【作者】 张伟；

【导师】 王金星；

【作者基本信息】 山东大学 ， 动物学， 2008， 博士

【摘要】 家蝇（Musca domestica）从幼虫到成虫均生活在杂菌横生的环境里,其体表带有多种病原菌,可以传播多种疾病。据报道约有100多个可引起人类和动物疾病的病原体与家蝇有关,包括伤寒、霍乱、杆菌性痢疾、肺结核、炭疽热、婴儿腹泻和一些寄生虫病等。虽然家蝇没有脊椎动物那样高度专一的免疫系统,但是在细菌感染或者其它物理、化学因子的诱导下,其体内可以快速生成免疫活性物质。这些活性物质主要包括多种抗菌肽、溶菌酶、酚氧化酶等。研究表明,家蝇体内的活性物质不仅对多种病毒、细菌、真菌、寄生虫具有杀伤和抑制作用,同时对多种癌细胞也具有杀伤和抑制作用,但是大部分对人体是比较安全的。昆虫体内活性物质的抗菌、抗癌作用是无脊椎动物先天免疫研究的热点之一。目前这些研究主要集中在果蝇（Drosophila melanogaster）、冈比亚按蚊（Anopheles gambiae）棕尾别麻蝇（Boettcherisca peregrinas）等中,已经分离出了多种具有抗菌和抗癌作用的抗菌肽及其它活性物质。在家蝇中的相关研究相对较少。因此,对家蝇的免疫系统的研究具有重要的理论意义和应用价值。本研究利用差异蛋白质组学的方法研究分析了家蝇幼虫血淋巴在细菌刺激前后蛋白质表达的变化。除了在昆虫中已报道的免疫反应蛋白,还确定了几种新的参与免疫反应的蛋白质。本研究还利用抑制性消减杂交的方法构建了家蝇cDNA消减文库,得到数个免疫相关基因和氧化还原酶系相关基因。为进一步克隆家蝇免疫相关基因提供了有价值的信息。然后通过同源克隆的方法从家蝇幼虫体内克隆得到了铜/锌超氧化物歧化酶基因,分析了它在细菌刺激不同时间后的转录模式,并通过原核表达的重组蛋白制备了家兔多克隆抗体,研究了该蛋白在不同组织中翻译模式,最后检测了该蛋白的活性。同时,通过RT-PCR方法分析了另一个免疫相关的泛素融合蛋白基因的转录模式,并通过原核表达获得了纯化的泛素融合蛋白,拟进一步分析其性质。这些研究使我们对家蝇的先天免疫反应有了进一步的认识,为了解家蝇独特的免疫防御机制提供了理论依据。本研究取得的实验结果如下:1.运用差异蛋白质组学的方法分析了细菌免疫刺激后家蝇幼虫血淋巴中蛋白质表达水平的变化。用大肠杆菌和金黄色葡萄球菌混合菌免疫家蝇幼虫后,用双向凝胶电泳的方法来检测血淋巴中蛋白质表达。在双向电泳图谱中共检测到350个蛋白质斑点,288个蛋白质斑点在免疫刺激前后没发生变化,11个蛋白质斑点免疫刺激后上调,37个蛋白质斑点免疫刺激后下调,14个蛋白质斑点只在免疫刺激后幼虫血淋巴样品中能检测到。MALDI-TOF-MS共鉴定了35个差异表达的蛋白质,包括7个免疫相关的蛋白质,如革兰氏阴性菌结合蛋白1,肽聚糖识别蛋白LF,核因子NF-κB p110亚基和溶菌酶X。除了在昆虫中已报道的免疫反应蛋白,还确定了几种新的可能与免疫反应相关的蛋白质,包括类DCN1样蛋白和cAMP依赖的蛋白激酶。另外还有一些参与解毒反应的蛋白质也被鉴定出来,如超氧化物歧化酶,谷胱甘肽硫转移酶,细胞色素P450等。RT-PCR分析了溶菌酶X,谷胱甘肽硫转移酶和超氧化物歧化酶在mRNA水平上的变化,结果显示这些蛋白质在细菌刺激之后转录水平的调节变化与翻译水平的变化是基本一致的的。上述结果表明,这些蛋白质可能参与了家蝇的先天免疫反应,为了解家蝇独特的免疫防御机制提供了理论依据。2.应用抑制性消减杂交技术,构建家蝇幼虫差异表达cDNA消减文库。以大肠杆菌和金黄色葡萄球菌混合菌液刺激的家蝇三龄幼虫与未刺激的同龄家蝇幼虫为消减杂交对象,分别提取mRNA,合成cDNA,刺激的为测试子（tester）,不刺激的为驱赶子（driver）,经两次消减杂交及两次抑制性PCR后就分离出测试子中差异表达cDNA片段,将差异表达cDNA片段与T载体连接,转化大肠杆菌,获得cDNA消减文库,经蓝白斑筛选和菌落PCR筛选部分阳性克隆进行测序及GenBank BLASTx分析,发现差异表达cDNA片段大部分分布于200-750bp之间,并筛选出数个免疫相关基因和氧化还原酶系相关基因的EST,如防御素基因、细胞色素氧化酶基因、溶菌酶基因、丝氨酸蛋白酶基因、过氧化氢酶基因、氧化还原酶基因,Dicer等。从而为家蝇免疫相关基因的克隆奠定了基础。3.根据GenBank上检索的Cu/Zn SOD mRNA序列,设计基因特异性引物克隆得到基因全长。Cu/Zn SOD基因全长为734bp,包含462bp的开放阅读框。基因的5’端含有9bp的非编码区,3’端含有263bp的非编码区,同时还有一个17 bppolyA尾巴。这一基因编码的蛋白质由153个氨基酸构成。蛋白质经过SMART预测,其中包含一个为Sod Cu结构域,没有信号肽,预测分子量为15.6kDa,理论等电点为5.67。为了进一步确认家蝇幼虫Cu/Zn SOD基因的转录模式,还利用RT-PCR的方法研究了在混合菌刺激后不同时间段这一基因的表达差异。结果显示在细菌刺激后3 h,Cu/Zn SOD表达开始增加,6 h时增至最高值,至12 h又有明显的下降。这一变化趋势说明在家蝇幼虫受到外界刺激后Cu/Zn SOD基因上调表达。这一结果与家蝇幼虫血淋巴的差异蛋白质组学分析所得到的结果一致。根据Cu/Zn SOD基因序列设计表达引物,将基因片段克隆到pGEX-4T-1载体中,转化大肠杆菌BL21（DE3）进行原核表达。重组表达的蛋白在上清中,没有形成包涵体,用Glutathione Sepharose 4B亲和柱直接进行纯化。利用重组表达并纯化的Cu/Zn SOD作为抗原免疫家兔,制备多克隆抗体。利用SOD多克隆抗体研究了SOD蛋白在不同组织中的表达模式。将细菌刺激前后家蝇血淋巴、中肠、脂肪体蛋白进行SDS-PAGE并转硝酸纤维素膜后,用SOD抗血清作为一抗,再用辣根过氧化物酶标记的羊抗兔IgG作为二抗进行Western blot分析。结果表明,天然的SOD蛋白主要存在于中肠和脂肪体中,在血淋巴也有较弱的表达。细菌刺激之后,在各组织中的表达都上调,但是在血淋巴中的上调较微弱。10%非变性聚丙烯酰胺凝胶进行酶的活性检测。实验结果表明,Cu/Zn SOD活性在IPTG诱导3 h以后,显示出明显的酶活性条带,纯化的Cu/Zn SOD也具有明显的酶活性条带。诱导前也有较弱的活性,可能转化菌株存在少量渗漏表达。4.本研究还对家蝇泛素融合基因(Mub_S27)在细菌刺激之后的表达模式进行分析,并进行了重组表达研究,拟进一步分析其性质。RT-PCR研究发现,在大肠杆菌和金黄色葡萄球菌刺激家蝇幼虫之后Mub_S27表达上调。通过蛋白质组分析在家蝇体内鉴定出了免疫相关蛋白NF-κB,它在细菌免疫之后诱导表达。Mub_S27可能在激活NF-κB的过程中起了重要的作用,而NF-κB参与了对细菌的防御过程,这表明Mub_S27可能在机体细菌防御过程中也发挥着重要功能。根据基因序列设计成熟肽表达引物,将基因片段克隆到载体pET-30a（+）中,转化其感受态细胞进行原核表达。并用His-Bind亲和柱纯化得到纯度较高的重组蛋白。本研究增强了对家蝇免疫防御体系的了解,丰富了对昆虫天然免疫机制的认识。更多还原

【Abstract】 The house flies（Musca domestica）live in surroundings full of various microbes. They can spread many diseases owing to a lot of pathogen on their body surface. House flies have been implicated in the spread of over 100 pathogens that may cause diseases in humans and animals,including typhoid,cholera,bacillary dysentery, tuberculosis,anthrax ophthalmia and infantile diarrhea,as well as parasitic worms. But they can thrive without causing infection.Therefore it is presumed that the house flies have unique immune defense mechanism.House flies lack adaptive immune system like the vertebrate.But they can produce immune active materials quickly when infected by bacteria and other pathogens.The active materials contain some antimicrobial peptides and lysozymes etc.They can kill and inhibit not only various viruses,bacteria,fungi and parasites but also some cancer cells.Most of them are safe to humans.The anticancer effect of the active materials from flies are the focus of studies.Some antibacterial peptides and other active materials have been separated from Drosophilas,Boettcherisca peregrinas and Anopheles gambiae.Therefore,studies in the immune system of the house flies have important academic value and good application prospect.In this study,comparative proteomic analysis was performed to detect the change of the protein expression in hemolymph of bacterial challenged and unchallenged house fly larvae.We also identified several novel immune response proteins in addition to those already reported immune response proteins in insects.And a suppression subtractive hybridization cDNA library was constracted.Differentially expressed genes were identified in larvae of house fly challenged for 12 hours with Escherichia coli and Staphylococcus aureus versus those from unchallenged house fly. A superoxide dismutase gene was cloned from the house flies by homology cloning. The transcriptional pattern of Cu/Zn SOD gene in house fly chanllenged with bacteria for different periods was analyzed using RT-PCR.The antibody was obtained with purified recombinant Cu/Zn SOD,and then the translational pattern of this gene was characterized using Western blot.Furthermore,the expression patterns of the gene encoding ubiquitin were studied.And the recombinant protein was purified for further analysis of its characteristic.The results of the studies are as follows:1.To understand immune response of the house fly（Musca domestica）to bacterial infection at the protein level,we performed comparative proteomic analysis. The protein expressional profiles in hemolymph of bacterial challenged and unchallenged larvae were examined by two-dimensional gel electrophoresis（2-DE）. Among～350 reproducibly detected protein spots on each gel,288 present at uniform levels in all samples,11 were up-regulated,37 were down-regulated in hemolymph from immune-challenged larvae,and 14 spots detected exclusively in challenged larvae.Mass spectrometry identified 35 differentially expressed proteins,including immune related proteins,such as Gram-negative bacteria-binding protein 1, Peptidoglycan-recognition protein- LF,Nuclear factor NF-κB p110 subunit and Lysozyme X.Proteins involved in detoxification and proteins with a potential role in the immune response were also identified,such as superoxide dismutase,glutathione S-transferase,cytochrome P450.We also identified several novel immune response proteins including DCN1-like protein and cAMP-dependent protein kinase,in addition to those already reported immune response proteins in insects.The results suggest that these proteins may be involved in innate immunity,providing new insights for the innate immune mechanisms of the house fly.Semi-quantitative reverse-transcription polymerase chain reaction（RT-PCR）analysis confirmed the changes of Lysozyme X,Glutathione S-transferase D5 and Superoxide dismutase [Cu-Zn]at mRNA levels,indicating that these proteins were also regulated at transcriptional levels.2.A suppression subtractive hybridization cDNA library of Musca domestica was constructed for identifing differentially expressing genes in larva of house fly challenged for 12 hours with Escherichia coli and Staphylococcus aureus versus those from unchallenged house fly.First,total RNAs were isolated from these different house flies.Then,mRNAs were purified from these total RNAs.After that, Single-strand cDNAs and double-strand cDNAs were synthesized.After digestion with restriction enzyme of Rsa,cDNAs were obtained,cDNAs from challenged house fly,named tester,then were divided into two groups and ligated to the specific adaptor 1 and adaptor 2R respectively.The cDNAs from unchallenged house fly, named driver,didn’t ligated the adaptors.The cDNAs from tester and driver hybridized twice,then underwent two times of nested PCR,subtractive cDNAs with adaptor 1 and adaptor 2 in tester were obtained and enriched.The subtractive cDNAs were used to constructed the library with pGEM-T Easy vector and transfored into DH5α,the subtractive library of Musca domestica was set up and amplificated. Screening positive clones by PCR shows that they all contain recombined plasmids with 200bp-750bp inserts.After sequence alignment by BLASTx,several ESTs of immunity related genes and oxidoreductase protein genes were found,such as defensin,cytochrome oxidase subunitⅡ,lysozyme,serine protease,catalase, oxidoreductase.Thus,the cDNA subtractive library of Musca domestica would provide sold basis for screening immunity related genes of the house fly.3.The full length cDNA of the Cu/Zn superoxide dismutase（Cu/Zn SOD）was cloned from house fly.The full-length Cu/Zn SOD cDNA（734bp）of Musca domestica was included a 9 bp 5’ untranslated region（UTR）,a 462 bp open reading frame and a 263 bp untranslated region in the 3’ UTR with a 17 bp poly A tail.The ORF encoded a 153 amino acid protein with no signal peptide.It has a calculated molecular mass of 15.6 kDa and a predicted isoelectric point of 5.67.The mature protein included one Sod_Cu domain.To further confirm the expression pattems of the gene encoding Cu/Zn SOD, RT-PCR was used to study the differences in gene expression after bacterial challenged in different time.The result demonstrated that the expression of Cu/Zn SOD increase from 3h after bacterial challenged,reached to a maximum value at 6h and was decreased post 12h challenged.This result was consistent with the result derived from comparative proteomic analysis of the house fly larvae to bacterial infection.This result suggested that the Cu/Zn SOD protein may have the function in the innate immunity of the house fly.The sequence coding for Cu/Zn SOD protein was amplified and ligated into expression vector pGEX-4T-1.The recombinant vector was transformed into competent E.coli BL21（DE3）host cells.The recombinant Cu/Zn SOD was epressed in the supernatant and was purified with Glutathione Sepharose 4B chromatography. Polyclonal antiserum was obtained from rabbit after continuous injection of recombinant Cu/Zn SOD.With polyclonal antibodies we also studied the expression patterns of the nature protein in different tissues including hemolymph,midgut and fatbody after bacterial challenged.After SDS-PAGE,different proteins were transfered to the Nitrocellulose membrane.After incubation with polyclonal antibodies and second antibody （HRP-conjugated goat anti-rabbit IgG）and visualization the natural Cu/Zn SOD was characterized as a 16kDa protein in hemolymph,midgut and fatbody,After bacterial challenged,the expressions in hemolymph,midgut and fatbody were all increased,but was increased weakly in hemolymph.The activity of recombinant Cu/Zn SOD was detected with Native-PAGE（10%）, and the result indicated that the specific band of Cu/Zn SOD with high activity was found after the induction with IPTG for 3h.Furthermore,purified Cu/Zn SOD showed high activity.Besides,weak activity was also found before induction,which suggested that a small amount of leakage expression of Cu/Zn SOD maybe take place in the transformed strain.4.The expression pattern of Mub_S27in the house fly challenged with bacteria was analyzed by RT-PCR.The result showed that after Staphylococcus aureus and E.coli infection,the expression of ubiquitin-fusion gene was upregulated from 2 to 8 h and then returned to the original level.We have also identified the NF-κB protein in the house fly larvae by proteomic analysis.It was inducing expression after bacterial challenge.Hence,we can speculate that the up-regulated Ub could cause TRAF6 ubiquitination and eventually activate the transcription factor NF-κB.The activated NF-κB would then induce the expression of a number of antimicrobial peptides. Therefore Mub_S27maybe involved in the defense against bacteria.The sequence coding for mature protein was amplified and ligated into expression vector pET-30a（+）.The recombinant vector was transformed into competent E.coli BL21（DE3）host cells.The recombinant Mub_S27was in the supernatant and was purified with His Bind resin chromatography.This study will enhance the understanding of the immune system in the house fly; enrich the knowledge of the innate immunity in insect.更多还原