construction of tenecteplase coding sequence by a rapid megaprimer pcr method and its cloning in a mammary gland tissue specific expression vector

tenecteplase is a variant of tissue plasminogen activator (t-pa) which has better pharmacokinetic properties and more selective thrombolytic activity. in the present study, we describe a rapid method to introduce three sets of mutation into defined positions in t-pa cdna by a site-directed mutagenesis based on a megaprimer pcr approach to produce tenecteplase coding sequence where amino acids at positions 103, 117 and 296-299 in polypeptide sequence were modified. this sequence was cloned in ptz57r by t/a cloning method to introduce suitable restriction sites at both ends of the amplified fragment. vectors containing tenecteplase cdna were propagated in one shot top10 chemically competent e. coli and positive clones were selected by blue/white screening method. after being isolated and purified, the recombinant plasmids were digested by suitable restriction enzymes. acquired tenecteplase coding sequence was subcloned into a tissue specific expression vector. upon expression, it is expected that tenecteplase will be expressed exclusively in lactating mammary glands during milk production in transgenic animal. finally, the recombinant vector was isolated and verified by restriction digestion and sequence analysis to confirm that the tenecteplase coding sequence has been inserted in the proper orientation.