Construction And Expression Of Mouse β-Casein Gene Recombinant, And Purification Of Expressive Protein And Immunity Research Of Its Hydrolysate

Research backgroud There are many bioactive peptides in beta-casein (β-CN), which play many physiological functions in vivo, so beta-casein is called strategic and active protein. To prepare and purify beta-casein, it is important to research beta-casein and bioactive peptides. Traditional method to obtain beta-casein was isolating and purifying from milk. By traditional method, there are low output and complicated program in preparing and isolating beta-casein. With the development of biotechnology, gene engineering is used to prepare beta-casein. There is very consistent character among animals and human beta-casein genes, especially between mouse and human, so the mouse beta-casein gene was chosed in our research.Object To construct the recombinate plasmid pGEX-KG -β-CN, transfer into host E.coli DH5αto express, purify the expressive protein and study some immune function of its hydrolysate by enzymes.Methods The total RNA was extracted in the mouse mammary gland in lactation. The exon of beta-casein was obtained by the method of RT-PCR. Then exon are connected with the expression vector to form the expression plasmid, which was transferred into the expression host E.coli DH5α. Then E.coli DH5αwas induced with the low concentration of IPTG for overnight in 20℃. The bacteria were broken up by ultrasonic wave, and its proteins were analyzed by SDS-PAGE. In bacterium hydrolysate, GST-beta-casein protein was purefied by GST beads affinity chromatography. The beta-casein produced by gene engineering was hydrolyzed by trysin and chyomtry together for 60min, 90min, 120min, 150min, respectively. By two series of experiments both, in vivo and in vitro, in rat and mice, the effect of the hydrolysates of beta-casein on lymphocyte transformation (LT) were investigated.Results The sequence ofβ-CN gene was successfully cloned and the target gene was correctly inserted into pGEX-KG. The beta-casein with GST-tag in its N terminal was expressed in E.coli DH5α, its molecular weight being 52 KD, and the output accounted by 12.4% of the whole protein of the bacteria. The fussion protein of beta-casein was dissolved in supernatant of bacterium hydrolysates. The fussion protein was successfully purefied by GST beads affinity chromatography. The hydrolysates of beta-casein for 90min and 120min can promote lymphocyte transformation.Conclussion The expressed plasmid ofβ-CN (pGEX-KG-β-CN) was successfully constructed. The soluble fussion protein of beta-casein with GST-tag in its N terminal was successfully expressed in E.coli and purified by GST beads affinity chromatography. The hydrolysates of beta-casein have immune function.