Study On The Clone And Expression Of Human Lactoferritin Gene And Production Of Transgenic Animal

Lactoferrin, also called lactotransferrin, is the main ferritin in milk existing in various tissues and cells. Lactoferrin has functions in unspecific immunity, cell development regulation and ferrum metabolism. Compared with other lactoferrin, human lactoferrin is more suitable for people uptake. A suitable expression system is required for heterologous expression of human lactoferrin. Among expression systems used, mammary gland is the most suitable organ for human lactoferrin expression. Human lactoferrin cDNA was cloned for construction of mammary gland expression vector which then was expressed in mice mammary cancer cells and mice organism. Transgenic rabbits and caprines were generated in trial by sperm vector.RT-PCR was adopted for amplification of lactoferrin cDNA from human mammary tissue. 800bp caprine β-lactoglobulin gene 5' end regulation sequence was amplified by PCR. Sequence amplified by homologous sequence-PCR was cloned into expression vector after digestion. Thus human lactoferrin gene mammary gland specific expression vector pLNCXHLF was constructed.Mice mammary cancer cell MA-782 was transfected by pLNCXHLF vector coated with liposome. Positive monoclonal cells were screened by G418 and PCR. Positive cells were embedded with sodium salt of alginic acid and hormone was added for induction. Human lactoferrin of 34K.D was_____________________________________Abstract______________________found in supernatant with highest content as 65mg/L/105 cells. The recombinant human lactoferrin could inhibit growth of E.Coli JM109 and could combine with ferrum ion.Recombinant vector coated by liposome was injected into mice testis followed by copulation with normal female mice. PCR and Southern blotting showed that exogenous gene was found in some of progenies. Human lactoferrin was found in positive female progenies' milk (4.8~6.7g/L) with the same migration velocity.Recombinant vector coated by liposome was injected into testis and spermaduct of rabbit and caprine. PCR and Southern blotting showed that exogenous gene was found in some of progenies. Testis injected rabbit could generate transfected sperm for pretty long time and exogenous gene was found in epdidymidis sperm 7 months after injection. Three caprine embryos were obtained by spermaduct injection and exogenous gene was distributed differently in 11 tissuses which showed that the embryos were chimera.