| Reg3y is a member of regenerating gene which contains 149 amino acid residues and it belongs to C-type lectin IV family. Reg3y is expressed mainly in the epithelial tissue of the mammalian gut and skin, with antibacterial, anti-inflammatory and promoting cell proliferation and other functions. It plays an important role in the immune process of pathogens. In view of the multiple functions of this protein, Reg3? has a very broad prospect in the prevention and treatment of neonatal piglets. In this study, the recombinant Reg3y protein was expressed in Escherichia coli BLR strain using prokaryotic expression vector the pEI-Reg3? plasmid. The recombinant Reg3? protein was purified with two steps of ammonium s ?lfate precipitation and self-cleavage. The antibacterial activity was analysized by radial diffusion. To enclose the expression characterisitics of Reg3? in intestine of piglets, 24 healthy 0 to 5 weeks of newborn piglets were euthanized and duodenum, jejunum, ileum, as well as colon were sampled for extracting the protein. The protein level of Reg3? were determined by western blotting. The main contents of this paper include the following aspects:1. Reg3? gene expression levels in different parts of the intestinal tract of newborn pigletsIn order to compare reg3? gene expression levels in newborn piglets during different periods in different parts, we take 0 to 5 weeks healthy newborn piglets of different parts of the intestine (duodenum, jejunum, ileum and colon tissue), adding RIPA strong lysis buffer and protease inhibitors, lysising tissue with electric homogenizer and ultrasonic cell disrupter and getting the protein solution.Finally,we use western blotting detect the expression levels of Reg3?, results as followings:In the jejunum, the expression of Reg3? is close to ileum, with age increasing protein expression increased gradually, and in 3 to 5 weeks of piglets in vivo protein expression relative newborn piglets expression was significantly different. The piglet intestine relative to other parts, and duodenal proteins expression variation is very small, in the colon, the expression of 3-4 weeks of protein relative 0 week piglets weight difference was significant, and in the fifth week relative to the previous two weeks down. In addition, duodenal expression is the highest among others in the same period. In 0 to 2 weeks, the expression of jejunum was only inferior to that of duodenum. But the expression of the ileum increased more than the jejunum by 3 weeks. And in the whole period of 0 to 5 weeks, the expression of Reg3? protein in the colon is relatively low.2. Using ELP-intein system to express and purify the recombinant Reg3? proteinTo get the purified recombinant Reg3? protein with a simple and economical method. The experiment using limit endonuclease Hind ? and Bsr G1 cut down the pET-EI vector and construct pET-EI-Reg3? plasmid with T4 ligase to connect the vector and synthesized Reg3? gene fragment. Transformed the plasmid into E.coli BLR strain in prokaryotic expression, using salt precipitation analysis characteristics and self shear properties of ELP-intein expression system,we get the purifed recombinant Reg3? protein. The purified supernatant were analyzed by SDS-PAGE electrophoresis and there is a band close to 16 kD after Coomassie brilliant blue stain and western blotting test. At the same time, the concentration of Reg? in supernatant was determined by dot blot hybridization method is 0.36mg/ml.3. Determination of the antibacterial activity of recombinant Reg3? protein in vitroIn order to determine the antimicrobial spectrum of recombinant Reg3? protein,we determine the antimicrobial effect of purified recombinant Reg3? protein and antibiotics (ampicillin and kanamycin). The results showed that the recombinant protein had no inhibitory effect on Gram negative Escherichia coli, and had obvious antimicrobial effect on Gram positive Staphylococcus aureus. |
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