Expression And Purification And Activity Analysis Of Human β₂-Defensin And MRNA Detection In Gingival Epithelium

Defensin that can be found in more microbial in the nature is animportant member of the endogenesis microorganism peptides families,which can be expressed in almost all type of cells including the plant andinsects cell.Recently, more and more concerns about the function of thedefensins families in nature immune system were given by the researches.Defensin is a cysteine and arginine rich positive ion antimicrobialpolypeptide, which has a low molecular weight, 6 conservativeaminothiopropionic acid residues which formed three intramoleculardisulfide bonds , the disulfide bonds can fold the peptide chain to tree beamβlamellar structure.Intramolecular disulfide bond is very important inmaintaining the stability of the Defensins molecular structure and it is also animportant basic structure in the antimicrobial activities and cell toxicity.Experiment in vitro testified that the defensins has the antimicrobial activityon the bacteria, eumycete, some enveloped virus, and cell killing activity ontumor. Drug resistance was not fond in the microbials up to now. With therevelation of βdefensins family genetic structure ,function and study ofpolypeptide biologic activity, the medical value of βdefensins has arise theattention of researcher .The powerful bacteriostasis function and distinctmechanism of action will bring hope to treat infection of drug resistancecausative organism. Oral cavity is not infected while suffering the challengeof various germ and stimuli of physico-chemical factor. In conventional ideamechanical barrier of mucosa epithelium, adaptive immunity of host, restricteffect of normal flora on pathogenic bacteria and opportunist, these allcontribute to it .Furthermore, it is correlated to antimicrobial polypeptide andprotein produced by organism .Oral epithelium provid the first line of defensebetween cavitas oris tissue and environment. βdefensins expressed inepithelial tissue andαdefensins in human neutrophil are important ingredientof natural defence reaction, they safeguard oral cavity health with otherdefence factor. Study indicated that HBD-1,HBD-2 play an important role inmaintaining health of the oral mucosa and gum tissue as composition ofepithelial tissue natural defence system . It has become a serious global issuethat bacterium is resistant to traditional antibiotic. Therefore it is imperativeto develop new high technology Antimicrobial.Defensins has the effect ofanti causative organism and can kill the clinic drug resistant strain.So it willbecome a new type of broad-spectrum antibacterials. Defensins are expressedvery lowly or not under natural condition .It can be expressed only under theinduction of microorganism or proinflammatory cytokines.But the chemicalsynthesis defensins can not ensure polypeptide to pair correctly, for it has 3-4pairs of disulfide bond which affect its biological activity. Defensin has greatpotentiality in gene engineering. But it worth further studying about how toraise expression level and stability of gene expression product, synthesizedefensins of more powerful sterilization vigor and broader sterilizationspectra. Therefore, researcher attempt to obtain defensin through geneengineering. At present scholars all over the world did a lot of research ongene engineering of defensin. So they lay a solid foundation for thedevelopment and application of defensin. But until now there is no researchpublication of systematic gene engineering production which is far fallbehind of the research of mechanism of action and function. The study ofgene expression of defensin was initially undertook in prokaryotic expressionsystem. But the antimicrobial peptide gene can only be expressed in the formof fusion protein, because of the lethal effect of antimicrobial peptide onbacterium and the lack of oyl-ammonification function of prokaryoticexpression.E.coli has been used as host cell usually in gene engineering forits rapid growth velocity and definite expression system .But there are twokey issue to solve if we want to establish E.coli as host cell ,that is theanti-Gram-negative-bacterium activity of HBD-2 and the degradation ofsmall peptid segment by enzyme.The above problem can be solved byselecting E.coli strain of low protease activity and inhibiting theanti-Gram-negative-bacterium activity of HBD-2 through plasmid.Thus it isfeasible to express defensin using prokaryotic expression system. This studydo some research on whether we should construct and express βdefensin interms of HBD-2 and observe the expression condition of HBD-2 inparadentitis ,so we can do some basic work on the local application of βdefensin. Method: 1.The samples from 14 chronic periodontitis patients and10 normal gingival epithelium were detected for HBD-2 mRNA using theRT-PCR method. 2. The human-defensin gene fragment was synthesized invitro, a Nco I and a EcoR I restriction enzyme sites were introduced into theupstream and downstream of the gene. To develop a human defensins 2fusion prokaryotic expression vector, firstly, clone the gene into the pMD18Tvector in orientation, then subclone the fragment into the pET32a, aprokaryotic expression vector, result in a defensin fusion expression genecontaining original TrX-A gene in frame. Transform the constructed vectorinto the BL21(λDE3) TrX-B plysS E.coil, induced by the IPTG, theexpression of the fusion protein and the biological activities of the expressedprotein were detected. The defensins gene obtained by PCR was confirmedby the DNA sequencing after cloning into the pMD18T. 3. Simulate the lowdose fermentation, change different culture medium, temperature,time, pH,get a optimizing fermentation condition, the concentration and time of theinductor was also optimized. DNA STAR was used to analyze the HBD-2hydrophilicity and epi-position. 4.The BL21(λDE3) TrX-BplysS/pET32a/HBD2 was ferment under the optimized condition. The richyield of the expressed protein was fractional precipitation saturatedammonium sulfate, desalt and purification was through the Cu ChelatingSepharose Fast Flow. the elute was assayed by electrophoresis and digest bythe enterokinase. 5. The purified and enterokinase digested HBD-2 was usedto detect the biological activity of various strains and cultural oral carcinomaeukaryotic cell.To observe the inhibiting effect of HBD-2. Result: 1.All thesamples from the 14 patients and normal cells were positive after theRT-PCR assay, the variance between them is statistical significance( P < 0.05). 2.The synthesized HBD-2 gene cloning in the pMD18T was confirmedby the DNA sequencing, digested with the NcoI and EcoR I subclone into thepET32a, the ligation was transformed into the E.coil competent, arecombinant plasmid pET32a-HBD2 was obtained. PCR and restrictionenzyme digestion were used to confirm the plasmid, a 137 bp band appearedafter double digestion with the EcoRI and NcoI and by PCR assay. Theexpression of the pET32a-HBD2 in the BL21(λDE3) TrX-B plysS wasdetected by the SDS-PAGE, about 21 kD(the molecular weight theoreticallyof the recombinant protein) band was observed compared with the originalvector pET32a. 3. There are different expressions of recombinant plasmidpET32a-HBD2 under the different conditon of engineering bacteria. Theexpression of the pET32a-HBD2 in the BL21(λDE3) TrX-B plysS wasdetected by the SDS-PAGE,about 21 KD band was observed. The yield of theexpression protein is different in the different condition, a rich yield canobtained in the M9B2 culture medium,pH 7.3-7.5,32℃,after the 1.2mmol/LIPTG induced 5-6h. The hydrophilicity of the recombinant protein wasboosted up by the TrXA, HisTag and enterokinase added in the N-termianl,illustrating the epi-position was not diminished. 4.pH7.2, dissolved oxygen28%, final concentration 1.2mmol/L IPTG, 32℃,6h were determined as theoptimum condition after a series screening optimized condition in theindustrial product. 165g recombinant bacteria was obtained in the 10Lfermentation culture. The recombinant protein existed mainly in the20%-40% saturated ammonium sulfate by fractional precipitation ofmycoprotein, interest protein exist in the 100-200mmol/Limmidazolbuffer .The concentration of the purified recombinant protein is1.5347mg/mL, and a 4.5 KD band appeared after the digested with theenterokinase on the Tricine-SDS-PAGE gel. 5. Inhibition zone assays wereperformed to check the bacteriostasis of purified HBD-2 protein, it hasbactericidal activity to Staphylococcus aureus, salmonella Pasteurella.Idicating it has different growth inhibiting effect on different strain. And thesize of zone is positive correlate with the protein dose. The growth of oralcarcinoma cell aslo be inhibited by the HBD-2.With the increasing actiontime of HBD-2, most of the oral carcinoma cell turn round and dim , and theproperty of refraction decreasing ,some even split and die. Conclusion: 1.Expression of the HBD-2 was detected in the normal gingival epithelium, butof significant high level in the chronic periodontitis patients gingivalepithelium .This indicate that cytokine involved in chronic periodontitis startHBD-2 genetic transcription and translation .Thus the HBD-2 expressionincrease. We confirm that HBD-2 has both inherent and inducible expressionin gum tissue. 2. The synthesized HBD-2 gene was cloned into thepMD18T, the sequences of the gene were completely matching with thereported sequences after DNA sequencing assay. 3. HBD-2 fragmentderived from restriction enzyme digestion was ligated with the pET32a underthe T4 DNA ligase control, a pET32q/HBD2 containing the HBD-2 fusiongene was obtained. The HBD-2 gene inserted into the pET32a in frame wasconfirmed by the DNA sequencing assay. 4. Transform recombinantplasmid respectively to BL21(λ DE3)TrxB pLysS .The expressed fusionprotein was dissolved and 16.12% of total bacterial protein is the fusionprotein by SDS-PAGE assay. 5. The analysis of the hydrophilicity andepi-position were predicted by the DNA star software. The result show thatHBD-2 have high hydrophilicity and epi-position. 6. We studuy severalfactor including medium composition, culture condition amd IPTG inductioncondition and time, which affect the expression of interest protein.BL21(λDE3) TrxB pLysS is Strain of HBD-2 engineering bacteria.The yieldof the expression protein is different in the different condition, a rich yieldcan be obtained in the M9B2 culture medium,pH 7.3-7.5,32℃,after the1.2mmol/L IPTG induced 5-6h. 7. Fermentation parameters of pH 7.2,dissolved oxygen 28%, rotation speed 400r/min, ventilatory capacity20L/min ,final concentration 1.2mmol/L IPTG, 32℃ ,6h were determined asthe optimum condition after a series screening optimized condition in theindustrial product. Recombinant bacteria was obtained in the 10Lfermentation culture. 8.The HBD-2 was purified by the fractionalprecipitation saturated ammonium sulfate, and Cu Chelating Sepharose FastFlow, the 94% purified protein was obtained at last. 9. The inhibition zoneassays were performed to check the bacteriostasis of purified protein , it hasbactericidal activity to Staphylococcus aureus , salmonella, and so on. Thesize of zone is positive correlate with the protein dose. The growth of oralcarcinoma cell aslo can be inhibited by the HBD-2. 10.The most creative ofthis study is that we construct TrxA,His?Tag,enterokinase and HBD-2 fusionprotein expression .So the effect of expression product endangering host canbe screened. At the same time it can decrease the procedure of purifyingprotein, reduce the cost and enhance recovery rate of interest protein .Up tonow, there is no similar report appeared in the Chinese journals.