Construction And Cell Expression Of Mammary Gland-Specific Expressional Vector Of Human Lactoferrin Gene

The key resolution for improving the expressional level of foreign gene in transgenic animal is to select appropriate regulation elements, which can regulate foreign gene specifically and high efficiently expression in gland epithelial cell. In this research, we used the 5ˊflanking regulation element of goat beta-lactoglobulin gene as promoter and human Matrix attachment region as enhancer to construct mammary gland specific expressional vector of human lactoferrin gene and transfected into mammary gland epithelial cells cultured in vitro with lipofemintin . Postive cells were selected and purified by the expression of antibiotics gene (neo). So the cells can be used as sources to create transgenic animal by nuclear transplant techniques. The results of research is following: 1.For the purpose of constructing mammary gland specific expressional vector, the goatβ-lactoglobulin gene 5'flanking fragment was cloned by PCR amplification. It consisted in part of the gene upstream region ,the first exon and the first intron . After PCR product was recovered and purified, the aim fragment was cloned at T site of pMD 18-T Vector. The fragment was sequenced, and it was compared with original gene ,The results indicated the homology were 99.70%.In addition, base C was missed at -684 and base G was mutated A. it was compared with bovine BLG gene variant A, bovine BLG gene variant B and sheep BLG gene by Blast. The results indicated the homology were 90.55%,91.79% and 96.09%respectively. 2.The cloned fragment was cloned in expression vector pEGFP-C1 and constructed vector pBLG-GFP. It wan transfected into mammary gland epithelial cells cultured in vitro with lipofemintin and screened cell under UV microscope after 48h. we found that GFP gene expressed well which indicated the cloned fragment is useful. 3.Vector pBLG and pEGFP-C1 were digested by Ase I and Hind III . After products were recovered and purified, the aim fragments were connected with T4 DNA ligase and original expression vector pEB was acquired.Vectror pEB and pLF were digested by Hind III and Sal I . Middle expression vector pBL was got with same way. Vector pBL and pMR were digested by Sal I and BamH I. Final expression vector pBLM was received which includes promoter , aim gene, antibiotics gene 4.pBLM was transfected into mammary gland epithelial cells that were selected two weeks with G418. After lived cells were cultured through limited diluted way, which was identified by PCR. It indicates that aimed gene has conformitied into genome.