Expression Of T-PA Gene Delivered By Attenuated Salmonella And Detection Of Its Fibrinolysis Activity In Vivo And In Vitro

Salmonellae are Gram-negative facultative intracellular bacteria. AttenuatedSalmonella could be considered an ideal vector for mucosal vaccination. LiveSalmonellae bacteria use strategies that allow them to cross the epithelial barrier of thegut to the immune cells, stimulate specific humoral and cellular immune responseagainst both Salmonella and heterologous antigen, it also stimulate mucosal andsystemic immune at the same time. As an intracellular invasive bacterium, attenuatedSalmonella has been used as vehicles to delivery recombinant plasmid DNA in vivointo prokaryotic and eukaryotic cells. In addition, Salmonella has fibrinolysis activity,which has a relationship with infectivity. Tissue-type plasminogen activator （t-PA） isthe secretion of vascular endothelial cells, it work by converting plasminogen to thenatural fibrinolytic agent plasmin. Plasmin lyses clot by breaking down the fibrinogenand fibrin contained in a clot. Live attenuated Salmonella, equipped with adhesion andinvasion properties but unable to cause clinical disease, can be administered orally.This convenient and inexpensive delivery makes application of heterologous proteinssimpler and more acceptable. In this study, we constructed plasmids by cloning t-PAgene into prokaryotic and eukaryotic expression vectors, attenuated Salmonella wereused as a vehicle for oral delivery of the plasmids to mice. This is the first attempt touse attenuated Salmonella as an oral delivery system for t-PA to prevent thromboticdiseases and rehabilitation treatment in mice.Three specific t-PA gene fragments were amplified by PCR using primers which weredesigned according to the published t-PA sequences. pET28-tPA and PYA3493-tPAprokaryotic expression plamids, pcDNA₃-tPA eukaryon expression plasmid wereconstructed,respectively. Here, attenuated Salmonella strain crpSL1344andΔcrpΔasdSL1344were used as vehicles for oral delivery of the expression plasmids to mice. We transformed the plasmids pET28-tPA and pcDNA₃-tPA to the bacteriacrpSL1344and PYA3493-tPA to ΔcrpΔasdSL1344by electroporation. BHK cellsand mice were transfected by the resulting recombinant attenuated Salmonella bacteriaΔcrpΔasdSL1344（PYA3493-tPA）,ΔcrpSL1344（pET28-tPA）and crpSL1344（pcDNA₃-tPA）. The expression efficiency in BHK cells were evaluated by SDS-PAGE andELISA, and the biological activity of t-PA expressed in BHKcells were evaluated byfibrinolysis test. Prothrombin time （PT）, Activated partial thromboplastin time （APTT）, Total time （TT）and Focused ion beam （FIB） were detected in mice. SDS-PAGE and Western blotconfirmed that the protein was expressed in BHK cells and the molecular weight of theexpressed protein was about66kDa, the expression yield were197μg/L,135μg/L and118μg/L measured in ELISA on the96hour postinfection. Fibrinolysis of t-PA in vitroindicated that the expressed protein had the activity to activate tissue-typeplasminogen, eukaryon expression plasmid is higher than prokaryotic expressionplamid of fibrinolysis activity, and ΔcrpΔasdSL1344（PYA3493-tPA） is the highestfibrinolysis activity. Basised on the detection of blood coagulation in mice, fourexperimental groups compared to the control group have different degrees of extensionof blood coagulation. Experimental group C extended very significantly of PT, APTT,TT in mice （P<0.01）, and FIB reduce very significantly （P<0.01）.The result show thatthe biological activity of t-PA gene in eukaryotic and prokaryotic plasmid delieveredby attenuated Salmonella is higher than attenuated Salmonella, t-PA protein wasmodified in eukaryotic cells and expressed in prokaryotic cell. It indicated that t-PAgene could stabile carried and highly expressed in attenuated Salmonella withaspartate semialdehyde dehydrogenase （asd-） balanced-lethal host vector system tomaintain multicopy plasmids that are devoid of antibiotic resistance genes. A new ideaof new biological agents have provided for the prevention of thrombotic diseases.