Cecrop-m2A10融合基因的构建、原核表达、生物学活性分析及转基因体系的构建
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摘要
研究背景:蚊媒可传播多种疾病,如:疟疾(malaria)、流行性乙型脑炎(epidemic encephalitis B)、丝虫病(filariasis)、登革热(dengue fever)等,是严重威胁人民生命健康的重要疾病。一些蚊媒病如登革热的发病率呈逐年递增的趋势并常有局部暴发流行。疟疾是世界上感染率最高,死亡人数最多的疾病之一。每年有超过30亿人受到疟疾的威胁,死亡人数达200余万。造成这种严重局面的最主要原因是缺乏预防疟疾的有效措施以及蚊媒和病原不断产生耐药性。因此探索并开发新的防治蚊媒疾病的措施已成为当前迫切需要解决的问题,控制昆虫媒介再次成为最有效且实用的抗疟手段。
疟原虫是人体疟疾的病原体,共有四种类型,在我国主要是间日疟原虫和恶性疟原虫。人体疟原虫需要人和雌性按蚊做宿主,它在蚊体内的发育包括在蚊胃腔内进行有性生殖即配子生殖(gametogony)和在蚊胃壁进行的无性生殖即孢子增殖(sporogony)两个阶段。子孢子是疟原虫感染人体的阶段。恶性疟原虫环子孢子蛋白(Circumsporozoite protein,CSP)覆盖于子孢子表面,是其重要保护性抗原及粘附入侵靶细胞的物质基础。恶性疟CSP的鼠源单克隆抗体2A10在体外能够阻止子孢子进入蚊唾液腺和人肝细胞。2A10scFv(Single-chainFV antibody fragment)是2A10单链抗体可变区片段。在以鼠源性2A10scFv为靶基因的转基因蚊中,蚊体内所表达的2A10scFv能够与子孢子结合,从而阻断其钻入唾液腺。但由于蛋白的表达量不高,因此未能达到完全阻断子孢子入侵的效果。
近年来,国际上提出了利用转基因技术对蚊媒进行遗传修饰以控制蚊媒疾病的新策略。外源基因在转基因蚊体内的表达对于转基因蚊的抗病原效应至关重要,它受到启动子、插入部位及外源基因本身特性等诸多因素的影响。其中,外源基因的密码子在宿主蚊体内的使用频率,即遗传密码子的偏嗜性被认为是一个重要影响因素。物种间编码同一氨基酸的不同密码子的使用频率存在差别,一些在外源基因中出现的密码子很少为表达宿主所使用,相对应的tRNA在宿主体内稀少,由此影响了外源基因mRNA的翻译过程,从而降低了效应蛋白的表达量。在对于大肠杆菌偏嗜性人胰岛素样生长因子1的研究中,实验人员依据大肠杆菌遗传密码子使用频率表,人工合成3条DNA单链,PCR拼接扩增出适于在大肠杆菌中表达的人IGF-1基因。所构建的大肠杆菌偏嗜性人IGF-1基因可显著提高rhIGF-1的产率。此外将异源的免疫原基因密码子优化为免疫接种动物偏嗜性密码子能够提高抗原表达效率并改进DNA疫苗免疫效果也已被许多研究所证实。本实验中经对比分析,鼠源性2A10scFv基因中有些密码子在蚊体内的使用频率确实很低,例如,编码丝氨酸的密码子UCU在冈比亚按蚊的使用频率仅为4.1%。那么我们能否通过2A10scFv基因密码子的蚊偏嗜性改造,使其更适于在转基因蚊体内表达从而提高其表达量、增加其稳定性及生物学活性呢?
抗菌肽(antimicrobial peptides,AMP)是抗菌活性短肽的总称。目前已发现抗菌肽或类似的小分子肽类广泛存在于生物界,包括细菌、动植物和人类。内源性的抗菌肽经诱导合成,在机体抵抗病原的入侵方面起着重要的作用,被认为是缺乏特异性免疫功能生物的重要防御成分。抗菌肽具有广谱杀菌作用,大多数对革兰氏阳性菌有较强的杀灭作用,有些则对革兰氏阴性菌和革兰氏阳性菌均起作用。例如,Reed等将Shiva-Ia转入小鼠中,转基因小鼠对布鲁氏杆菌的抵抗力显著增强;Possani等的研究表明,在蚊体内表达Shiva-3可以抑制疟疾的传播。与传统抗生素相比,某些抗菌肽的抗菌活性还不够理想,改造已有抗菌肽和设计新抗菌肽分子是提高其产量及活性的有效途径。例如,Christsnen B等研究中得到的融合抗菌肽的抗菌活性比其任何一个供体抗菌肽的活性都高;Durasula等通过在长红猎蟋的共生菌中表达Cecropin A显减少了其体内锥虫的数量。Cecropins是第一个被发现的动物抗菌肽,1975年瑞典科学家从惜古比天蚕(Hyatophoracecropia)蛹中诱导分离得到。其中Cecropin A对各种大肠杆菌和其他革兰氏阴性菌如沙门氏菌具有很强的活性,对部分革兰氏阳性菌也具有一定的杀伤活性。其作用机理不同于传统抗生素,不会导致抗药菌株的产生,且对正常的真核生物细胞及霉菌不起作用。由于抗菌肽的分子小,对蛋白酶非常敏感,因此很难直接大量表达而多采用融合表达策略以降低其对宿主细胞的毒性。综上,能否通过基因融合配伍增强Cecropin A及2A10scFv的抗病原作用及Cecrop-m2A10在蚊体内的稳定性呢?
目的:
1.对鼠源性恶性疟原虫环子孢子蛋白单链抗体2A10基因进行改造,构建融合基因Cecrop-m2A10。
2.原核表达Cecrop-m2A10基因,并对其表达产物进行生物学活性分析。
3.构建以蚊眼特异性启动子(3xP3)驱动的红色荧光蛋白(DsRed)作为筛选标志物,Vg启动子驱动Cecrop-m2A10序列及高度特异性转座元件piggyBac组成的具有完整调控元件的重组转基因载体质粒pBac-AsVg-Cecrop-m2A10。
方法:
1.根据按蚊偏嗜性密码子对鼠源性恶性疟原虫环子孢子蛋白单链抗体2A10基因进行改造,并融合按蚊抗菌肽Cecropin A编码基因,构建融合基因Cecrop-m2A10及重组质粒pBSK(+)-Cecrop-m2A10。
2.用PCR方法以pBSK(+)-Cecrop-m2A10为模板克隆Cecrop-m2A10基因,构建表达重组质粒pET32a(+)-Cecrop-m2A10,并对重组质粒进行测序鉴定。
3.将重组质粒pET32a(+)-Cecrop-m2A10转入BL21(DE3)中,用IPTG诱导表达。
4.优化工程菌pET32a(+)-Cecrop-m2A10的表达条件,大量诱导表达后对重组蛋白进行纯化。
5.Western blotting鉴定重组表达蛋白。
6.采用琼脂糖扩散法检测融合蛋白的体外抑菌活性。
7.运用分子克隆技术,以pBSK(+)-Cecrop-m2A10及pSLfall80fa-AsVg-CFP-3'VTR载体质粒为基础质粒,构建以蚊眼特异性启动子(3xP3)驱动的红色荧光蛋白(DsRed)作为筛选标志物,Vg启动子驱动Cecrop-m2A10序列及高度特异性转座元件piggyBac组成的具有完整调控元件的重组转基因载体质粒pBac-AsVg-Cecrop-m2A10。
结果:
1.对鼠源性环子孢子蛋白单链抗体2A10中6种氨基酸的170个核苷酸进行了改造并融合Cecropin A编码基因,全基因合成法获得融合基因Cecrop-m2A10。并成功构建重组质粒pBSK(+)-Cecrop-m2A10。
2.成功构建表达重组质粒pE/32a(+)-Cecrop-m2A10,测序结果显示读码框架正确。通过IPTG诱导得到以包涵体形式高效表达的重组蛋白。包涵体经过尿素洗涤,变性,透析复性后纯度达83.7%。Western blotting分析显示重组蛋白与抗6-His抗体可产生59KD大小的特异性反应条带。
3.琼脂糖扩散法结果显示,纯化的重组蛋白具备抗大肠杆菌DH5α的活性。
4.成功构建了以蚊眼特异性启动子(3xP3)驱动的红色荧光蛋白(DsRed)作为筛选标志物,Vg启动子驱动Cecrop-m2A10序列及高度特异性转座元件piggyBac组成的具有完整调控元件的重组转基因载体质粒pBac-AsVg-Cecrop-m2A10。
结论:
1.成功构建融合基因Cecrop-m2h10。
2.成功构建表达重组质粒pET32a(+)-Cecrop-m2A10。在大肠杆菌中以包涵体形式高效表达,纯化后纯度达83.7%。
3.初步鉴定显示纯化的重组蛋白具备抗大肠杆菌DH5α的活性。
4.构建了以蚊眼特异性启动子(3xP3)驱动的红色荧光蛋白(DsRed)作为筛选标志物,Vg启动子驱动Cecrop-m2A10序列及高度特异性转座元件piggyBac组成的具有完整调控元件的重组转基因载体质粒pBac-AsVg-Cecrop-m2A10。
Background:Mosquitoes transmit numerous human diseases,such as malaria, epidemic encephalitis B,fiariasis,dengue fever,etc,severely harm to human health. An increasing trend on the rate of dengue fever has appeared year by year,occasionally,it might burst out and spread in some regions of the world.Malaria is one of the attacks that result in the highest infection ratio and mortality,taking a heavy toll on the human population in many parts of the world by infecting more than 300 million and killing more than 200 million people each year.The major reasons for this tragic situation are the unavailability of effective vaccines for malaria and the development of insecticide and drug resistance by the vectors and pathogens,respectively.Therefore,there is an urgent need to explore every possible avenue for developing novel control strategies against these mosquito-borne menacing diseases,and at the same time,drug resistance of malaria means that controlling of mosquito becomes the most effective and pragmatic way to ralieve the burden caused by malaria.
Plasmodium,the causative agent of malaria,has to complete a complex developmental program in its mosquito host for transmission to occur.Malaria sporozoites exhibit infectivity for mosquito salivary glands,human body and other vertebrate host tissue.The surface membrane of sporozoites of Plasmodium berghei—circumsporozoite protein(CSP) which covers the entire surface of mature,salivary gland sporozoites is a protective antigen of sporozoites.Meanwhile,it may play a role in the adhesion and invasion into the target cell.Mouse monoclonal antibodies against CSP of Plasmodium berghei—2A10 can affect mosquito salivary gland and human liver cell invasion by sporozoites in vitro assays and prevent malaria transmission.2A10scFv is a single chain antibody against CSP of Plasmodium falciparum.CHEN had successfully transformed a 2A10scFv gene into An.stephensi. Transgenic mosquitoes carrying 2A10scFv transgenes exhibited resistance to mosquito salivary gland invasion,the RT-PCR and Northern blot showed that the transgene transcribed well,but Western blot did not detect the protein(Unpublished data)which may be at very low levels in the transgenic mosquitoes.
In recent years,the prospect of using transgenic mosquitoes is rapidly gaining strength,owing to the development of molecular biology、immunology and genetics. The central event in transgenic mosquitoes is the heterologous expression of transgenes,expression levels of heterologous genes are crucial to their function. There are many factors may affect the expression level,such as the promoter,coding sequence,the copy number,insertion place and the nature of the expressed protein itself.The most frequently used codons among various species are different. Differences in codon usage can seriously hamper the expression of the heterologous transgenes.This is due,in part,to differences in codon usage,which can significantly result in the potential depletion of tRNAs encoding the same amino acid,decrease translation efficiency and thereby lower protein production in a heterologous system. Suggesting that codon optimization is a valuable strategy for improving the heterologous expression of native sequences,it has been widely and successfully used not only in P.pastoris,but also in other expression system such as E.coli BL21(DE3), Aspergillus niger,and mammalian cells.Various experimental procedures about PCR-based codon optimization have been developed,from the modification of a few codons about 5'codon adaptation to the extensive rewriting of up 1000 bp of DNA by overlap PCR.Likewise,the compatibility of codon usage between the 2A10scFv gene sequence and that of the expression host-mosquito are indeed respectively distinct. Then,if the DNA sequence encoding 2A10scFv gene was designed and synthesized based on the codon bias of mosqiuto,the transgenic expression may be at a higher level and the anti-pathogen effect of transgenic mosqiuto may be stronger.
Antimicrobial peptides(AMPs) are small molecular weight proteins with broad spectrum antimicrobial activity against a broad range of micro-organisms,including gram-negative and gram-positive bacteria,fungi,and viruses.They were found in plants,insects,bacteria and hunman.An expansive list of unique AMPs has been discovered in our environment,with well over 800 peptides known.Once in a target microbial membrane,the peptide kills target cells through diverse mechanisms. Cathelicidins and defensins are major groups of epidermal AMPs.Cecropins are one family of these peptides which were first isolated from the haemolymph of the giant silk moth,Hyalophora eecropia and may be idespread throughout the animal kingdom.The principle insect cecropins(A,B and D) are 35 to 37 residues,devoid of cysteine,with a strongly basic N-terminal linked to a neutral C-terminal by a flexible glycine-proline link.The overall structure deduced by NMR for cecropin A is two near perfect amphipathic segments joined by a Gly-Pro hinge.Antibacterial activity has been reported for cecropins A,B and P gainst Gram-positive and Gram-negative organisms.There are some problems in direct expression of AMPs for the following reasons:①In the case of direct expression of AMPs,undesirable degradation of such small sized mRNA and peptide was expected.②The direct expression of an antimicrobial peptide in bacteria may be cytotoxic to the host or subjected to degradation by host-derived peptidases.To overcome these potential problems,a common strategy to achieve an efficient expression of a protein or antimicrobial peptide is to express the molecule in a microbial system,either de novo or as a fusion partner with another protein.The gene fusion was successfully translated in a bacterial expression system.The fusion protein was non-toxic to the host bacteria. Our researches suggest that the strategy investigated in this study is useful to produce a large amount of AMPs and it might be applicable to other cationic small peptides especially to those which are difficult to synthesize chemically and have no available affinity purification methods.Consequently,in order to increase the anti-pathogen effect and stability of 2A 10scFv gene,we designed to linked 2A 10scFv gene with the coding sequence from A.gambiae cecropin A and constructed a fusion gene Cecrop-m2A10.
Objective:
1.To modify coding sequence of a single chain antibody(2A10) against circumsporozoite protein(CSP) of Plasmodium falciparum and construct fusion gene Cecrop-m2A10.
2.To express Cecrop-m2A10 in E.coli BL21(DE3) and analyze the bioactivity of the expression products.
3.To construct pBac-AsVg-Cecrop-m2A10 plasmid which contains a full regulation sequence,3xP3-DsRed eye specific promoter,vitellogenin(Vg) promoter and Cecrop-m2A10 gene.
Methods:
1.The coding sequence of a single chain antibody(2A10) against circumsporozoite protein(CSP) of Plasmodium falciparum were modified according to Anopheles gambiae preferred codons and then linked with the coding sequence from A.gambiae Cecropin A.The fusion gene Cecrop-m2A10 were produced by whole gene synthesis.To follow it,recombinant cloning plasmid pBSK(+)-Cecrop-m2A10 was constructed.
2.Completed Cecrop-m2A10 gene was cloned from pBSK(+)-Cecrop-m2A10 plasmid by PCR,Cecrop-m2A10 gene fragment was cloned into pET32a(+) vector to construct recombinant plasmid pET32a(+)-Cecrop-m2A10 which was identifed by sequence analysis.
3.The plasmid pET32a(+)-Cecrop-m2A10 was transformed into E.coli BL21(DE3) to express the recombinant proteins after the induction of IPTG.
4.Optimize expression conditions and purify expression products.
5.Identify the recombinant proteins by Western blotting.
6.The bioactivity of the expression products was assayed with Argarose-Diffusion-Method.
7.The pBac-AsVg-Cecrop-m2A10 plasmid which contains 3xP3-DsRed eye specific promoter,vitellogenin(Vg) promoter,Cecrop-m2A10 gene was constructed by techniques of molecular cloning such as digestion and ligation methods with the basic plasmid:pBSK(+)-Cecrop-m2A 10 and pSLfall80fa-AsVg-CFP-3'VTR.
Results:
1.The coding sequence of a single chain antibody(2A10) against circumsporozoite protein(CSP) of Plasmodium falciparum were modified according to Anopheles gambiae preferred codons.Totally,170 nucleotides in six kinds of amino acids were replaced and then linked with the coding sequence from A.gambiae Cecropin A.Subsequently,the fusion gene,Cecrop-m2A10 were produced by whole gene synthesis.To follow it,recombinant cloning plasmid pBSK(+)-Cecrop-m2A10 was successfully constructed.
2.Recombinant expression plasmids pET32a(+)-Cecrop-m2A10 was successfully constructed and its open frame was corrected by sequence analysis.After IPTG inducting,recombinant protien was expressed in inclusion body form in E.coli BL21(DE3).After the collection of inclusion body,denaturing lysis and refoding dialysis,the purity of the expression product was over 83.7%.Western blotting showed the recombinant protien could be specifically recognized by anti-6-His antibody.
3.The argarose-Diffusion-Method showed bacteriostasis inhibition to the growth of E.coli DH5α.
4.The pBac-AsVg-Cecrop-m2A10 plasmid which contains 3xP3-DsRed eye specific promoter,vitellogenin(Vg) promoter,Cecrop-m2A10 gene was successfully constructed.
Conclusion:
1.The fusion gene Cecrop-m2A10 was successfully constructed.
2.Recombinant expression plasmids pET32a(+)-Cecrop-m2A10 was successfully constructed and expressed in inclusion body form in E.coli BL21(DE3). After the collection of inclusion body,denaturing lysis and refoding dialysis,the purity of the expression product was over 83.7%.
3.The preliminary evaluation showed bacteriostasis inhibition to the growth of E.coli DH5α.
4.The pBac-AsVg-Cecrop-m2A10 plasmid which contains 3xP3-DsRed eye specific promoter,vitellogenin(Vg) promoter,Cecrop-m2A10 gene was successfully constructed.
疟原虫是人体疟疾的病原体,共有四种类型,在我国主要是间日疟原虫和恶性疟原虫。人体疟原虫需要人和雌性按蚊做宿主,它在蚊体内的发育包括在蚊胃腔内进行有性生殖即配子生殖(gametogony)和在蚊胃壁进行的无性生殖即孢子增殖(sporogony)两个阶段。子孢子是疟原虫感染人体的阶段。恶性疟原虫环子孢子蛋白(Circumsporozoite protein,CSP)覆盖于子孢子表面,是其重要保护性抗原及粘附入侵靶细胞的物质基础。恶性疟CSP的鼠源单克隆抗体2A10在体外能够阻止子孢子进入蚊唾液腺和人肝细胞。2A10scFv(Single-chainFV antibody fragment)是2A10单链抗体可变区片段。在以鼠源性2A10scFv为靶基因的转基因蚊中,蚊体内所表达的2A10scFv能够与子孢子结合,从而阻断其钻入唾液腺。但由于蛋白的表达量不高,因此未能达到完全阻断子孢子入侵的效果。
近年来,国际上提出了利用转基因技术对蚊媒进行遗传修饰以控制蚊媒疾病的新策略。外源基因在转基因蚊体内的表达对于转基因蚊的抗病原效应至关重要,它受到启动子、插入部位及外源基因本身特性等诸多因素的影响。其中,外源基因的密码子在宿主蚊体内的使用频率,即遗传密码子的偏嗜性被认为是一个重要影响因素。物种间编码同一氨基酸的不同密码子的使用频率存在差别,一些在外源基因中出现的密码子很少为表达宿主所使用,相对应的tRNA在宿主体内稀少,由此影响了外源基因mRNA的翻译过程,从而降低了效应蛋白的表达量。在对于大肠杆菌偏嗜性人胰岛素样生长因子1的研究中,实验人员依据大肠杆菌遗传密码子使用频率表,人工合成3条DNA单链,PCR拼接扩增出适于在大肠杆菌中表达的人IGF-1基因。所构建的大肠杆菌偏嗜性人IGF-1基因可显著提高rhIGF-1的产率。此外将异源的免疫原基因密码子优化为免疫接种动物偏嗜性密码子能够提高抗原表达效率并改进DNA疫苗免疫效果也已被许多研究所证实。本实验中经对比分析,鼠源性2A10scFv基因中有些密码子在蚊体内的使用频率确实很低,例如,编码丝氨酸的密码子UCU在冈比亚按蚊的使用频率仅为4.1%。那么我们能否通过2A10scFv基因密码子的蚊偏嗜性改造,使其更适于在转基因蚊体内表达从而提高其表达量、增加其稳定性及生物学活性呢?
抗菌肽(antimicrobial peptides,AMP)是抗菌活性短肽的总称。目前已发现抗菌肽或类似的小分子肽类广泛存在于生物界,包括细菌、动植物和人类。内源性的抗菌肽经诱导合成,在机体抵抗病原的入侵方面起着重要的作用,被认为是缺乏特异性免疫功能生物的重要防御成分。抗菌肽具有广谱杀菌作用,大多数对革兰氏阳性菌有较强的杀灭作用,有些则对革兰氏阴性菌和革兰氏阳性菌均起作用。例如,Reed等将Shiva-Ia转入小鼠中,转基因小鼠对布鲁氏杆菌的抵抗力显著增强;Possani等的研究表明,在蚊体内表达Shiva-3可以抑制疟疾的传播。与传统抗生素相比,某些抗菌肽的抗菌活性还不够理想,改造已有抗菌肽和设计新抗菌肽分子是提高其产量及活性的有效途径。例如,Christsnen B等研究中得到的融合抗菌肽的抗菌活性比其任何一个供体抗菌肽的活性都高;Durasula等通过在长红猎蟋的共生菌中表达Cecropin A显减少了其体内锥虫的数量。Cecropins是第一个被发现的动物抗菌肽,1975年瑞典科学家从惜古比天蚕(Hyatophoracecropia)蛹中诱导分离得到。其中Cecropin A对各种大肠杆菌和其他革兰氏阴性菌如沙门氏菌具有很强的活性,对部分革兰氏阳性菌也具有一定的杀伤活性。其作用机理不同于传统抗生素,不会导致抗药菌株的产生,且对正常的真核生物细胞及霉菌不起作用。由于抗菌肽的分子小,对蛋白酶非常敏感,因此很难直接大量表达而多采用融合表达策略以降低其对宿主细胞的毒性。综上,能否通过基因融合配伍增强Cecropin A及2A10scFv的抗病原作用及Cecrop-m2A10在蚊体内的稳定性呢?
目的:
1.对鼠源性恶性疟原虫环子孢子蛋白单链抗体2A10基因进行改造,构建融合基因Cecrop-m2A10。
2.原核表达Cecrop-m2A10基因,并对其表达产物进行生物学活性分析。
3.构建以蚊眼特异性启动子(3xP3)驱动的红色荧光蛋白(DsRed)作为筛选标志物,Vg启动子驱动Cecrop-m2A10序列及高度特异性转座元件piggyBac组成的具有完整调控元件的重组转基因载体质粒pBac-AsVg-Cecrop-m2A10。
方法:
1.根据按蚊偏嗜性密码子对鼠源性恶性疟原虫环子孢子蛋白单链抗体2A10基因进行改造,并融合按蚊抗菌肽Cecropin A编码基因,构建融合基因Cecrop-m2A10及重组质粒pBSK(+)-Cecrop-m2A10。
2.用PCR方法以pBSK(+)-Cecrop-m2A10为模板克隆Cecrop-m2A10基因,构建表达重组质粒pET32a(+)-Cecrop-m2A10,并对重组质粒进行测序鉴定。
3.将重组质粒pET32a(+)-Cecrop-m2A10转入BL21(DE3)中,用IPTG诱导表达。
4.优化工程菌pET32a(+)-Cecrop-m2A10的表达条件,大量诱导表达后对重组蛋白进行纯化。
5.Western blotting鉴定重组表达蛋白。
6.采用琼脂糖扩散法检测融合蛋白的体外抑菌活性。
7.运用分子克隆技术,以pBSK(+)-Cecrop-m2A10及pSLfall80fa-AsVg-CFP-3'VTR载体质粒为基础质粒,构建以蚊眼特异性启动子(3xP3)驱动的红色荧光蛋白(DsRed)作为筛选标志物,Vg启动子驱动Cecrop-m2A10序列及高度特异性转座元件piggyBac组成的具有完整调控元件的重组转基因载体质粒pBac-AsVg-Cecrop-m2A10。
结果:
1.对鼠源性环子孢子蛋白单链抗体2A10中6种氨基酸的170个核苷酸进行了改造并融合Cecropin A编码基因,全基因合成法获得融合基因Cecrop-m2A10。并成功构建重组质粒pBSK(+)-Cecrop-m2A10。
2.成功构建表达重组质粒pE/32a(+)-Cecrop-m2A10,测序结果显示读码框架正确。通过IPTG诱导得到以包涵体形式高效表达的重组蛋白。包涵体经过尿素洗涤,变性,透析复性后纯度达83.7%。Western blotting分析显示重组蛋白与抗6-His抗体可产生59KD大小的特异性反应条带。
3.琼脂糖扩散法结果显示,纯化的重组蛋白具备抗大肠杆菌DH5α的活性。
4.成功构建了以蚊眼特异性启动子(3xP3)驱动的红色荧光蛋白(DsRed)作为筛选标志物,Vg启动子驱动Cecrop-m2A10序列及高度特异性转座元件piggyBac组成的具有完整调控元件的重组转基因载体质粒pBac-AsVg-Cecrop-m2A10。
结论:
1.成功构建融合基因Cecrop-m2h10。
2.成功构建表达重组质粒pET32a(+)-Cecrop-m2A10。在大肠杆菌中以包涵体形式高效表达,纯化后纯度达83.7%。
3.初步鉴定显示纯化的重组蛋白具备抗大肠杆菌DH5α的活性。
4.构建了以蚊眼特异性启动子(3xP3)驱动的红色荧光蛋白(DsRed)作为筛选标志物,Vg启动子驱动Cecrop-m2A10序列及高度特异性转座元件piggyBac组成的具有完整调控元件的重组转基因载体质粒pBac-AsVg-Cecrop-m2A10。
Background:Mosquitoes transmit numerous human diseases,such as malaria, epidemic encephalitis B,fiariasis,dengue fever,etc,severely harm to human health. An increasing trend on the rate of dengue fever has appeared year by year,occasionally,it might burst out and spread in some regions of the world.Malaria is one of the attacks that result in the highest infection ratio and mortality,taking a heavy toll on the human population in many parts of the world by infecting more than 300 million and killing more than 200 million people each year.The major reasons for this tragic situation are the unavailability of effective vaccines for malaria and the development of insecticide and drug resistance by the vectors and pathogens,respectively.Therefore,there is an urgent need to explore every possible avenue for developing novel control strategies against these mosquito-borne menacing diseases,and at the same time,drug resistance of malaria means that controlling of mosquito becomes the most effective and pragmatic way to ralieve the burden caused by malaria.
Plasmodium,the causative agent of malaria,has to complete a complex developmental program in its mosquito host for transmission to occur.Malaria sporozoites exhibit infectivity for mosquito salivary glands,human body and other vertebrate host tissue.The surface membrane of sporozoites of Plasmodium berghei—circumsporozoite protein(CSP) which covers the entire surface of mature,salivary gland sporozoites is a protective antigen of sporozoites.Meanwhile,it may play a role in the adhesion and invasion into the target cell.Mouse monoclonal antibodies against CSP of Plasmodium berghei—2A10 can affect mosquito salivary gland and human liver cell invasion by sporozoites in vitro assays and prevent malaria transmission.2A10scFv is a single chain antibody against CSP of Plasmodium falciparum.CHEN had successfully transformed a 2A10scFv gene into An.stephensi. Transgenic mosquitoes carrying 2A10scFv transgenes exhibited resistance to mosquito salivary gland invasion,the RT-PCR and Northern blot showed that the transgene transcribed well,but Western blot did not detect the protein(Unpublished data)which may be at very low levels in the transgenic mosquitoes.
In recent years,the prospect of using transgenic mosquitoes is rapidly gaining strength,owing to the development of molecular biology、immunology and genetics. The central event in transgenic mosquitoes is the heterologous expression of transgenes,expression levels of heterologous genes are crucial to their function. There are many factors may affect the expression level,such as the promoter,coding sequence,the copy number,insertion place and the nature of the expressed protein itself.The most frequently used codons among various species are different. Differences in codon usage can seriously hamper the expression of the heterologous transgenes.This is due,in part,to differences in codon usage,which can significantly result in the potential depletion of tRNAs encoding the same amino acid,decrease translation efficiency and thereby lower protein production in a heterologous system. Suggesting that codon optimization is a valuable strategy for improving the heterologous expression of native sequences,it has been widely and successfully used not only in P.pastoris,but also in other expression system such as E.coli BL21(DE3), Aspergillus niger,and mammalian cells.Various experimental procedures about PCR-based codon optimization have been developed,from the modification of a few codons about 5'codon adaptation to the extensive rewriting of up 1000 bp of DNA by overlap PCR.Likewise,the compatibility of codon usage between the 2A10scFv gene sequence and that of the expression host-mosquito are indeed respectively distinct. Then,if the DNA sequence encoding 2A10scFv gene was designed and synthesized based on the codon bias of mosqiuto,the transgenic expression may be at a higher level and the anti-pathogen effect of transgenic mosqiuto may be stronger.
Antimicrobial peptides(AMPs) are small molecular weight proteins with broad spectrum antimicrobial activity against a broad range of micro-organisms,including gram-negative and gram-positive bacteria,fungi,and viruses.They were found in plants,insects,bacteria and hunman.An expansive list of unique AMPs has been discovered in our environment,with well over 800 peptides known.Once in a target microbial membrane,the peptide kills target cells through diverse mechanisms. Cathelicidins and defensins are major groups of epidermal AMPs.Cecropins are one family of these peptides which were first isolated from the haemolymph of the giant silk moth,Hyalophora eecropia and may be idespread throughout the animal kingdom.The principle insect cecropins(A,B and D) are 35 to 37 residues,devoid of cysteine,with a strongly basic N-terminal linked to a neutral C-terminal by a flexible glycine-proline link.The overall structure deduced by NMR for cecropin A is two near perfect amphipathic segments joined by a Gly-Pro hinge.Antibacterial activity has been reported for cecropins A,B and P gainst Gram-positive and Gram-negative organisms.There are some problems in direct expression of AMPs for the following reasons:①In the case of direct expression of AMPs,undesirable degradation of such small sized mRNA and peptide was expected.②The direct expression of an antimicrobial peptide in bacteria may be cytotoxic to the host or subjected to degradation by host-derived peptidases.To overcome these potential problems,a common strategy to achieve an efficient expression of a protein or antimicrobial peptide is to express the molecule in a microbial system,either de novo or as a fusion partner with another protein.The gene fusion was successfully translated in a bacterial expression system.The fusion protein was non-toxic to the host bacteria. Our researches suggest that the strategy investigated in this study is useful to produce a large amount of AMPs and it might be applicable to other cationic small peptides especially to those which are difficult to synthesize chemically and have no available affinity purification methods.Consequently,in order to increase the anti-pathogen effect and stability of 2A 10scFv gene,we designed to linked 2A 10scFv gene with the coding sequence from A.gambiae cecropin A and constructed a fusion gene Cecrop-m2A10.
Objective:
1.To modify coding sequence of a single chain antibody(2A10) against circumsporozoite protein(CSP) of Plasmodium falciparum and construct fusion gene Cecrop-m2A10.
2.To express Cecrop-m2A10 in E.coli BL21(DE3) and analyze the bioactivity of the expression products.
3.To construct pBac-AsVg-Cecrop-m2A10 plasmid which contains a full regulation sequence,3xP3-DsRed eye specific promoter,vitellogenin(Vg) promoter and Cecrop-m2A10 gene.
Methods:
1.The coding sequence of a single chain antibody(2A10) against circumsporozoite protein(CSP) of Plasmodium falciparum were modified according to Anopheles gambiae preferred codons and then linked with the coding sequence from A.gambiae Cecropin A.The fusion gene Cecrop-m2A10 were produced by whole gene synthesis.To follow it,recombinant cloning plasmid pBSK(+)-Cecrop-m2A10 was constructed.
2.Completed Cecrop-m2A10 gene was cloned from pBSK(+)-Cecrop-m2A10 plasmid by PCR,Cecrop-m2A10 gene fragment was cloned into pET32a(+) vector to construct recombinant plasmid pET32a(+)-Cecrop-m2A10 which was identifed by sequence analysis.
3.The plasmid pET32a(+)-Cecrop-m2A10 was transformed into E.coli BL21(DE3) to express the recombinant proteins after the induction of IPTG.
4.Optimize expression conditions and purify expression products.
5.Identify the recombinant proteins by Western blotting.
6.The bioactivity of the expression products was assayed with Argarose-Diffusion-Method.
7.The pBac-AsVg-Cecrop-m2A10 plasmid which contains 3xP3-DsRed eye specific promoter,vitellogenin(Vg) promoter,Cecrop-m2A10 gene was constructed by techniques of molecular cloning such as digestion and ligation methods with the basic plasmid:pBSK(+)-Cecrop-m2A 10 and pSLfall80fa-AsVg-CFP-3'VTR.
Results:
1.The coding sequence of a single chain antibody(2A10) against circumsporozoite protein(CSP) of Plasmodium falciparum were modified according to Anopheles gambiae preferred codons.Totally,170 nucleotides in six kinds of amino acids were replaced and then linked with the coding sequence from A.gambiae Cecropin A.Subsequently,the fusion gene,Cecrop-m2A10 were produced by whole gene synthesis.To follow it,recombinant cloning plasmid pBSK(+)-Cecrop-m2A10 was successfully constructed.
2.Recombinant expression plasmids pET32a(+)-Cecrop-m2A10 was successfully constructed and its open frame was corrected by sequence analysis.After IPTG inducting,recombinant protien was expressed in inclusion body form in E.coli BL21(DE3).After the collection of inclusion body,denaturing lysis and refoding dialysis,the purity of the expression product was over 83.7%.Western blotting showed the recombinant protien could be specifically recognized by anti-6-His antibody.
3.The argarose-Diffusion-Method showed bacteriostasis inhibition to the growth of E.coli DH5α.
4.The pBac-AsVg-Cecrop-m2A10 plasmid which contains 3xP3-DsRed eye specific promoter,vitellogenin(Vg) promoter,Cecrop-m2A10 gene was successfully constructed.
Conclusion:
1.The fusion gene Cecrop-m2A10 was successfully constructed.
2.Recombinant expression plasmids pET32a(+)-Cecrop-m2A10 was successfully constructed and expressed in inclusion body form in E.coli BL21(DE3). After the collection of inclusion body,denaturing lysis and refoding dialysis,the purity of the expression product was over 83.7%.
3.The preliminary evaluation showed bacteriostasis inhibition to the growth of E.coli DH5α.
4.The pBac-AsVg-Cecrop-m2A10 plasmid which contains 3xP3-DsRed eye specific promoter,vitellogenin(Vg) promoter,Cecrop-m2A10 gene was successfully constructed.
引文
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[1]Horn C,Schmid BG,Pogoda FS,et al.Fluorescent transformation markers for insect transgenesis[J].Insect Biochem Mol Biol,2002,32(10):1221 - 1235.
[2]Jasinskiene N,Coates CJ,Benedict MQ,et al.State transformation of the yellow fever mosquito,Aedes aegypti,with the Hermes element from the horsefly[J].Proc Natl Acad Sci U.S.A.,1998,95(7):3743-3747.
[3]Catteruccia F,Nolan T,Loukeris TG,et al.Stable transformation of malaria mosquito Anopheles stephensi[J].Nature,2000,405(6789):959-962.
[4]Moreira LA,Edwards MJ,Adhami F,et al.Robust gut-specific gene expression in transgenic Aedes aegypti mosquitoes[J].Proc Natl Acad Sci U.S.A.,2000,97(20):10895-10898.
[5]Kokoza VA,Martin D,Mienaltowski MJ,et al.Transcriptional regulation of the mosquito vitellogenin gene via a blood meal-triggered cascade[J].Gene,2001,274(1):47-65.
[6]Coates CJ,Jasinskiene N,Pott GB,et al.Promoter-directed expression of recombinant fire-fly luciferase in the salivary glands of Hermes-transformed Aedes aegypti[J]. Gene,1999, 226(2):317-325.
[7] Catteruccia F, Benton JP, Crisanti A, An Anopheles transgenic sexing strain for vector control[J]. Nat Biotechnol,2005,23(11):1414-1417.
[8] Kim W, Koo H, Richman AM, et al. Ectopic expression of a cecropin transgene in the human malaria vector mosquito Anopheles gambiae (Diptera:Culicidae): effects on susceptibility to Plasmodium[J]. J Med Entomol,2004,41(3):447-455.
[9] Conde R, Zamudio FZ, Rodriguez MH, et al. Scorpine, an anti-malaria and anti-bacterial agent purified from scorpion venom[J]. FEBS Lett,2000,471 (2-3): 165-168.
[10] de Lara Capurro M, Coleman J, Beerntsen BT, et al. Virus-expressed,recombinant single-chain antibody blocks sprozoite infection of salivary glands in Plasmodium gallinaceum-infected Aedes aegypti[J]. Am J Trop Med Hyg,2000,62(4):427-433.
[11] Ghosh AK, Ribolla PE, Jacobs-Lorena M, Targeting Plasmodium ligands on mosquito salivary glands and midgut with a phage display peptide library[J].Proc Natl Acad Sci U.S.A.,2001,98(23):13278-13281.
[12] Maragathavally KJ, Kaminski JM, Coates CJ, Chimeric Mosl and piggyBac transposases result in site-directed integration[J]. FASEB J,2006,20(11):1180-1182.
[13] Nimmo DD, Alphey L, Meredith JM, et al. High efficiency site-specific genetic engineering of the mosquito genome[J]. Insect Mol Biol,2006,15(2):129-136.
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[15] Kokoza V, Ahmed A, Cho WL, et al. Engineering blood meal-activated systemic immunity in the yellow fever mosquito, Aedes aegypti[J]. Proc Natl Acad Sci U.S.A., 2000,97(16):9144-9149.
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[17] Yoshida S ,Watanabe H, Robust salivary gland-specific transgene expression in Anopheles stephensi mosquito[J]. Insect Mol Biol,2006, 15(4):403-410.
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[21] Nirmala X, Marinotti O, Sandoval JM, et al. Functional characterization of the promoter of the vitellogenin gene, AsVg1, of the malaria vector, Anopheles stephensi[J]. Insect Biochem Mol Biol,2006,36(9):694-700.
[22] Ito J, Ghosh A, Moreira LA, et al. Transgenic anopheline mosquitoes impaired in transmission of a malaria parasite[J]. Nature,2002,417(6887):452-455.
[23] Moreira LA, Ito J, Ghosh A, et al. Bee venom phospholipase inhibits malaria parasite development in transgenic mosquitoes[J]. J Biol Chem,2002,277(43):40839-40843.
[24] Abraham EG, Donnelly-Doman M, Fujioka H, et al. Driving midgut-specific expression and secretion of a foreign protein in transgenic mosquitoes with AgAperl regulatory elements[J]. Insect Mol Biol,2005,14(3):271-279.
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