| Mammalian defensins have received widespread attention due to their strong antibacterial ability,extensive antibacterial effects,and resistance to pathogenic microorganisms.Therefore,they are considered to be a new type of antimicrobial agent that can replace traditional antibiotics and have broad application prospects.However,the expression of natural defensins in pigs is low,the molecules are small,separation and purification are difficult,and the cost of chemical synthesis of defensins is too high.Therefore,genetic engineering technology has become the preferred method for obtaining large amounts ofβ-defensins.This experiment aims to construct a prokaryotic expression vector of porcineβ-defensin 2(PBD2)through gene cloning and recombination technology,in vitro expression to obtain large quantities of recombinant PBD2 and proved the antibacterial activity of recombinant defensins through in vivo and in vitro tests and also evaluated its safety in mice.It provides a certain theoretical basis for the application of defensins in clinical research and production,and lays a foundation for the further research and development of new therapeutic preparations.The main research contents and results are summarized as follows:1.Prokaryotic expression of recombinant protein PBD2Obtain PBD2 mature peptide gene sequence through Genbank.According to the preference of E.coli codons,optimize the gene sequence of PBD2,construct the p ET-32a-PBD2 recombinant expression vector,and transform it into E.coli BL21(DE3)p Lys S competent cells to induce the expression of recombinant protein,and verify the expressed protein by SDS-PAGE and Western blot.The results showed that the p ET-32a-PBD2 recombinant expression vector was successfully constructed,and the recombinant protein PBD2 was obtained after induction.The recombinant protein was subjected to affinity purification using a nickel column,and the concentration of the purified recombinant protein measured by the BCA protein concentration determination kit was 1.5μg/μL.2.Analysis of antibacterial activity of recombinant protein PBD2 in vitroIn order to verify the in vitro antibacterial activity of recombinant protein PBD2,standard strains of Escherichia coli(CMCC 44102),standard strains of Salmonella(ATCC9150),standard strains of Staphylococcus aureus(ATCC 25923)and Porcine extra-intestinal pathogenic Escherichia coli(Ex-P22,Ex-P27,Ex-P30)six kinds of pathogenic bacteria are indicator strains.The micro-antibacterial test shows that PBD2 has antibacterial activity against the above indicator bacteria.Among them,the minimum inhibitory concentrations against pig-derived extra-intestinal pathogenic Escherichia coli(Ex-P22,Ex-P27,Ex-P30)are 75μg/μL,and the minimum inhibitory concentration against the standard strain of Escherichia coli(CMCC 44102)is 37.5μg/μL,the minimum inhibitory concentrations for the standard strain of Salmonella(ATCC 9150)and the standard strain of S.aureus(ATCC 25923)are 150μg/μL.PBD2 was co-cultured with the standard strain of E.coli(CMCC 44102)and S.aureus(ATCC 25923)to prepare samples,and the ultrastructure of the bacteria was observed by electron microscopy.The results showed that the bacteria in the control group were intact,the appearance was plump and round,the surface was smooth and straight,and there was no phenomenon of cell membrane puncture and shrinkage and leakage of contents;the bacterial cells of the test group showed obvious shrinkage and were accompanied by obvious symptoms such as the leakage of contents on the surface of the cell wall.Intuitively demonstrated that PBD2 can destroy the integrity of bacteria,which proves that it has antibacterial activity.3.The protection test and safety test of recombinant protein PBD2 on BALB/c miceIn order to verify the antibacterial activity of recombinant protein PBD2 in vivo,through the infection test of pig-derived extra-intestinal pathogenic Escherichia coli Ex-P30 on mice,it was determined that the optimal infection concentration of mice on Ex-P30 was 1×10~8 CFU/m L.After the mice were infected and treated,the mice were dissected and pathological tissue sections were made.From the results of the pathological tissue sections,the inflammation in the infected group was obvious,the prevention group had a certain degree of inflammation,and the treatment group had a milder inflammation,indicating that PBD2 has a certain therapeutic effect on mice infected with pathogenic bacteria.Observation and analysis results of inoculation test of recombinant protein PBD2in mice showed that there were no obvious abnormal changes in the behavioral characteristics of mice in each group.Compared with before vaccination,the mental state,activity and food intake of the mice in the experimental group and the control group were normal,without adverse clinical reactions.The pathological tissue sections of the mouse kidney,heart,liver,spleen and lungs showed no obvious organic lesions.The results show that the recombinant PBD2 expressed in this study has no obvious toxic and side effects on the experimental mice and has good safety.In summry,this study successfully used the Escherichia coli expression system to obtain recombinant PBD2 expressed in the supernatant at a high level,and verified that it has biological activity against both Gram-negative bacteria and Gram-positive bacteria in vivo and in vitro.The inoculation test on mice proved that PBD2 has no toxic and side effects on experimental rats and is safe.This has certain reference value for the application of PBD2 in clinical research and production. |
PDF Full Text Request