Cloning, Expressing And Functional Analysis Of β-casein From Chinese Human

Spleen is maximal immune organ in hunman body, contains many immunocompetent cells, the proliferation of lymphocyte is the indicator of immune. Thl/Th2cells together with cytokine secreted by other immune cells kept the Thl/Th2balance, and activate the normal or pathological immune responses, Thl/Th2cells balance is characterized by immune protection, imbalance can cause immune mediated diseases. IECs is known as the strongest function of the first defense barrier and a major immune response initiator of intestinal mucosal immunity in vivo. IECs is a bridge and link of contaction between innate and adaptable immunity, playing a key role in the induction of immune responses. But there’s less further study about HCSN2enzymatic hydrolysate reflecting the immune system.We cloned the CDS region of HCSN2gene, which came from a Chinese female’s breast tissue for the first time, and the amino acids deduced by the nucleotide sequence was used for bioinformatic analyses. Recombinant HCSN2was expressed by prokaryotic expression vector pET-30a-GST-CSN2. The enzymatic hydrolysate of recombinant HCSN2and HCSN2our research team got from human milk previously, were cocultivation with primary lymphocytes and IECs previously, to detect the proliferation and cytokine production.Through the study, we get the following results:(1) The human β-casein CDS region we reported and the same gene published in NCBI database shows that the homology was100%, such highly homology between different races suggests that this gene is conserved.(2) The protein contained no span membrane structures, and has a strong hydrophobic regions, the amino acids sequence of the β-casein of Homo Sapiens had the nearest relationship with Pan troglodytes.(3) The recombinant plasmid PET-30a-GST-HCSN2, induced by IPTG at37degrees Celsius for4h, the fusion protein expressed in Escherichia coli (E. coli) BL21(DE3) mainly existing in the inclusion bodies. The recombinant human β-casein was purified using NI-NTA affinity chromatography, and amount of recombinant protein could be1.10mg/mL.(4) The two kind of enzymatic hydrolysate could accelerate the proliferation of spleen cells induced by ConA in a dose and time dependent manner, and can significantly up-regulate the INF-y/IL-4value, improve Thl/Th2cells balance toward Th1dominance.(5) The two kind of enzymatic hydrolysate could significantly up-regulate the proliferation of IECs, which was dose-dependent and time-dependent, and up-regulated the content of IL-10, down-regulated the content of IL-8and TNF-a.