Prokaryotic Expression And Preliminary Study On The Function Of Rhesus Monkey Theta Defensin 1(RTD-1)

Since the 19 th century,antibiotics have played a vital role in the prevention and treatment of bacterial diseases in China’s farming industry.With the widespread use of antibiotics,the corresponding abuse and residue problems have also appeared one after another.Food safety and healthy farming have gradually been affected by people.Therefore,it is urgent for animal husbandry and related industries to explore products that can replace antibiotics.Therefore,it is the only effective way to find antibacterial drugs that have no drug resistance,no residue,anti-inflammatory and improve the body’s immunity.Theta-defensin 1(RTD-1)is the only cyclic peptide of animal origin,its molecular weight is generally around 2 k Da,has a broad-spectrum antibacterial,no residue,no drug resistance,stable physical and chemical properties,and anti-inflammatory and influenza suppression Virus adhesion and other functions.However,the cost of defensins from biological extraction or chemical synthesis is too high,the yield is low,and the purification steps are cumbersome,which has become a major obstacle to its clinical application.Increasing the yield of θ-defensin 1 through biorecombination synthesis technology is the current economic and efficient way,and has good application prospects.In this study,the base sequence of θ-defensin 1(RTD-1)was designed according to the codon bias of E.coli,and splicing cyclization of RTD-1 was mediated by the highly efficient intein Npu Dna E.Add the His tag to separate and purify the target protein.Using E.coli expression system to induce the expression of RTD-1,preliminary study on its physical and chemical properties,and to explore its antibacterial effect in vivo and in vitro,the main research and content are as follows:1.Using DNAMAN,Primer Premier 5 and other molecular cloning software,design and synthesize the gene sequence based on the amino acid sequence of θ-defensin(RTD-1)and intein Npu Dna E.Gel electrophoresis and sequencing analysis showed that the band size was consistent with the theoretical size(498 bp).The fusion fragment was cloned into the large intestine expression vector p ET28 a,and the NRTD-1-p ET28 a recombinant expression vector was constructed.E.coli DH5α competent cells were then transformed.2.After the NRTD-1-p ET28a-DH5α recombinant strain was identified as positive by bacterial liquid PCR,the PCR product was sequenced for double verification to ensure that there were no mutations or deletions in the fragment.The NRTD-1-p ET28 a plasmid was then extracted to transform E.coli BL21(DE3)competent cells.3.After the expression product of the recombinant expression strain NRTD-1-p ET28a-BL21 induced by IPTG was broken,Tricine-SDS-PAGE electrophoresis analysis showed that most of the fusion protein existed in the lysed supernatant,and its expression form was soluble expression.The expression level was about 239 mg/L.The fragmented supernatant was purified by Ni-NTA column affinity chromatography,and a specific band with a molecular weight of about 2 k Da was found in the product,which was consistent with the theoretical size,indicating that RTD-1 had been successfully spliced by intein.4.The purified sample was further separated and purified by dextran gel chromatography analysis.The identification results by thin layer chromatography showed that the expressed RTD-1 had been successfully cyclized and the structure was relatively stable.The stability,hemolytic activity,and bactericidal ability of RTD-1 were measured.The results show that it has good resistance to protease,acid and alkali,and heat resistance.It has strong bactericidal activity against multiple strains of pathogenic bacteria,high safety,and no hemolytic activity.5.Using RTD-1 crude and pure product as a sample,preliminary study was conducted on the protective effect of RTD-1 on mice infected with E.coli.The experiment was divided into the infusion group and the injection group.The results showed that the protection rate of the injection group was higher than that of the infusion group.After 48 hours,the mouse survival rates were 37.5% and 12.5%,respectively.Both groups have protective effects,indicating that RTD-1 has a certain protective effect on mice infected with E.coli.The above research shows that this study successfully constructed the θ-defensin 1(RTD-1)E.coli expression system engineering bacteria,and conducted a preliminary study of its function,through in vivo and in vitro tests to prove that RTD-1 against Gram-positive bacteria He Gram-negative bacteria have a certain bactericidal activity,which provides a certain theory and basis for the development of θ-defensin 1 as a new anti-disease drug.