses the fibrin in thrombus matrix and recovers the perfusion of the blocked vessels. As one of the most effective therapy drugs on acute myocardial infarction (AMI), ht-PA has been intensively studied over the last years. To establish a stable cell line highly expressing t-PA, its cDNA was cloned to the downstream of CMV promoter and an eukaryotic vector pCPA was obtained. Liposome- mediated cotransfection of pCPA and pSV2-dhfr was carried out on CHO-dhfr cells. After being screened with G4 18 and cloned by endpoint dilution assay, four t-PA expressing CHO cell clones were gained. The t-PA expression positive cell clones were ampliiied and harvested for determination of expression level by ELISA. The results showed that ht-PA was stably expressed at a level of 0.6-2. 1~.tg/106 cells.day. Modified fibrin agarose plate assay(FAPA) showed that 30-1 OOIU/ml oft-PA were confirmed to be in the supematant of thawed CHO cells. This laid a foundation for further co- amplification with metrotexate(MTX). To make clear whether a 5?untranslated region(UTR) has am effect on t-PA expression in eukaiyotic cells, tl* 5?UTR sequence of 13-casein gene was cloned from caprine genome by PCR using a pair of primers which was designed according to the sequence in GenBank. An 111 bp fragment was amplified and sequenced and then subcloned to the upstream of the t-PA coding region and the downstream of cytomegalovirus promoter in pCPA. The transient expression in COS-7 indicated that the introduction of the ~3-casein 5?UTR led to a 2/3 decrease in lit-PA expression level. The probable reason may be that the 13-casein 5?UTR lengthened the leader sequence of ht-PA mRNA and this unfavored its translation. For improving the sensitivity and eliminating the false positive background of FAPA, a modified FAPA method was re-erected by regulating the parameters. The sensitivity of FAPA was raised by 30-folds after the introduction of plas.minogen as an additional supplement. The main parameters, including the time of incubation, the concentration of fibrin in agarose, the thickness of the agarose plate, were investigated and standardized. 38 The modified FAPA provides a convenient method for detection of low activity PAs and their mutants.