【作者】 王艾平；

【导师】 粟永萍；

【作者基本信息】 第三军医大学 ， 军事预防医学， 2008， 博士

【摘要】 当前临床上出现了大量针对传统抗生素的耐药细菌株和一些强致病性病毒毒株,人类健康常常受到病原微生物的威胁。面对感染防治的严峻形势,我们不得不努力寻找新型的抗感染物质。经过近二十年的研究,发现在植物、昆虫和包括人在内的高等动物机体内存在近千种内源性阳离子肽——防御素（Defensin）。具有活性的防御素分子一般为3～6 kD,多数分子内存在6～8个保守半胱氨酸残基形成的3～4对链内二硫键外,还富含精氨酸、脯氨酸等特殊残基,整个分子在中性溶液中带正电荷;晶体衍射揭示防御素分子具有α螺旋、β折叠、二硫键桥、无规卷曲等单种或多种二级结构所形成的单体或多聚体空间结构。根据二硫键等结构的组成方式,防御素被分为α、β和θ等类型。研究已证实,有805种防御素不仅具有快速、强效地杀灭多种细菌的活性,并且还具有不诱导产生耐药菌株的特性;有261种防御素具有抗白色念珠菌等真菌和螺旋体的作用;有48种防御素具有抗HIV、HPV和或HSV等病毒的效果;有54种防御素可产生抗癌细胞的生物效应。此外,防御素作为机体免疫系统的重要组成部分,参与了免疫调节等多种生物功能。因此,防御素成为当前研究的热点领域之一。生物学家们认为防御素可能成为一种新型的抗生素,在作为抗感染制剂、食品防腐剂等方面具有广阔的应用前景。国外有数家制药公司在进行防御素的开发研究,但因防御素分子太小、含特殊氨基酸较多及具有的抗微生物活性等原因,目前制约防御素产品走上市场的主要瓶颈依然是尚未建立成本低廉、高效稳定的制备工艺。人α防御素5是由小肠潘氏细胞、泌尿生殖道粘膜等上皮细胞中表达的一种生物活性肽。通过对组织提取和化学合成肽的活性检测发现人α防御素5既可以高效杀灭多种细菌,又具有抗病毒的生物活性。基于人α防御素5在小肠隐窝上皮细胞中组成性或诱导性高表达的特点和肠道是人体最大内源性感染病原菌贮存库的关联性,我们认为α防御素5是感染防治研究的重要靶分子。因人α防御素5的组织提取和化学合成存在产量低、成本高等问题,所以基因工程制备应该是获得大量活性肽的理想途径。鉴于缺乏制备重组防御素相关工艺报道的现状,为满足肠道损伤修复与抗感染等研究的需求,本课题率先探索了人α防御素5的生物工程制备过程及重组产品的生物活性。现将研究中所采用的主要方法、获得的主要结果和结论简述如下:1.DEFA5基因的克隆与生物信息学分析采用RT-PRC技术,从经LPS诱导表达的LoVo细胞总RNA中克隆出DEFA5的阅读框序列,并构建pUCm-T-DEFA5重组载体。利用DNAStar、Antheprot 5.0等生物学软件与信息平台分析了DEFA5的信号肽、生化性质等特点,筛选出生物活性可能最强的mHD5序列。2.重组原核表达载体及其表达工程菌株的构建基于第一章所获得的mHD5序列和有关DEFA5的信息,模拟DEFA5信号肽与前片段的性质,筛选并克隆用于融合表达的承载分子mtrxA、trxA和malE的序列及其表达载体;同时,根据表达载体的特点设计特异引物,扩增出带有不同克隆位点的mHD5片段,分别构建了pQE-mHD5、pQE-mtrxA-mHD5、pET-mHD5和pMAL-mHD5表达载体及pQE-mHD5/M15、pQE-mtrxA-mHD5/M15、pET-mHD5/Rosetta-gami B和pMAL-mHD5/BL21等工程菌株。3.融合蛋白和rmHD5的表达、纯化与活性检测经IPTG诱导和表达条件的优化,获得了mtrxA-mHD5包涵体蛋白（MW.17135.724Da）和trxA-mHD5（MW.20646.552 Da）、malE-mHD5（MW.48602.468Da）2种可溶性融合蛋白的高效表达,表达量占菌体总蛋白的比例分别为40%、26.9%和26.7%,融合蛋白中目的肽所占百分比分别为21.2%、17.4%和7.4%。分别采用亲和层析、离子交换层析等不同策略对3种融合蛋白进行纯化、裂解与鉴定。从malE-mHD5酶解体系中分离纯化获得2.67 mg重组目的肽,并证实对大肠杆菌标准株（ATCC 25922）和金黄色葡萄球菌标准株（ATCC 25923）均具有抗菌活性。4.酵母表达载体的构建利用Leto软件优化出酵母细胞偏好的mHD5密码子,并设计合成1对长链引物,采用重叠延伸PCR方法扩增mHD5优化序列DNA。成功构建含有mHD5天然序列和优化序列的酵母表达载体pPIC9K-nmHD5和pPIC9K-omHD5。5.高拷贝转化酵母克隆的筛选与发酵工艺的建立选用SacⅠ限制性内切酶线型化重组表达质粒,利用载体与GS115酵母菌株基因组在5’AOX区进行单交换同源重组可产生多拷贝外源基因整合的原理,采用电击转化、筛选和再次电击转化的方法,将多拷贝目的基因整合到酵母细胞基因组中。通过PCR鉴定、G418抗性筛选和表型鉴定,分别获得了可耐受8mg/ml G418,表型为His⁺/Mut⁺的5株pPIC9K-omHD5/GS115高拷贝转化克隆（O05,O07,O08,O09,O10）和3株pPIC9K-nmHD5/GS115高拷贝转化克隆（N06、N08、N09）。挑选O05、O08、O09、N06、N08和N09 6株克隆在摇瓶中进行甲醇诱导发酵,采用RT-PCR技术鉴定所挑选的6株克隆均能在mRNA水平上成功表达目的肽的基础上,采用Western blot分别对其中的4个克隆株（O05、O09、N06、N08）进行了蛋白水平的表达鉴定,结果显示密码子经优化的mHD5表达量获得了显著性提高。筛选出表达量最高的克隆（O09）进行发酵罐高密度发酵实验。经过2次发酵罐发酵过程的探索,综合采用DO值监测、酵母细胞生长活力检测和目的蛋白表达的检测等措施,建立pPIC9K-omHD5/GS115种子工程菌的发酵罐发酵工艺,并取得不同时间段中DO值、pH值、空气/氧化传送速率、甘油/甲醇流加速率等参数的优化值。获得重组目的肽含量约10%的表达上清3.9L。6.重组肽的纯化与鉴定采用反相层析、亲和层析、离子交换层析和分子筛层析等分离方法,建立起重组肽回收率较高的纯化工艺:发酵上清→Ni柱亲和层析→阳离子交换层析→浓缩脱盐层析→冻干。共获得纯化重组肽152.9871mg,产品得率为39.2275 mg/L发酵上清。产品经HPLC分析,纯度为81.73%,经SDS-PAG、Western Blot和质谱鉴定证实产品成分是重组人α防御素5成熟肽。7.生物活性检测与作用机制探讨①抗菌活性检测应用平板法检测了重组肽的抗菌活性,结果表明重组产品对3种标准菌株ATCC25922、ATCC25923和ATCC 27853均具有较强的杀灭活性。采用稀释法测定了重组肽对14种菌株（包括3种标准菌株和13种临床分离菌株）的MIC,结果显示产品具有很强的杀灭G^-菌活性,对G⁺菌的作用稍弱。②抗菌机制初探应用扫描电镜和透射电镜观察了ATCC25922和ATCC25923 2种菌株与重组肽作用前后的形态变化。发现了重组人α防御素5成熟肽引起G^-菌细胞外膜脂质突起,内膜结构模糊,进而导致菌体肿胀、破碎的现象;而G⁺菌细胞膜结构未发现有明显改变。结果显示,人α防御素5是通过“地毯机制”作用模型发挥的抗菌活性。③抗病毒活性检测首先采用MTT法检测了重组产品对HeLa细胞增殖的影响,结果表明在100ug/ml以下浓度,重组肽对细胞无毒性作用。通过基因转染和病毒扩增与浓缩、纯化,成功制备基因组中含荧光蛋白阅读框的重组HPV5（含红色荧光蛋白基因）和HPV16（含绿色荧光蛋白基因）病毒液,测得后者的感染性滴度为5×10¹⁰。以HeLa细胞为靶细胞进行重组肽抑制HPV感染的实验,经荧光显微镜观察和流式细胞仪分析表明重组肽对HPV具有很强的抑制活性,且活性与浓度呈依赖关系,当重组肽浓度达66.7ug/ml时,抑制率达90%以上。抗病毒活性的时间-效应关系实验表明,人α防御素5抗HPV的活性发生在病毒感染细胞的早期过程,作用机制可能包括防御素作用于细胞后提高了靶细胞的抗病毒能力。总之,本课题完成了人α防御素5的开发性研究,建立了从基因克隆、载体及其表达系统的选择与构建、表达种子工程菌的筛选、发酵表达条件的优化与放大,直至产物的分离纯化、生物活性测定等整套工艺。为大量制备具有生物活性的人α防御素5及其它类似多肽搭建了平台,促进了防御素临床前研究的进程,并为其走向临床应用打下了基础。更多还原

【Abstract】 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.更多还原