Construction Of Genetic Engineering Bacteria For Expression Of Antimicrobial Hybrid Peptide Attacin-Thanatin Gene And The Biological Effectiveness Of The Recombinant Peptide

There were two aims for this study:(1) to construct the genetic engineering bacteria for expression of the hybrid peptide AT gene;(2) to investigate the biological effectiveness of the recombinant antimicrobial peptide by antibacterial test and in vivo test taking rats as the model.To achieve the above aims,firstly,the full-length open reading frame(ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template.The hybrid peptide gene Attacin-Thanatin(abbreviated as AT) was generated by using the technology of gene splicing by overlap extension and touchdown PCR.The genetic engineering bacteria for expression of the antimicrobial peptide AT and mA in Escherichia coli and mammalian cell were constructed.According to the principle that host strain is sensitive to antimicrobial peptide,that is to say,the expressed antimicrobial peptide would inhibit the growth of the host strain,thereby the host strain was caused to "suicide",we developed the method to inspect the antimicrobial activity in vivo and the antimicrobial activity of hybrid peptide AT was detected by this method.Meanwhile,the products of prokaryotic expression were purified and antimicrobial test was carried out in vitro.The biological effectiveness of the recombinant peptide AT was studied by in vivo test taking rats as the model,the ability to regulate the growth performance,immune functions and intestinal morphology of weanling rats were inspected.The results are as followings:1.Attacin A gene was cloned by two-step RT-PCR from Drosophila stimulated by E.coli.Results of agarose gel electrophoresis indicated that it included 732 bp in length and encodes 221 AA which was consistent to the predicted size.The sequence analysis revealed that the nucleotide sequence of Attacin A gene had high similarity to other Attacin genes(96%-99%),which indicated that we have cloned Attacin A gene successfully.The nucleotide sequence of Attacin A gene had been submitted in GenBank(Accession No. EU008826).2.The hybrid peptide gene AT with the 660bp in length was generated using the technology of splicing by overlap extension and touchdown PCR using the recombinant plasmid pMD-AttA as the template.AT gene included Attacin A(189 amino acids in N terminal) and Thanatin(18 amino acids in C terminal).In order to avoid affecting their structure,a specific short peptide(-G-G-S-G-S-G-) was added between them.3.The prokaryotic expression vector pET-mAT(pe),pET-mA(pe) were construted successfully and transformed into Rosetta-gami^TM(DE3)pLysS.E.coli Rosetta cells harbouring the recombinant pET-mAT(pe) and pET-mA plasmid were induced with IPTG and analyzed by SDS-PAGE.Results showed that the molecular mass of recombinant fusion proteins were in agreement with the calculated value from deduced amino acid sequence,which indicated that Rosetta-gami^TM(DE3)pLysS could express the antimicrobial protein AT and mA. 4.To develop the method of inspecting the antimicrobial activity in vivo,the secombinant expression plasmid pET-mAT(pe) was transformed into E.coli DH5α,BL21(DE),Rosetta-gami^TM(DE3)pLysS respectively.IPTG induction was carried out.In order to optimize the method for assaying bacteriostatic activity in vivo,different factors were tested,including the susceptible strain(E.coli DH5α,BL21(DE3),Rosetta-gami^TM (DE3) pLysS),concentration of IPTG(0.0-1.0mM) and the initial bacterial cell density of the host strain(D(600nm) = 0.2-1.0) and so on.Optimal method was obtained at an initial cell density of D(600nm) = 0.3 with 0.1mM IPTG when the most sensitive strain E.coli DH5αwas taken as the host strain.After the recombinant plasmid pET-mAT(pe) was transformed into competent E.coli DH5αand induced with IPTG,cell growth was monitored by measuring the optical density at D(600nm) for continuous 14h.The growth curve demonstrated that the generation of E.coli DH5αharboring recombinant plasmid of pET-mAT(pe) was inhibited obviously compared with the normal E.coli DH5α.5.The His-tag of the fusion protein made it convenient for protein purification by Ni²⁺-NTA agarose affinity chromatography.The antibacterial activity of antimicrobial peptide AT and mA were determined by agarose diffusion assay.The results demonstrated that the recombinant antimicrobial peptide AT and mA were active to E.coli DH5α,E.coli BL21(DE3),Salmonella choleraesuis and Staphylococcus aureus.Among these test bacteria,E.coli was most susceptible.The toxicities of the peptide to eukaryotic cells were tested by its ability to lyse porcine red blood cells.We found the peptide had almost no detectable hemolytic activity.6.To express AT and mA in mammalian cell,firstly,we carried out the fusion expression with the enhanced green fluorescent protein(EGFP) to determine whether it is feasible to express in mammalian cell through observing the green fluorescent with fluorescent microscope.The results showed that after the recombinant eukaryotic expression vector pEGFP-mAT(ee),pEGFP-mA(ee) were transfected into Vero cells. There were green fluorescent in Vero cells and the fluorescent amount reached the maximum 72h later.The localization of green fluorescent indicated that the expressed fusion protein could secrete ecto-cell.The eukaryotic expression vector pCI-mAT(ee) and pCI-mA(ee) were constructed subsequently and transfected into Vero cells. According to the above results,SDS-PAGE was conducted after 72h of transfection. Results showed that there were predominant bands with apparent molecular weight of 23.54 kDa and 20.9 kDa which was consistent with the molecular weight calculated from the amino acid sequence.So,eukaryotic expression in Vero cells were carried out successfully.The supernatant of cell culture fluid were collected for the following usage.7.By testing the antibacterial activity to E.coli DH5α,E.coli JM109,Streptococcus, Staphylococcus aureus and Salmonella choleraesuis of the supernatant of cell culture medium containing the recombinant peptide AT or mA,we found that the higher concentration of the supernatant,the more inhibition of the bacteria,which indicated that the supernatant of cell culture medium contained the antimicrobial peptide AT or mA and could be as the material for the further study on rats.8.Eighty weanling rats with an average initial weight of 14.2±1.03g(means±SD) were randomly allocated to four treatments with five replicate per treatment.The supernatant of cell culture medium containing the recombinant peptide AT or mA was administered by intraperitoneal injection into rats for 7 days consecutively.At the same time,the supernatant of cell culture medium of normal cell and Ampicillin were as control.On day 8, the fresh E.coli was administered by intraperitoneal injection into rats and the rats were reared for another week.The results showed that the supernatant of cell culture medium containing the recombinant peptide AT slightly increased the average daily gain(ADG) of rats by 14.7%,13.8%and 12.1%compared with the supernatant of cell culture medium containing the recombinant peptide mA,the supernatant of cell culture medium of normal cell and Ampicillin.But the difference was not significant.There was no different effect on ADG among the supernatants of cell culture medium containing the mA and of normal cell and Ampicillin.After infected with E.coli,the growth performance of rats treated with the supernatant containing the recombinant peptide AT increased 34.18%,53.62%(p＜0.05) and 17.78%compared with the supernatant containing the recombinant peptide mA and of normal cell and Ampicillin.From the whole experiment period,the ADG of rats treated with the supernatant containing the recombinant peptide AT increased significantly compared with the supernatant containing the mA and of normal cell(p＜0.05),but there was no significant difference compared with Ampicillin.The supernatant containing the recombinant peptide AT or mA did not affect the relative weights of immune organ thymus gland and spleen,villus height of duodenum,but the villus height of jejunum increased by 74.2%(p＜0.01) and 77.4%compared with the supernatant of normal cell.Meanwhile,crypt depths of duodenum were decreased by 11.12%(p＞0.05) and 16.99%(p＜0.01) compared with the supernatant of normal cell.After infected with E.coli,the crypt depth of duodenum of rats treated with the supernatant containing the recombinant peptide AT or mA were decreased by 11.3%(p＜0.05),15.38%(p＜0.01) while the crypt depth of jejunum decreased by 4.8%,7.14%compared with Ampicillin.In addition,the level of IgM,IL-1βin serum of rats treated with the supernatant containing the recombinant peptide AT or mA increased obviously while the level of IL-1βdecreased after infected with E.coli.Conclusions:The genetic engineering bacteria for expression of the antimicrobial peptide AT and mA in Escherichia coli and mammalian cell were constructed successfully. Antibacterial test indicated that antimicrobial peptide AT and mA were active to E.coli, Salmonella choleraesuis,Staphylococcus aureus and so on.Among these test bacteria, E.coli was most susceptible.In vivo experiments taking the rats as the model demonstrated that the recombinant antimicrobial peptide AT possessed the biological effectiveness on regulating the growth performance,immune functions and intestinal morphology of weanling rats.