中华按蚊防御素基因的克

发布时间：2018-01-26 03:50

  本文关键词： 中华按蚊 防御素 克隆 显微注射 转基因　出处：《第一军医大学》2006年硕士论文　论文类型：学位论文

【摘要】：研究背景：蚊媒可传播许多人类疾病，WHO公布的“十大重点防治的疾病”中，疟疾和丝虫病均位于其中，疟疾更是世界上感染率最高、死亡人数最多的疾病之一，每年约有超过30亿人受疟疾威胁，死亡人数达200余万，2004年全国报告疟疾发病38972例，发病率为0.38／万。其他蚊媒病如登革热日趋严重；美国西部西尼罗河脑炎病毒也在迅速蔓延。造成这些悲惨局面最主要的原因是缺乏有效预防疟疾和其他蚊媒病的措施，以及蚊媒及病原不断产生抗药性。因此，探索并开发新的防治蚊媒病措施已成为当前迫切需要解决的问题，而疟原虫耐药性的出现意味着控制昆虫媒介再一次成为最有效且实用的减轻疟疾负担的方法。 几个世纪以来，昆虫、疟原虫和哺乳动物宿主间的关系已达到了一种协调的状态，使得疟原虫能够在一定程度逃避人类和昆虫免疫系统的杀伤而幸存。昆虫是世界上最大的生物种群，据估计其种类多于1×10~6，个体数量超过1×10~(18)，占整个动物数量的80％。除海洋外，其余的生态环境均有昆虫分布，这表明昆虫有极强的适应和防御能力。但研究发现，昆虫并不具备高等动物高度专一的免疫体系，即昆虫缺乏B和T淋巴细胞系统，也无免疫球蛋白及补体。但昆虫能在自然界占据如此优势，表明其先天性或获得性免疫能力非常惊人，其防御系统也必有独到之处。 大量研究表明，在感染病菌或可能导致病菌感染(注射细菌或真菌，体壁损伤等)的情况下，昆虫能快速合成大量抗菌肽，迅速杀灭已侵入的病菌，并阻止病菌继续侵染。目前为止，在昆虫中已发现大量的抗细菌肽、抗真菌肽以及既抗真菌又抗细菌的抗菌肽。这些抗菌肽不仅对细菌、真菌有广谱抗菌能力，对病毒、原虫及癌细胞也有作用。很多可诱导的抗菌肽类蛋白质，根据其各自特点，可分为四个主要大类：(1)形成两性分子α-螺旋的抗细菌肽类，如大蚕素等；(2)有分子内二硫桥的抗细菌肽类，如防御素或sapecins等；(3)富含脯氨酸的抗细菌肽类及富含甘氨酸的抗细菌多肽类，如攻击素样蛋白Attacins、SarcotoxinsⅡ、Diptericins等；(4)富含脯氨酸的蛋白，如Apidaecins和abaecin。天蚕素能够溶解并杀死多种革兰氏阳性菌和阴性菌。攻击素样蛋白可阻断大肠杆菌主要外膜的合成。而富含脯氨酸蛋白的抗菌机制，如apidaecines和abaecins尚不清楚。防御素(Defensins)是一类具有广谱抗微生物与细胞毒活性的可诱导阳离子抗菌肽，为昆虫体内产生的最广泛的抗微生物蛋白；在受到外来病原侵袭时，蚊虫激活其内源的防御素基因并在其脂肪体(相当于哺乳动物的肝脏)中大量表达，从而形成有效的防御系统。蚊虫防御素基因首先从埃及伊蚊(Aedes aegypti)中克隆出来；2000年，Eggleston P等报道了冈比亚按蚊(Anopheles gambiae)防御素基因的DNA结构及其免疫调节作用。2002年，支国舟等在中国首先克隆了埃及伊蚊和白纹伊蚊(Aedes albopictus)的防御素基因并进行序列分析。2002和2003年，刘先凯等进一步对埃及伊蚊和白纹伊蚊的defensin A基因进行了研究。而中华按蚊作为我国疟疾的重要传播媒介，其防御素基因的全长cDNA序列和基因组序列及相关的序列鉴定和生物信息学分析目前国内外均未见报道。 昆虫媒介的基因调控为蚊媒病提供了一种新的防治方法，此方法主要通过调控昆虫的染色体组，最终产生转基因昆虫。本研究尝试将中华按蚊的防御素全长cDNA序列及基因组DNA序列进行体外克隆，并将其编码序列连接入带有眼特异性启动子(3xP3)驱动的红色荧光蛋白(DsRed)、蚊卵黄蛋白原(vitellogenin，Vg)启动子以及SV40 poly A尾的具有完整调控元件的重组载体质粒后，用显微注射的方法将其注入新鲜蚊卵中制备转基因蚊，使其血餐后在Vg特异性启动子的驱动下令防御素基因得到高效表达，从而使侵入蚊虫体内的病原不能继续存活，则有望对蚊媒病的传播起到一定阻断作用。 目的：克隆中华按蚊防御素基因全长cDNA序列及基因组序列，并对其进行鉴定和生物信息学分析；构建出由眼特异性启动子(3xP3)驱动的红色荧光蛋白(DsRed)作为筛选标志物、Vg启动子驱动的中华按蚊防御素全长cDNA序列、SV40 poly A尾以及具有高度特异性转座元件piggyBac组成的具有完整调控元件的重组载体质粒pBac-Vg-Defensins-SV40；运用显微注射技术进行转基因蚊的制备并对孵化出的幼虫进行了初步的定性分析。 方法： 1．采用分子生物学技术方法及美国Clontech Laboratories公司的基因组文库构建试剂盒构建中华按蚊基因组文库。 2．用巢式PCR(Nested-PCR)方法，以中华按蚊基因组文库为模板，克隆中华按蚊全长基因组DNA序列。 3．根据设计的多对引物，以中华按蚊总RNA为模板，用RT-PCR，克隆中华按蚊防御素基因全长cDNA序列。 4．用GenBank和ExPasy网站等提供的软件，对克隆出的中华按蚊防御素全长基因组DNA序列及全长cDNA序列进行深入的生物信息学分析。 5．运用分子克隆技术，通过设计酶切位点，酶切，载体与目的基因的连接等方法，以美国加州大学河边分校虫媒病研究中心Alexander S．Raikhel教授馈赠的转移载体质粒pBac[3xP3-DsRed afm]、pSLfa1180fa、pVg-SV40及辅助载体质粒phsp-pBac为基础质粒，构建由眼特异性启动子(3xP3)驱动的红色荧光蛋白(DsRed)作为筛选标志物、Vg启动子驱动的中华按蚊防御素全长cDNA序列、SV40 poly A尾以及具有高度特异性转座元件piggyBac组成的具有完整调控元件的重组载体质粒pBac-Vg-Defensins-SV40。 6．运用显微注射技术注射新鲜的蚊卵进行转基因蚊的制备。 7．将注射完的蚊卵在27℃、湿度85％的条件下放置16-20 h后转移至39℃孵箱中，热休克60 min，，然后转移至27℃、湿度85％的环境中待孵化。在蚊卵孵化后进行转基因蚊初步定性分析。 结果： 1．成功构建中华按蚊基因组文库。 2．克隆了中华按蚊全长基因组DNA序列。 3．克隆了中华按蚊防御素基因全长cDNA序列。 4．生物信息学分析结果显示：中华按蚊全长cDNA序列为324 bp，其开放阅读框共编码107个氨基酸，其中1-28位氨基酸残基为信号肽部位，29-68位氨基酸残基为前导肽部位，成熟肽部分位于68-107位氨基酸残基，其中α-螺旋占36.45％，无规卷曲占29.91％，β-转角占12.15％；为分泌性蛋白，跨膜方式为由胞里到胞外，具有一个跨膜超螺旋；理论分子量为11.1898 kD，理论等电点pI为6.05，酸性氨基酸残基总数为9，碱性氨基酸残基总数为10，脂质参数88.32，亲水性(GRAVY)0.160。将其全长编码氨基酸序列与其他节肢动物比对发现：中华按蚊防御素基因编码的蛋白质与节肢动物编码保守区域一致，与黑花蝇属同一性达到76.19％，成熟肽部位相对保守，都具有6个半胱氨酸的保守区域，二硫键结合方式为C1-C4，C2-C5，C3-C6，序列变异主要发生在信号肽和前导肽部位。另外，通过中华按蚊全长基因组序列测定及分析鉴定得出：该序列长为2256 bp，具有两个外显子，由大小为85bp的内含子所分隔；序列上游区域包括核心启动子元件，如TATA box、节肢动物通用起始序列TCAGT，以及一些上游免疫反应元件，如核因子NF-κB和GATA因子、核因子白介素6(NF-IL6)、干扰素一致反应元件(ICRE)、白细胞内皮黏附分子5(HNF-5)和SP1，此外，其3′端还具有聚腺苷酸(poly A)尾转录的终止信号核苷酸序列。 5．构建出由眼特异性启动子(3xP3)驱动的红色荧光蛋白(DsRed)作为筛选标志物、Vg启动子驱动的中华按蚊防御素全长cDNA序列、SV40 poly A尾以及具有高度特异性转座元件piggyBac组成的具有完整调控元件的重组载体质粒pBac-Vg-Defensins-SV40。其中Vg启动子为卵黄蛋白原编码基因的启动子序列，从理论上而言，在蚊血餐后可以用来驱动防御素基因通过吸血链激活并得到高效表达。 6．在荧光显微镜下观察到孵化的转基因蚊幼虫眼部有3xP3启动子驱动的DsRed表达出的眼特异性红色荧光。 结论：克隆出中华按蚊防御素全长基因组DNA序列和中华按蚊防御素基因全长cDNA序列，cDNA序列被GenBank收录(登陆号：DQ002892)，并对其进行了深入的生物信息学分析。用由眼特异性启动子(3xP3)驱动的红色荧光蛋白(DsRed)作为筛选标志物、Vg启动子驱动的中华按蚊防御素全长cDNA序列、SV40 poly A尾以及具有高度特异性转座元件piggyBac组成的pBac-Vg-Defensins-SV40以及phsp-pBac辅助载体质粒，显微注射制备出转基因蚊，并在荧光显微镜下观察到转入到转基因蚊体内的眼特异性启动子驱动的3xP3-DsRed表达出了红色荧光。
[Abstract]:Background: mosquitoes can spread many human diseases, WHO announced the "ten focus of disease prevention and control of malaria and filariasis are located in which malaria is the world's highest infection rate, one of the most disease deaths, about more than 3 billion people threatened by malaria each year, the death toll reached more than 200, 2004 the 38972 malaria cases were reported, the incidence rate was 0.38 per million. Other mosquito borne diseases such as dengue fever has become increasingly serious; the western United States West Nile encephalitis virus is spreading rapidly. The cause of these tragic situation the main reason is the lack of effective prevention of malaria and other mosquito borne diseases and mosquitoes and pathogen and drug resistance therefore, the exploration and development of the prevention and control of mosquito borne disease of new measures has become an urgent need to solve the problem, and the emergence of drug resistance of malaria control means that insect vectors once again become the most effective and practical The way to reduce the burden of malaria.
For centuries, insects, and the relationship between the mammalian host parasite has reached a harmonious state, which can avoid the human and Plasmodium killing insect immune system in a certain extent and survived. The insect is the world's largest population of organisms, estimated the types of more than 1 * 10~6, the number of individuals over 1 * 10~ (18), accounted for 80%. in addition to the sea outside the whole animal population, ecological environment has the rest of this show that the distribution of insects, insects have strong adaptability and defensive ability. But the study found that the insects do not have the higher the high specificity of the immune system of the animal, they are lack of B and T lymphocytes, but also immunoglobulin and complement. But insects can occupy such advantages in nature, shows that the congenital or acquired immune ability is very impressive, the defense system will have originality.
A large number of studies show that in bacteria infection or may lead to bacterial infections (bacterial or fungal injection, wall injury) under the condition that the insects can quickly produce a large amount of antimicrobial peptides, rapidly kill bacteria and has invaded, prevent bacteria from infecting. So far, a large number of anti microbial peptides have been found in insects, antifungal peptide both antifungal and antibacterial peptides and anti bacteria. These antimicrobial peptides not only against bacteria, fungi have broad-spectrum antibacterial ability against viruses, parasites and cancer cells have a role in many antibacterial peptide protein can be induced, according to their characteristics, can be divided into four main categories: (1) the formation of amphiphilic alpha helix anti bacterial peptides, such as cecropin; (2) there are two intramolecular disulfide bridges of antibacterial peptides, such as defensins or sapecins; (3) bacterial polypeptide anti bacterial peptide and glycine rich proline rich, such as aggressin like protein A Ttacins, Sarcotoxins II, Diptericins; (4) proline rich proteins, such as Apidaecins and abaecin. cecropin can dissolve and kill many gram positive and negative bacteria. Aggressin like protein can block the synthesis of Escherichia coli outer membrane. The antibacterial mechanisms of proline rich peptides, such as apidaecines and abaecins defense is not clear. Prime (Defensins) is a kind of broad-spectrum antimicrobial and cytotoxic activity induced by cationic antimicrobial peptides, the most extensive anti microbial protein produced in insects; under pathogen invasion, the activation of endogenous mosquito defensin genes in the fat body (the equivalent of the mammalian liver) in expression thus, the formation of an effective defense system. The first mosquito defensin gene from Aedes aegypti (Aedes aegypti) was cloned; 2000, P reported that Eggleston (Anopheles in Gambia Anopheles gambiae) DNA structure and immune regulation of defensin gene.2002, a Guozhou in China first cloned Aedes aegypti and Aedes albopictus (Aedes albopictus) sequence analysis of.2002 and 2003 defensin genes, Liu Xiankai further defensin A gene on Aedes aegypti and Aedes albopictus were studied. As an important media and Anopheles malaria in China, analysis at home and abroad were reported to the defensin gene full-length cDNA and genomic DNA sequences and related sequence identification and bioinformatics.
Gene regulation of insect vectors provides a new method for the prevention and control of mosquito borne diseases, this method mainly through the regulation of insect genome, eventually produce transgenic insects. This study attempts to Anopheles sinensis defensin full-length cDNA and genomic DNA sequences were cloned in vitro, and its encoding sequence connected with eye specificity promoter (3xP3) red fluorescent protein (DsRed), drive the mosquito vitellogenin (vitellogenin, Vg) SV40 poly promoter and A tail with complete regulatory elements of recombinant plasmid, by microinjection method into the transgenic mosquitoes for fresh mosquito eggs in the blood after a meal in the specific Vg promoter driven ordered defensin gene was highly expressed, so that the pathogen cannot invade mosquitoes to survive, is expected to be blocking effect on the spread of mosquito borne disease.
Objective: to clone the full-length cDNA gene of Anopheles sinensis defense sequence and genome sequence, and its identification and bioinformatics analysis; constructed by eye specific promoter (3xP3) red fluorescent protein (DsRed) driven as screening marker, Vg promoter in Chinese Anopheles defensin full-length cDNA sequence. SV40 poly A tail and highly specific transposable elements composed of piggyBac with complete regulatory elements of recombinant plasmid pBac-Vg-Defensins-SV40; transgenic mosquitoes using microinjection technique and preparation of the hatched larvae were analyzed qualitatively.
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