Study On Gene-engineering Preparation Of Human α-defensin 5 And It's Bioactivity Against Several Pathogens

Today,more and more drug-resistance bacteria against conventional antibiotics and infectious virus strains emerge in clinical practices.Human health is being affected by pathogens.Under this situation,more light shoud be put on the seeking of new antimicrobials.In the past twenty years,about a thousand of peptides with cation,named as Defensins, were found in the bodies of plants,insects and mammals including human.Molecular weight of active defensins in general is 3.0～6.0 kilodalto,most of which has 3～4 intra-molecular disulfide bond formed by 6～8 cysteines,and with much special aminoacid such as arginine, praline etc.and with positive charge in pH7.0 solution.Crystal diffraction picture of defensin demonstrates that its spatial structure is monomer or polymers formed byα-helix,β-folding, disulfide bridge and/or irregular coiling.Confirmed by experiments,805 defensins are not only powerful to kill various bacteria,but also do not induce pathogen to generate resistance, and 261 defensins can inhibit fungi such as Blastomyces albicans,and sprirochete to infect, and 48 defensins block HIV,HSV and/or HPV to infect cells,and 54 defensins have biological function against cancer cell.Additionally,defensins take part in various biological reactions such as immunity adjust,as important component of immunologic system.So defensin becomes one of hot fields of research.Biologists predict that defensins would be a kind of new antibiotics in the near future with broad application promising in the fields of medicine,food conservation,etc.Nowadays,there are about ten oversea pharmaceutical companies takeing part in exploitation of defensins.However,the efficient,stable and cheap process for defensin product is not yet established for that defensins is small molecular,and active to kill various microbacteria,with many special aminoacids.Humanαdefensin 5 mature peptide(mHD5) is bioactive peptide mainly expressioned by epithelial cell of jejunum,ileum and genital tract,etc.It was confirmed that various bacterials and some kinds of virus are sensitive to mHD5 synthesised or extracted from tissue.Based on the relationship between mHD5-expressed constitutely and/or inducedly cells localize in the digestion tract and which is the biggest bank of in vivo pathogen in the body,Human defensin 5 should be a critical targeted molecule for research on prevention and treatment of infectious diseases.It may be an ideal method to product mHD5 by Biotechnology,due to the low productivity and high cost of mHD5 obtained from tissue extracting or chemical synthesis.On basis of scarce reports of gene-engineering product of defensin,and in order to satisfy the need required by studies on impairment and repair of digest tract and anti-infection et al.,this study was carried out to explore the process of gene-engineering production of mHD5 and to test its bioactivity.The methods,results and conclusion are as follows:1.clone and bioinformatics analysis of DEFA5The sequence of DEFA5' open reading frame was successfully inserted into the pUCm-T, which was cloned by RT-PCR from LoVo cell strain induced by LPS.Characteristics such as signal peptide,bio-chemical nature of DEFA5 was analyzed by bioinformatics working station and softwares such as DNAStar,Antheprot 5.0 etc.The amino sequence of mHD5, which is possibly the strongest bioactive peptide,was singled out.2.Construction of recombinant prokaryotic expression vectors and its bacteriaCarriers of mHD5 such as mtrxA,trxA and malE were selected,and/or cloned,including the suitable vectors through modeling the nature of prepropiece of DEFA5 obtained in the first chapter of this study,mHD5 with different clone sites was cloned by specific primers designed based on the characteristics of corresponding vectors,was linked with carrier,and was inserted into pQE-80L,pET-32a(+) and pMAL-p2x respectively.Prokaryotic expression bacteria such as pQE-mHD5/M15,pQE-mtrxA-mHD5/M15,pET-mHD5/Rosetta-gami B and pMAL-mHD5/BL21 were obtained successfully.3.Expression & purification and test of bioactivity of fusion protein and recombinant mHD5(rmHD5)Under the optimal situation,inclusion protein of mtrxA-mHD5(MW.17135.724 Da) and soluble protein of trxA-mHD5(MW.20646.552 Da)and malE-mHD5(MW.48602.468 Da) were expressed efficiently induced by isopropy-β-D-thiogalactoside(IPTG) with 40%, 26.9%,26.7%expression level,in which rmHD5 amounts to 21.2%,17.4%and 7.4% respectively。According to the nature of different proteins and peptides,different strategies such as affinity chromatography,iron-exchange chromatography were adopted to purify, cleavage,and/or to identify those recombinant protein and peptides.2.67mg rmHD5 was obtained through purifying the solution of purified fusion protein of malE-mHD5 cleavaged by Factor Xa.Results of experiment confirmed that rmHD5 showed different effects against E.Coli((ATCC 25922) and Staphylococcus aureus(ATCC 25923).4.Construction of Pichia pastoris expression recombinant vectorDNA fragment containing mHD5 coding sequence with biased codons of Pichia pastois designed by Leto software was amplified by PCR.Two vectors of pPIC9K-omHD5 and pPIC9K-nmHD5(with optimized and natural sequence of mHD5 respectively) were constructed successfully by molecule clone techniques.5.Screen of GS115 clones with multiple-mHD5 gene and establishment of fermentation processThe recombinant vectors was linearized by restriction endonuclease SacI which could create a single freedon end at 5' AOX region of the vector.Utilizing the mechanism of single-exchange homo-recombination between the vector and the genome of pichia pastoris cell,the methods of electro-transformation,screening and second electro-transformation were adopted to make the multiple heterologous targeted gene integrate into the genome of GS115,a strain of Pichia pastois,with His~+/Mut~+,Five transformed clones(O05,O07,O08, O09,O10) containing multiple omHD5 were singled out identified by PCR and G418, meanwhile three transformed strains(N06,N08,N09) containing multiple nmHD5 with the same phenotype were obtained.In bottle-culturing system,six clones(O05,O08,O09,N06, N08,N09) were confirmed by PT-PCR to transcribe targeted gene successsully induced by methanol.Four clones(O05,O09,N06,N08) were identified by western blot to express rmHD5,in which the clone with optimized sequence of mHD5 has significantly higher expression level of rmHD5 than that with natural sequence of mHD5.The clone(O09) with highest expression level of rmHD5 was chosed as engineering strain for following study of fermentation in tank-culturing system.Fermentation process with optimal parameters,which include the value of dissolved oxgen(DO),pH,transferring rate of air/oxgen and adding rate of glycerol/methanol in different fermentation stages,was establised through two trials of fermentation in 16L tank.About 3.9L fermention supernatant in which targeted rmHD5 occupies 10%of total soluble protein was obtained.6.Purification and identification of recombinant peptide Better purification process was developed,namely:supernatant of fementation→affinity chromatography(Ni)→cation exchange chromatography→condensation and desalting→lyophilization.152.9871 mg recombinant peptide was obtained,namely 39.2275 mg product could be recoved from 1L fermentation supernatant.The purity of product tested by HPLC is about 81.73%.The product is rmHD5 identified by SDS-PAGE and MS.7.Bioactivity test and mechanism explore of rmHD5①anti-bacterial activityTest results show 3 strains including E Coli ATCC25922,Staphylococcus aureus ATCC 25923 and P.aeruginosa ATCC 27853 are all sensitive to rmHD5 by Kirby-Bauer method. Minimal inhibition concentration(MIC) of rmHD5 to 14 strains of bacterial(including 3 international standard strains and 13 clinical isolated strains) were measured by Dilution method.In brief,rmHD5 shows a better bactericidal effect to G~- bacteria than G~+ bacteria.②anti-bacteria mechanismUnder scanning/transmission electron microscope,the membrant structure change of G~-bacterial caused by rmHD5 was observed,namely cell surface seems more rough and adheres to much something like hair.③anti-virus activityThe result of MTT test shows＜100ug/ml rmHD5 is nontoxic to HeLa cell.Virusinhibited test tells us that rmHD5 possesses good anti-HPV activity,66.7ug/ml rmHD5 could block 90%HPV16 to infect HeLa cell.In a word,with humanαdefensin 5(HD5) as a representative molecule,this dissertation proposed a process of gene-engineering preparation of defensin,which includes the clone of gene,the selection and construction of expression vectors & systems,the screening of expression-engineering clone,the optimization and scaling up of the fermention system,and the purification and bioactivity determination of the product.The platform for bulk product of bioactive HD5 was established,which facilitates the procedure of pre-clinical research of defensin,and make a base for its clinical application.