The Construction Of Bovine αS1casein-htPA Hybrid Locus Vector And It Confer HtPA Expression In Milk Of Transgenic Mice

The human tissue plasminogen activator (htPA) is a serine protease secreted byvascular endothelial cell and excreted to cycling system at an endogenous level of about7.1ng/ml in the human plasma.Tissue plasminogen activator can activate theplasminogen in the blood, and lysis the blood clots.It is widely used to treat myocardialinfarction,coronary thrombosis,pulmonary thrombosis and other cardiovasculardiseases.tPA is the only thrombolytic drug that FDA authorized to treat acute ischemicstroke nowadays.Because of the rapid increase of cardiovascular diseases as the topharmful factor to human being,tPA has a promising market and its price reached as to2900RMB/20mg.For the production of rhtPA,there are some shortcomings for thetraditional genetic engineering way,the quality of tPA produced by bacteria and yeast isquite poor,and it's quite expensive to produce rhtPA by mammalian cell culturesystem.Comparing with the above traditional genetic engineering system,the mammarygland bioreactor is an approproate candidate for the large scale production of rhtPA,thetransgenic goat explored by the GTC company can secrete rhtPA in the milk,and theirrhtPA purified from the milk is now in the third stage of clinical trial.Mamary gland bioreactor is the transgenic animal that can produce valuablesubstances in the mammary gland.The genes of valuable substances are transmitted intothe germ cell and the following transgenic animals are established. In which theexogenous proteins are expressed specifically in the mammary gland and secreted intothe milk. During the past thirty years many groups had explored the feasibility ofproducing human therapeutic proteins in the milk of transgenic animals,with oneobjective being the reduction of costs inherently associated with conventionalproduction of complex recombinant proteins in mammalian cell culture systems. Butunfortunately most of the efforts failed to give transgene expression at desired highamount.The transgene expression level mainly depend on the ability of the expressionvector. In order to dissolve the problem of expression vector construction, here wepresent the idea of hybrid gene locus, in which the genomic coding sequence oflactoprotein is substituted by that of rhtPA exactly from the start coden to the end coden.There are at least three advantages for the design as compared with the previously usedtraditional expression vector: 1)Much longer 5'and 3'flanking sequences were includedin the vector, which increase the possibility of high level expression; 2) The genomic coding sequence of tPA was used,while not the cDNA coding sequence, there werealready many reports support the idea that genomic coding sequence is much better forhigh level expression;3)The ligation between the 5'&3'regulatory elements and rhtPAgenomic coding sequence is completely seamless, no any additional basepairs wereintroduced or deleted.Here in the paper, the bovineαS1-casein gene locus was chosed as regulatoryelements to direct the expression of rhtPA genomic coding sequencee, and aαS1-casein-htPA hybrid locus was constructed, followed by expression abilityidentification in the transgenic mice model.A successive three-step method wasdeveloped to achieve the construction of the above hybrid gene locus. Totally sixhomologous arms T1-T6 were amplified by PCR with BAC as template, and thenseamlessly ligated together in order and cloned into the pBR322 vector to construct thesuccessive three-step gap repair vector. For the first step gap-repair, homologous armsT5&T6 were dissociated by restriction enzyme digestion to catch the 9Kb 3'flankingregion of bovineαS1 casein gene locus with bovineαS1 casein BAC as template, thesecond step gap-repair started from the product of the first step, the arms T3&T4 weredissociated to catch the 17.4Kb htPA genomic coding sequence with htPA BAC astemplate,exactly from the start coden to the end coden. And the last step of gap-repairstarted from the product of the second step, the arms T1&T2 were dissociated to catchthe 20Kb 5'flanking region of bovineαS1 casein gene locus with bovineαS1 caseinBAC as template. Finally we get the 46.4KbαS1 casein-htPA hybrid gene, andcorresponding three lines of transgenic mice were established with the above hybridgene locus as expression vector. The genotype of transgenic mice were identified byPCR and southern-blot methods, and the expression of rhtPA in the milk of transgenicmice were identified by western-blot and the expression level of rhtPA was assayed withELISA method, with the highest level attained as to 0.247g/L.Our results demonstrated that the bovineαS1 casein-htPA hybrid gene locus cangive efficient expression of rhtPA in the milk of transgenic mice, the constructed vectorcan be used in the establishment of future mammary gland bioreactor such as transgenicgoat or bovine.