| The widespread use of poultry and animal antibiotics had caused a series of problems such as bacterial resistance and drug residues,which seriously threaten the quality and safety of food and human health,and are important hygiene issues.Therefore,focusing on bacteriostasis and sterilization functions and developing new green antibiotic substitutes have become an urgent problem to be solved.Porcine β-defensin-1(PBD-1)has a wide range of biological activities,a special antibacterial mechanism,is not easy to produce drug residues and drug resistance,and is considered to be one of the current green antibiotic alternatives.However,porcine β-defensin-1(PBD-1) has shortcomings such as low expression level and difficulty in extraction,and cannot meet the demand.The use of genetic engineering methods to reorganize defensins has become one of the main methods.Recombinant defensins are easily degraded by various enzymes in the gastrointestinal tract of animals after oral administration,and it is difficult to exert their functions.Microcapsule technology can coat the recombinant defensins to maximize the protection of their biological activity and bioavailability.Providing a new reference for the oral administration of protein drugs.In this study,an E.coli expression system was constructed in vitro to obtain biologically active recombinant PBD-1,and it was prepared into microcapsules with sodium alginate to provide a reference for the oral administration route of defensin.The specific research content and results are as following :1.Construction of recombinant porcine β-defensin-1(PBD-1)vector and expression and purification of proteinWe obtained the mature peptide gene sequence of porcine β-defensin-1(PBD-1)through Genbank,optimized the gene sequence of PBD-1 according to the preference of E.coli codons,constructed p ET-32a-PBD-1 recombinant expression vector,and it was transformed into E.coli BL 21(DE3)p Lys S competent cells to induce the expression of recombinant protein,which was verified by SDS-PAGE and Western blot.The results showed that the p ET-32a-PBD-1 recombinant expression vector was successfully constructed.After induction,the recombinant protein PBD-1 with a size of about 24.5 k D was obtained.The recombinant protein was purified by a nickel column,and the concentration of the purified recombinant protein measured by the BCA protein concentration determination kit was 320 μg/mL.2.Evaluation antibacterial activity of recombinant porcine β-defensin-1(PBD-1)in vitroThe agar hole diffusion method was used to analyze the purified recombinant protein in vitro bacteriostatic activity,the results showed that the diameter of the inhibition zone of recombinant PBD-1 against E.coli CMCC 44102 was 19.17±0.57 mm,and the inhibition zone of Staphylococcus aureus ATCC 25923 the diameter is 28.47±1.28 mm,the diameter of the inhibition zone of Salmonella ATCC 9150 is 21.97±0.98 mm,the diameter of the inhibition zone of pathogenic E.coli S5 is 21.99±0.94 mm,the inhibition of pathogenic E.coli S10 in the intestine the diameter of the bacteria circle is 24.91±0.76 mm,and the diameter of the inhibition zone of pathogenic E.coli S12 in the intestine is 27.45±0.88 mm.At the same time,the results of the microbial inhibition test showed that the recombinant PBD-1 had an inhibitory effect on E.coli CMCC 44102,Salmonella ATCC 9150,and Staphylococcus aureus ATCC 25923.The minimum inhibitory concentration of E.coli CMCC 44102 was 80 μg/L.The minimum inhibitory concentration for Salmonella ATCC9150 and Staphylococcus aureus ATCC 25923 are both 40 μg/L3.Preparation of recombinant porcine β-defensin-1(PBD-1)microcapsules and preliminary study on its release characteristics in vitroUsing sodium alginate as the wall material and purified recombinant PBD-1 as the core material,microcapsules were prepared by the injection drop method.The microcapsules were successfully prepared based on the indexes of the diameter,roundness and embedding rate of the microcapsules.Optimize the preparation conditions,and conduct preliminary study on the in vitro release characteristics of the prepared microcapsules.The results showed that when the concentration of sodium alginate was 1.5% and the concentration of calcium chloride was 1.5%,the prepared microcapsules had good roundness,a diameter of about 1.1±0.11 mm,and an embedding rate of 75.19%.The prepared microcapsules were relatively stable in simulated gastric fluid,and the release amount was about 15% at 5 h;the rapid and large amount release in simulated intestinal juice,the release amount reached about 96% at 5 h.The results showed that the recombinant PBD-1 microcapsules could resist the damaging effect of gastric juice and have good enteric solubility.In summry,this study took the expression and purification of porcine β-defensin-1(PBD-1)vector and protein construction in vitro as the research direction.Successfully constructed p ET-32a-PBD-1 recombinant expression vector and transformed it into E.coli BL21(DE3)p Lys S competent cells to induce the expression of recombinant protein,obtained biologically active PBD-1,and successfully prepared it as micro The capsule has laid the foundation for its later industrial application... |
