本文關(guān)鍵詞： 中華按蚊 防御素 克隆 顯微注射 轉(zhuǎn)基因　出處：《第一軍醫(yī)大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景：蚊媒可傳播許多人類疾病，WHO公布的“十大重點(diǎn)防治的疾病”中，瘧疾和絲蟲病均位于其中，瘧疾更是世界上感染率最高、死亡人數(shù)最多的疾病之一，每年約有超過(guò)30億人受瘧疾威脅，死亡人數(shù)達(dá)200余萬(wàn)，2004年全國(guó)報(bào)告瘧疾發(fā)病38972例，發(fā)病率為0.38／萬(wàn)。其他蚊媒病如登革熱日趨嚴(yán)重；美國(guó)西部西尼羅河腦炎病毒也在迅速蔓延。造成這些悲慘局面最主要的原因是缺乏有效預(yù)防瘧疾和其他蚊媒病的措施，以及蚊媒及病原不斷產(chǎn)生抗藥性。因此，探索并開(kāi)發(fā)新的防治蚊媒病措施已成為當(dāng)前迫切需要解決的問(wèn)題，而瘧原蟲耐藥性的出現(xiàn)意味著控制昆蟲媒介再一次成為最有效且實(shí)用的減輕瘧疾負(fù)擔(dān)的方法。 幾個(gè)世紀(jì)以來(lái)，昆蟲、瘧原蟲和哺乳動(dòng)物宿主間的關(guān)系已達(dá)到了一種協(xié)調(diào)的狀態(tài)，使得瘧原蟲能夠在一定程度逃避人類和昆蟲免疫系統(tǒng)的殺傷而幸存。昆蟲是世界上最大的生物種群，據(jù)估計(jì)其種類多于1×10~6，個(gè)體數(shù)量超過(guò)1×10~(18)，占整個(gè)動(dòng)物數(shù)量的80％。除海洋外，其余的生態(tài)環(huán)境均有昆蟲分布，這表明昆蟲有極強(qiáng)的適應(yīng)和防御能力。但研究發(fā)現(xiàn)，昆蟲并不具備高等動(dòng)物高度專一的免疫體系，即昆蟲缺乏B和T淋巴細(xì)胞系統(tǒng)，也無(wú)免疫球蛋白及補(bǔ)體。但昆蟲能在自然界占據(jù)如此優(yōu)勢(shì)，表明其先天性或獲得性免疫能力非常驚人，其防御系統(tǒng)也必有獨(dú)到之處。 大量研究表明，在感染病菌或可能導(dǎo)致病菌感染(注射細(xì)菌或真菌，體壁損傷等)的情況下，昆蟲能快速合成大量抗菌肽，迅速殺滅已侵入的病菌，并阻止病菌繼續(xù)侵染。目前為止，在昆蟲中已發(fā)現(xiàn)大量的抗細(xì)菌肽、抗真菌肽以及既抗真菌又抗細(xì)菌的抗菌肽。這些抗菌肽不僅對(duì)細(xì)菌、真菌有廣譜抗菌能力，對(duì)病毒、原蟲及癌細(xì)胞也有作用。很多可誘導(dǎo)的抗菌肽類蛋白質(zhì)，根據(jù)其各自特點(diǎn)，可分為四個(gè)主要大類：(1)形成兩性分子α-螺旋的抗細(xì)菌肽類，如大蠶素等；(2)有分子內(nèi)二硫橋的抗細(xì)菌肽類，如防御素或sapecins等；(3)富含脯氨酸的抗細(xì)菌肽類及富含甘氨酸的抗細(xì)菌多肽類，如攻擊素樣蛋白Attacins、SarcotoxinsⅡ、Diptericins等；(4)富含脯氨酸的蛋白，如Apidaecins和abaecin。天蠶素能夠溶解并殺死多種革蘭氏陽(yáng)性菌和陰性菌。攻擊素樣蛋白可阻斷大腸桿菌主要外膜的合成。而富含脯氨酸蛋白的抗菌機(jī)制，如apidaecines和abaecins尚不清楚。防御素(Defensins)是一類具有廣譜抗微生物與細(xì)胞毒活性的可誘導(dǎo)陽(yáng)離子抗菌肽，為昆蟲體內(nèi)產(chǎn)生的最廣泛的抗微生物蛋白；在受到外來(lái)病原侵襲時(shí)，蚊蟲激活其內(nèi)源的防御素基因并在其脂肪體(相當(dāng)于哺乳動(dòng)物的肝臟)中大量表達(dá)，從而形成有效的防御系統(tǒng)。蚊蟲防御素基因首先從埃及伊蚊(Aedes aegypti)中克隆出來(lái)；2000年，Eggleston P等報(bào)道了岡比亞按蚊(Anopheles gambiae)防御素基因的DNA結(jié)構(gòu)及其免疫調(diào)節(jié)作用。2002年，支國(guó)舟等在中國(guó)首先克隆了埃及伊蚊和白紋伊蚊(Aedes albopictus)的防御素基因并進(jìn)行序列分析。2002和2003年，劉先凱等進(jìn)一步對(duì)埃及伊蚊和白紋伊蚊的defensin A基因進(jìn)行了研究。而中華按蚊作為我國(guó)瘧疾的重要傳播媒介，其防御素基因的全長(zhǎng)cDNA序列和基因組序列及相關(guān)的序列鑒定和生物信息學(xué)分析目前國(guó)內(nèi)外均未見(jiàn)報(bào)道。 昆蟲媒介的基因調(diào)控為蚊媒病提供了一種新的防治方法，此方法主要通過(guò)調(diào)控昆蟲的染色體組，最終產(chǎn)生轉(zhuǎn)基因昆蟲。本研究嘗試將中華按蚊的防御素全長(zhǎng)cDNA序列及基因組DNA序列進(jìn)行體外克隆，并將其編碼序列連接入帶有眼特異性啟動(dòng)子(3xP3)驅(qū)動(dòng)的紅色熒光蛋白(DsRed)、蚊卵黃蛋白原(vitellogenin，Vg)啟動(dòng)子以及SV40 poly A尾的具有完整調(diào)控元件的重組載體質(zhì)粒后，用顯微注射的方法將其注入新鮮蚊卵中制備轉(zhuǎn)基因蚊，使其血餐后在Vg特異性啟動(dòng)子的驅(qū)動(dòng)下令防御素基因得到高效表達(dá)，從而使侵入蚊蟲體內(nèi)的病原不能繼續(xù)存活，則有望對(duì)蚊媒病的傳播起到一定阻斷作用。 目的：克隆中華按蚊防御素基因全長(zhǎng)cDNA序列及基因組序列，并對(duì)其進(jìn)行鑒定和生物信息學(xué)分析；構(gòu)建出由眼特異性啟動(dòng)子(3xP3)驅(qū)動(dòng)的紅色熒光蛋白(DsRed)作為篩選標(biāo)志物、Vg啟動(dòng)子驅(qū)動(dòng)的中華按蚊防御素全長(zhǎng)cDNA序列、SV40 poly A尾以及具有高度特異性轉(zhuǎn)座元件piggyBac組成的具有完整調(diào)控元件的重組載體質(zhì)粒pBac-Vg-Defensins-SV40；運(yùn)用顯微注射技術(shù)進(jìn)行轉(zhuǎn)基因蚊的制備并對(duì)孵化出的幼蟲進(jìn)行了初步的定性分析。 方法： 1．采用分子生物學(xué)技術(shù)方法及美國(guó)Clontech Laboratories公司的基因組文庫(kù)構(gòu)建試劑盒構(gòu)建中華按蚊基因組文庫(kù)。 2．用巢式PCR(Nested-PCR)方法，以中華按蚊基因組文庫(kù)為模板，克隆中華按蚊全長(zhǎng)基因組DNA序列。 3．根據(jù)設(shè)計(jì)的多對(duì)引物，以中華按蚊總RNA為模板，用RT-PCR，克隆中華按蚊防御素基因全長(zhǎng)cDNA序列。 4．用GenBank和ExPasy網(wǎng)站等提供的軟件，對(duì)克隆出的中華按蚊防御素全長(zhǎng)基因組DNA序列及全長(zhǎng)cDNA序列進(jìn)行深入的生物信息學(xué)分析。 5．運(yùn)用分子克隆技術(shù)，通過(guò)設(shè)計(jì)酶切位點(diǎn)，酶切，載體與目的基因的連接等方法，以美國(guó)加州大學(xué)河邊分校蟲媒病研究中心Alexander S．Raikhel教授饋贈(zèng)的轉(zhuǎn)移載體質(zhì)粒pBac[3xP3-DsRed afm]、pSLfa1180fa、pVg-SV40及輔助載體質(zhì)粒phsp-pBac為基礎(chǔ)質(zhì)粒，構(gòu)建由眼特異性啟動(dòng)子(3xP3)驅(qū)動(dòng)的紅色熒光蛋白(DsRed)作為篩選標(biāo)志物、Vg啟動(dòng)子驅(qū)動(dòng)的中華按蚊防御素全長(zhǎng)cDNA序列、SV40 poly A尾以及具有高度特異性轉(zhuǎn)座元件piggyBac組成的具有完整調(diào)控元件的重組載體質(zhì)粒pBac-Vg-Defensins-SV40。 6．運(yùn)用顯微注射技術(shù)注射新鮮的蚊卵進(jìn)行轉(zhuǎn)基因蚊的制備。 7．將注射完的蚊卵在27℃、濕度85％的條件下放置16-20 h后轉(zhuǎn)移至39℃孵箱中，熱休克60 min，，然后轉(zhuǎn)移至27℃、濕度85％的環(huán)境中待孵化。在蚊卵孵化后進(jìn)行轉(zhuǎn)基因蚊初步定性分析。 結(jié)果： 1．成功構(gòu)建中華按蚊基因組文庫(kù)。 2．克隆了中華按蚊全長(zhǎng)基因組DNA序列。 3．克隆了中華按蚊防御素基因全長(zhǎng)cDNA序列。 4．生物信息學(xué)分析結(jié)果顯示：中華按蚊全長(zhǎng)cDNA序列為324 bp，其開(kāi)放閱讀框共編碼107個(gè)氨基酸，其中1-28位氨基酸殘基為信號(hào)肽部位，29-68位氨基酸殘基為前導(dǎo)肽部位，成熟肽部分位于68-107位氨基酸殘基，其中α-螺旋占36.45％，無(wú)規(guī)卷曲占29.91％，β-轉(zhuǎn)角占12.15％；為分泌性蛋白，跨膜方式為由胞里到胞外，具有一個(gè)跨膜超螺旋；理論分子量為11.1898 kD，理論等電點(diǎn)pI為6.05，酸性氨基酸殘基總數(shù)為9，堿性氨基酸殘基總數(shù)為10，脂質(zhì)參數(shù)88.32，親水性(GRAVY)0.160。將其全長(zhǎng)編碼氨基酸序列與其他節(jié)肢動(dòng)物比對(duì)發(fā)現(xiàn)：中華按蚊防御素基因編碼的蛋白質(zhì)與節(jié)肢動(dòng)物編碼保守區(qū)域一致，與黑花蠅屬同一性達(dá)到76.19％，成熟肽部位相對(duì)保守，都具有6個(gè)半胱氨酸的保守區(qū)域，二硫鍵結(jié)合方式為C1-C4，C2-C5，C3-C6，序列變異主要發(fā)生在信號(hào)肽和前導(dǎo)肽部位。另外，通過(guò)中華按蚊全長(zhǎng)基因組序列測(cè)定及分析鑒定得出：該序列長(zhǎng)為2256 bp，具有兩個(gè)外顯子，由大小為85bp的內(nèi)含子所分隔；序列上游區(qū)域包括核心啟動(dòng)子元件，如TATA box、節(jié)肢動(dòng)物通用起始序列TCAGT，以及一些上游免疫反應(yīng)元件，如核因子NF-κB和GATA因子、核因子白介素6(NF-IL6)、干擾素一致反應(yīng)元件(ICRE)、白細(xì)胞內(nèi)皮黏附分子5(HNF-5)和SP1，此外，其3′端還具有聚腺苷酸(poly A)尾轉(zhuǎn)錄的終止信號(hào)核苷酸序列。 5．構(gòu)建出由眼特異性啟動(dòng)子(3xP3)驅(qū)動(dòng)的紅色熒光蛋白(DsRed)作為篩選標(biāo)志物、Vg啟動(dòng)子驅(qū)動(dòng)的中華按蚊防御素全長(zhǎng)cDNA序列、SV40 poly A尾以及具有高度特異性轉(zhuǎn)座元件piggyBac組成的具有完整調(diào)控元件的重組載體質(zhì)粒pBac-Vg-Defensins-SV40。其中Vg啟動(dòng)子為卵黃蛋白原編碼基因的啟動(dòng)子序列，從理論上而言，在蚊血餐后可以用來(lái)驅(qū)動(dòng)防御素基因通過(guò)吸血鏈激活并得到高效表達(dá)。 6．在熒光顯微鏡下觀察到孵化的轉(zhuǎn)基因蚊幼蟲眼部有3xP3啟動(dòng)子驅(qū)動(dòng)的DsRed表達(dá)出的眼特異性紅色熒光。 結(jié)論：克隆出中華按蚊防御素全長(zhǎng)基因組DNA序列和中華按蚊防御素基因全長(zhǎng)cDNA序列，cDNA序列被GenBank收錄(登陸號(hào)：DQ002892)，并對(duì)其進(jìn)行了深入的生物信息學(xué)分析。用由眼特異性啟動(dòng)子(3xP3)驅(qū)動(dòng)的紅色熒光蛋白(DsRed)作為篩選標(biāo)志物、Vg啟動(dòng)子驅(qū)動(dòng)的中華按蚊防御素全長(zhǎng)cDNA序列、SV40 poly A尾以及具有高度特異性轉(zhuǎn)座元件piggyBac組成的pBac-Vg-Defensins-SV40以及phsp-pBac輔助載體質(zhì)粒，顯微注射制備出轉(zhuǎn)基因蚊，并在熒光顯微鏡下觀察到轉(zhuǎn)入到轉(zhuǎn)基因蚊體內(nèi)的眼特異性啟動(dòng)子驅(qū)動(dòng)的3xP3-DsRed表達(dá)出了紅色熒光。
[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.
Method錛

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