Construction Of Mammary Gland Dual-Expression Vector Containing Prolactin And T-PA Gene And Its Expression In Cells

In this research,prolactin gene was cloned from GuangZhong goat and the mammary gland dual-expression vector pPT was constructed which contained prolactin and t-PA gene .Then pPT was transfected into mice mammary gland cancer cells cultured in vitro and the expression cell strains were abtained. High-level expression vector was provided for the mammary gland bioreactor by nuclear transfer. This research can help to solve problem on how to increase the expression of foreign protein. The results of research is as following:1. Goat prolactin cDNA was cloned from Guanzhong milk goat anterior pituitary RNA with RT-PCR, which was 760 bp and comprised intact coding regions. Sequence analysis demonstrated that its homology with the relative region of prolactin on GenBank was 99 %. And mutations could not alter protein's structure and function .2. By the strategy of directional cloning, prolactin gene was inserted to mammary gland expression vector pEBT and mammary gland dual-expression vector pPT was constructed which contained prolactin and t-PA gene, which included resistant gene and EGFP report gene. And EGFP and aimed gene express fusion protein.3. The vector pPT was transfected into mice bovine ear fibroblast mammary gland cancer cells cultured in vitro using liposome. After G418 fastness screening, the positive cells low-level expressed stably. PCR identification demonstrated that the vector had integrated into mice mammary gland caner cellular chromosomes.The cells which transfected pPT expressed more EGFP protein than cells transfected Pebt...