Construction Of IPR1 Expression Vector With Macrophage Speciality And Production Of Boving Cloned Embryos Transfected With IPR1 Gene

Ipr1 (intracellular pathogen resistance 1)which contains full length code sequence was obtained from C57BL/6J by RT-PCR method in this study. pEGFP-C1-Ipr1 fusion expression Vector was constructed, Then the fusion expression vector pEGFP-C1-Ipr1 were transfected into RAW264.7 cells by using electroporation to Subcellular localization of Ipr1expression product.Macrophage special promoter(sp) was cloned and macrophage special expression vector (psp-EGFP-Ipr1)was constructed which could express in macrophage.Expresion of Ipr1 in macrophage of creature could prevent tuberculosis, on this basis of construction of macrophage special expression vector, psp-EGFP-Ipr1 was trasfected into bovine fetal fibroblasts by electroporation and positive cells was selected to produce transgenetic cloned embryos by Somatic Cell Nuclear Transfer(SCNT).Cattle of anti-tuberculosis will be produced by embryo transfer . breeding for special disease-resistant animals will come ture by transgene method, this method provides a new means for prevention of tuberculosis The results are as follows:1. Ipr1 gene was cloned from C57BL/6J lung with PCR, which was 1614 bp and comprised intact coding regions .Sequence analysis demonstrated that its homology with the relative region of Ipr1 gene sequence on GenBank was 99 %. There are 2 site-mutations which occurred in the region of intron, so they could not infuence protein's function.2. .By the strategy of blunt end cloning.Then the objective fragment Ipr1 on was cloned directionally into eukaryotic expression vector pEGFP-C1.then macrophage special promoter(sp) was subcloned into eukaryotic expression vector pEGFP-C1.Non-fusion expression vector which contains resistant gene neo and reporter gene EGFP was constructed.3.Fusion expression vector pEGFP-C1-Ipr1 was transfected into RAW264.7 culture in vitro by electroporation.After 24h,Ipr1 and EGFP fusion protein was observed in endonuclear. Fluorescence expression was not detected in the cytoplasmic4.Macrophage special expression vector psp-EGFP-Ipr1 was transfected into bovine fetal fibroblasts culture in vitro by electroporation. The expression of report gene EGFP was observed in positive cells derived from G418 selection under fluoroscope. Monoclonal cell was selected with sterile tip,and expand culture.Positive cells which were obtained via culture in vitro could express Green Fluorescence protein( GFP). PCR results showed that Ipr1has been integrated into genome of bovine fetal fibroblasts.Cattle for anti-tuberculosis will be gained because of Ipr1 expression with psp-EGFP-Ipr1 integrating to fibroblasts.genome.5. Trans- Ipr1 gene bovine fetal fibroblasts was gained by G418 resistance select and proliferation of Monoclonal cell Donor cell were transfered to enucleated oocyte.Clone embryos were achieved.The tansgenetic embryos successfully developmented into blastocysts in vitro and Ipr1gene was integrated into the genome of embryos which can be trasplanted into uterus of receptor to produce trans- Ipr1 gene cow.