Construction Of Mammapy Gland-Specific Expressional Vector And Expression In Mouse Breast Carcinoma Cells For Human Lysozyme

Human lysozyme(hLYZ)participate in the body defense mechanism. It acts as a shield against bacterial infection and it has effects on malignant growth and immunoloregulation. So it has wide application in medicine and foodstuff industry. hLYZ can be purified from human breast milk, neutrophils, and urine. Because of the limitation of its source and the complexity of its extraction, and the potential risk of transmitting pathogens to the nursing infants, we can not get it by large-scale. So development of safer recombinant hLYZ is needed. Animal mammary gland bioreactor is a novel alternative strategy for production of recombinantproteins with many advantages including high production capacities, easy purification, faithful translational modifications and authentic biological activities of the expressed products.In this research, the mammary gland-specific expression vector pEBH was constructed, which was consisted of regulating elements regulation region of bovineβ-casein and expression sequence human lysozyme cDNA. Then pEBH was transfected into mouse breast carcinoma cells cultured in vitro and the expression cell strains were abtained. The expressed products were analysed by SDS-PAGE and western blot. The results of research are as following:1.Human lysozyme cDNA was cloned from Human placenta totle RNA with RT-PCR, which was 853 bp and comprised intact coding regions. Sequence analysis demonstrated that its homology with the relative region of human lysozyme on GenBank was 99 %. Within the open reading frame, three site-mutations occured which could not alter protein's function.2.By the strategy of directional cloning, hLYZ was fused with the commonly primary expression vector pBCP, then was cloned directionally into expression vector pEGFP-C1, the mammary gland-specific expression vector(pEBH)for hLYZ was constructed, which included resistant gene and EGFP reporter gene. It could also ensure EGFP and aimed gene express respectively.3.The vector pEBH was transfected into mouse breast carcinoma cells cultured in vitro...