Research On Gene Cloning Deer Serum Albumin And Its Bioactive Peptides

Sika deer is a natural "Treasure of Traditional Chinese Medicine",Deer blood has a significant effect on the tonic,blood,essence,stomach.Serum albumin is the most abundant protein in plasma and albumin is a very important position in deer blood.However,there is no complete deer serum albumin gene sequence or complete protein sequence in NCBI,GenBank and a series of international famouse database.In this study,the fresh sika deer liver was used as experimental material,and the complete albumin gene of deer deer was cloned firstly.The effects bioactive peptides derived from serum albumin on cell proliferation and inhibition of intestinal inflammation were investigated.Firstly,Gene cloneing and protein translating of deer serum albumin.The 11-month-old healthy male sika deer was selected as the experimental material and injected anesthesia without pain.Total RNA was extracted from liver cells by Trizol.The primers were designed using software CLUSTW and Premier Primer 5.0 for RT-PCR,PCR,TA cloning and gene sequencing experiments.The 2019 bp serum albumin gene sequence were obtained and translated into protein sequenceSecondly,the serum albumin protein sequence of the deer serum was simulated and digested to obtain a series of peptide molecules,and functional alignment analyzed by AQS system of bioinformatics,15 kinds of active short peptides were predicted.Then,the deer serumalbumin active peptide was obtained by chemical synthesisThirdly,effect of target active peptide on cell proliferation in vitro.The effects of 15 target active peptides on 3T3 cells were investigated.The labeled 1,2,3 target peptides could promote the cells proliferation after 48 h culture and theproliferation rate were respectively 22%,29%,30%.Then the three peptides were prepared into five gradients with concentrations of 1,0.1,0.01,0.001 and 0.0001 mg/mL for 24 h,48 h culture.The results showed that the activity of the active peptides had concentration-dependent manner.After 48 h culture.The active peptide concentrations were 0.1 mg/mL.The cell proliferation rates of peptide 1,2,3 were relatively well to be,25%,30%,26%,respectively.These results showed that peptides 1,2 and 3 could promote the 3T3 cell proliferation,with the optimum concentration of 0.1 mg/mL.Fourthly,the three active peptides were tested for the efficacy of inhibition of intestinal inflammation in vivo.The three active peptide of 1,2 and 3,PBS and HS-6 active peptides were selected respectively as the experimental group,the negative control group and the positive control group.DSS model mice were treated by intragastric ad ministration.The results showed that the clinical symptoms of blood glucose,body weight and intestinal tissue of experimental group were similar to those of the control groups,which was better than that of the PBS group,so the deer serum albu min active peptide has a significant effect on anti-inflammatory.