Construction And Identification Of The Vector For Multiple Locus Gene Targeting And Galactophore Expressing

The Bos Taurus was the study object in this research. The vectors, which weremultiple locus genes targeting on the repetitive internal transcribed spacers betweenrRNA genes in Bos Taurus and could uniquely express medication proteins htPA ingalactophore, were constructed based on the BAC with Cre/loxP system. The keymaterial of Bos Taurus mammary biologic reactor would be got.The tPA a member of the serine family excreted mainly by blood vessel endothelialcell was one kind of physiological tissue-type plasminogen activator, which has beenused in clinic. The study followed Key's method make point mutations into tPA so as toprolong it's half life. After changing Asn103 into Thr, Asn117 into Gln, andLysHisArgArg in 296—299 into AlaAlaAlaAla,the mutational protein's rate ofdegradation in vivo would reduce 8 times.The galactophore expression vector of pYLSV-pBC I-htPA has been constructedfirstly. The plasmid of BAC-TDN-GD-SV-pBC I-tPA was attained by the co-transformof the selective vector of BAC-TDN-GD and the expression vector of pYLSV-pBCl-tPAto E.coli NS3529 through the action of Cre/loxP system. Then the galactophoreexpression vector of multiple locus genes targeting for Bos Taurus of BAC-TDN-PT-GDwas got by cutting the sequence of pYLSV with the Homing Endonucleas.es of PI-Sce Iand ligating with the linker of LS. The multiple locus gene targeting vectorBAC-TDN-PT-GD transfected embryo Bos Taurus fibred cell, then the expression statusof GFP could been observed in 24 hours late so as to preparatorily verify the genetargeting efficiency of the vector in vivo cell. There are some problems such as the lowefficiency and safe problem in the gene targeting. The technique of multiple locus genestargeting on. repetitive sequences will resolve a part of these problems. The join of Capra casein promoter would enable uniquely expression of medication proteins in galactophore.The study lay the foundation for establishing a series of Bos Taurus galactophoreexpression vectors system which were multiple locus genes targeting.