通过大肠杆菌thyA染色体-质粒平衡致死系统构建无抗性基因表达载体

克隆了大肠杆菌和霍乱弧菌胸腺嘧啶合成酶基因thyA ,并以pcDNA3质粒为基础 ,分别用两种来源的thyA基因替代其氨苄抗性基因Amp,构建了不含抗性基因 ,且可在thyA营养缺陷型大肠杆菌中基于染色体 质粒平衡致死系统稳定传代的真核表达载体。该载体可有效表达红色荧光蛋白报告基因。为核酸疫苗的制备提供一个无抗性的表达载体系统     

基于谷氨酸消旋酶基因(murI)构建杀香鱼假单胞菌减毒活疫苗株的染色体-质粒平衡致死表达系统

【目的】构建一种基于谷氨酸消旋酶(MurI)基因的染色体-质粒平衡致死系统,用于杀香鱼假单胞菌减毒活疫苗株(Pseudomonas plecoglossicida ΔtssD-1, Pp ΔtssD-1)中表达外源抗原,为开发多联活疫苗提供新的思路和方法。【方法】利用同源重组技术,将亲本株Pp ΔtssD-1中的murI基因敲除,构建murI基因缺失突变株;将广宿主穿梭质粒pBBR1MCS-2的卡那霉素抗性基因替换为murI基因,构建平衡致死质粒(即无抗性回补质粒);在平衡致死质粒的多克隆位点处插入绿色荧光蛋白以检测外源抗原是否稳定表达,对重组菌株进行生物学特性分析,包括生长曲线、质粒稳定性和外源抗原表达水平。【结果】murI基因缺失株在不含D-谷氨酸的LB培养基上无法生长;无抗性回补株在不含D-谷氨酸的LB培养基上恢复了生长能力,但生长速度低于亲本株;经鉴定外源抗原可在无抗性质粒中稳定表达,并可在荧光显微镜下观察到明显的绿色荧光信号;此外,平衡致死质粒在重组菌株中具有良好的遗传稳定性。【结论】本研究以murI为靶点构建了新型的染色体-质粒平衡致死系统,可在无抗性筛选条件下在Pp ΔtssD-1中表达外源抗原,为开发多联活疫苗提供了新的策略和方法。     

基于大肠杆菌受体菌DH5α △asd缺失株的构建及作为基因转化、表达操作系统的评估

基于大肠杆菌(E.coli)染色体上asd基因的已知序列,利用λ噬菌体的Red同源重组系统一步法构建E.coliDH5α的asd基因缺失突变株DH5α△asd::cat,在二次重组中利用携带能够表达FLP位点特异性重组酶的质粒pCP20介导二次同源重组,以去除上述缺失突变株中氯霉素抗性筛选基因。结合PCR扩增和测序结果,证明DH5α△asd缺失突变株的正确构建。该缺失突变株失去了在普通LB培养基上生长的能力,只有添加DAP或导入表达asd基因的质粒(asd基因互补试验)才能在LB培养基上生长,与原型DH5α比较,其生长速度和生长对数期、接受不同拷贝数质粒的转化效率几乎相一致。基于该缺失突变株构建出以asd营养基因为标志的大肠杆菌染色体-质粒平衡致死系统。体外培养连续传代50代次,pnirBMisL-fedF-asd质粒不丢失,并功能性表达F18大肠杆菌黏附素FedF。     

利用λ-Red重组系统和平衡致死系统改造抗药性致腹泻工程疫苗

质粒pMM085是含有猪毒素源性大肠杆菌(ETEC)的黏附素K88与无毒肠毒素LTA-B 基因的重组质粒,含氯霉素抗性基因,由此构建的菌苗株带有抗药性。利用平衡致死系统改建此疫苗株,即将质粒上的氯霉素抗性基因cat替换成asd基因,并把新构建的质粒转移到缺失asd基因的大肠杆菌X6097中。但由于质粒pMM085是一个23kD的大质粒,传统的基因工程操作不易进行,利用λ-Red重组系统,将表达Red重组蛋白的质粒pKD46转化含pMM085的大肠杆菌X6097,并用两端各带有39ntcat基因同源区、含全长asd基因的PCR产物电击转化此感受态细胞,在λ-Red重组系统的帮助下,成功实现了asd基因对cat基因的置换。     

鼠伤寒沙门氏菌asd基因的克隆及序列分析

以鼠伤寒沙门氏茵标准株基因组DNA作为模板,用PCR的方法扩增鼠伤寒沙门氏菌的asd基因并克隆入质粒pUCl9,并对其进行测序,序列与献报道一致。同时将质粒pYA248上的链球菌asd基因进行了置换,观察了分别含有链球菌asd基因与鼠伤寒沙门氏菌asd基因的质粒在减毒鼠伤寒沙门氏菌X4072中的生长情况,结果表明含有鼠伤寒沙门氏菌的asd基因的高拷贝质粒pUCl9的菌株生长情况更好。为完善染色体／质粒平衡致死系统,构建减毒鼠伤寒沙门氏活菌疫苗奠定了基础。     

新型生长抑素原核表达质粒的构建及表达鉴定

将生长抑素(SS)与乙肝表面抗原(S)融合基因插入平衡致死系统原核表达质粒pYA3493中, 转化至缺失asd基因的减毒猪霍乱沙门氏菌C500, 经酶切、测序筛选得到非抗性的目的克隆, 命名为pYA-SS。应用SDS-PAGE和Western blotting 技术分离并检测融合蛋白在宿主菌中的表达活性。结果表明, 本试验构建的非抗性筛选生长抑素原核表达质粒可以在宿主菌C500中稳定、正确表达。此研究为开发新型、高效、安全的促生长疫苗提供了可靠的研究材料。     

新型生长抑素原核表达质粒的构建及表达鉴定

将生长抑素(SS)与乙肝表面抗原(S)融合基因插入平衡致死系统原核表达质粒pYA3493中, 转化至缺失asd基因的减毒猪霍乱沙门氏菌C500, 经酶切、测序筛选得到非抗性的目的克隆, 命名为pYA-SS。应用SDS-PAGE和Western blotting 技术分离并检测融合蛋白在宿主菌中的表达活性。结果表明, 本试验构建的非抗性筛选生长抑素原核表达质粒可以在宿主菌C500中稳定、正确表达。此研究为开发新型、高效、安全的促生长疫苗提供了可靠的研究材料。     

通过大肠杆菌thyA染色体质粒平衡致死系统构建无抗性基因表达载体

克隆了大肠杆菌和霍乱弧菌胸腺嘧啶合成酶基因thyA,并以pcDNA3质粒为基础,分别用两种来源的thyA基因替代其氨苄抗性基因Amp,构建了不含抗性基因,且可在thyA营养缺陷型大肠杆菌中基于染色体-质粒平衡致死系统稳定传代的真核表达载体。该载体可有效表达红色荧光蛋白报告基因。为核酸疫苗的制备提供一个无抗性的表达载体系统。     

CS3菌毛抗原和霍乱毒素B亚基在人伤寒菌中的表达

通过体内外重组的方法,构建了人伤寒菌流行株Ｔｙ２的△ａｒｏＡ,△ａｒｏＣ和ａｓｄ＾－基因缺失突变体（ＲＳ４１７）作为抗原载体菌：同时,构建包含ａｓｄ基因的表达质粒ｐＹＸ１０２,与ＲＳ４１７一起,构成宿主－载全平衡致死系统,用于在没有抗生素条件选择的情况下,稳定表达克隆在表达质粒上的外源抗原基因,将肠毒素性大肠杆菌的菌毛抗原ＩＩＩ（ＣＳ３）基因和霍乱弧菌毒素Ｂ亚基（ＣＴＢ）基因分别克隆至ｐＹＸ１０２     

肠毒素性大肠杆菌CS6菌毛抗原与霍乱毒素B亚基在痢疾菌中的表达及其免疫学效果

构建了携带asd、霍乱毒素B亚基(CTB)基因的表达质粒pYX201,与福氏2a痢疾菌T32的△asd突变株FaD构成宿主质粒平衡致死系统,用于在没有抗生素选择压力的情况下,稳定表达CTB抗原基因。以此为基础,构建了单独表达肠毒素性大肠杆菌CS26菌毛抗原基因的重组质粒pYX202,以及同时表达CS6和CTB的共表达质粒pYX203。Western blotting和ELISA检测结果证实CS6及CTB在痢疾菌FaD中可以有效表达。重组菌免疫家兔后可诱生相应的血清抗体,特别是CTB的抗体效价较高,并持续较长时间。本研究为细菌性腹泻疫苗的研究提供了候选株。     

A Listeria ivanovii balanced-lethal system may be a promising antigen carrier for vaccine construction

Expressing heterologous antigens by plasmids may cause antibiotic resistance. Additionally, antigen expression via plasmids is unstable due to the loss of the plasmid. Here, we developed a balanced-lethal system. The Listeria monocytogenes (LM) balanced-lethal system has been previously used as an antigen carrier to induce cellular immune response. However, thus far, there has been no reports on Listeria ivanovii (LI) balanced-lethal systems. The dal and dat genes from the LI-attenuated LIΔatcAplcB (LIΔ) were deleted consecutively, resulting in a nutrient-deficient LIΔdd strain. Subsequently, an antibiotic resistance-free plasmid carrying the LM dal gene was transformed into the nutrient-deficient strain to generate the LI balanced-lethal system LIΔdd:dal. The resultant bacterial strain retains the ability to proliferate in phagocytic cells, as well as the ability to adhere and invade hepatocytes. Its genetic composition was stable, and compared to the parent strain, the balanced-lethal system was substantially attenuated. In addition, LIΔdd:dal induced specific CD4⁺/CD8⁺ T-cell responses and protected mice against LIΔ challenge. Similarly, we constructed an LM balanced-lethal system LMΔdd:dal. Sequential immunization with different recombinant Listeria strains will significantly enhance the immunotherapeutic effect. Thus, LIΔdd:dal combined with LMΔdd:dal, or with other balanced-lethal systems will be more promising alternative for vaccine development.     

The aspartate-semialdehyde dehydrogenase of Edwardsiella ictaluri and its use as balanced-lethal system in fish vaccinology

asdA mutants of gram-negative bacteria have an obligate requirement for diaminopimelic acid (DAP), which is an essential constituent of the peptidoglycan layer of the cell wall of these organisms. In environments deprived of DAP, i.e., animal tissues, they will undergo lysis. Deletion of the asdA gene has previously been exploited to develop antibiotic-sensitive strains of live attenuated recombinant bacterial vaccines. Introduction of an Asd(+) plasmid into a ΔasdA mutant makes the bacterial strain plasmid-dependent. This dependence on the Asd(+) plasmid vector creates a balanced-lethal complementation between the bacterial strain and the recombinant plasmid. E. ictaluri is an enteric gram-negative fish pathogen that causes enteric septicemia in catfish. Because E. ictaluri is a nasal/oral invasive intracellular pathogen, this bacterium is a candidate to develop a bath/oral live recombinant attenuated Edwardsiella vaccine (RAEV) for the catfish aquaculture industry. As a first step to develop an antibiotic-sensitive RAEV strain, we characterized and deleted the E. ictaluri asdA gene. E. ictaluri ΔasdA01 mutants exhibit an absolute requirement for DAP to grow. The asdA gene of E. ictaluri was complemented by the asdA gene from Salmonella. Several Asd(+) expression vectors with different origins of replication were transformed into E. ictaluri ΔasdA01. Asd(+) vectors were compatible with the pEI1 and pEI2 E. ictaluri native plasmids. The balanced-lethal system was satisfactorily evaluated in vivo. Recombinant GFP, PspA, and LcrV proteins were synthesized by E. ictaluri ΔasdA01 harboring Asd(+) plasmids. Here we constructed a balanced-lethal system, which is the first step to develop an antibiotic-sensitive RAEV for the aquaculture industry.     

Asd-based balanced-lethal system in attenuated Edwardsiella tarda to express a heterologous antigen for a multivalent bacterial vaccine

Edwardsiella tarda is an enteric Gram-negative invasive intracellular pathogen, which causes enteric septicemia in fish. It could be potentially used to develop a recombinant attenuated E. tarda vaccine for the aquaculture industry. Because live vaccine strains can potentially be released into the environment upon vaccination, medical and environmental safety issues must be considered. Deletion of the asdB gene in E. tarda resulted in a diaminopimelic acid (DAP)-dependent mutant. The wild type asdB gene was inserted in place of the antibiotic-resistance gene in the plasmid, and the resultant non-antibiotic resistant vector was transformed into the attenuated and DAP-dependent E. tarda vaccine strain (WEDΔasdB) to obtain a balanced-lethal system for heterologous antigen expression. The balanced-lethal expression system was further optimized by comparing plasmid replicons with different Shine–Dalgarno sequences and start codons for the asdB gene. Utilizing the optimized balanced-lethal expression system, the protective antigen gene gapA34 from the fish pathogen Aeromonas hydrophila LSA34 was expressed in the attenuated E. tarda to generate the multivalent vaccine candidate WEDΔasdB/pUTta4DGap. This vaccine was shown to evoke an effective immune response against both E. tarda and A. hydrophila LSA34 by vaccinating turbot via a simple immersion route. This multivalent E. tarda vector vaccine has great potential for broad applications in aquaculture.     

Cloning and characterization of the asd gene of Salmonella typhimurium: use in stable maintenance of recombinant plasmids in Salmonella vaccine strains

The asd mutants of Salmonella typhimurium have an obligate requirement for diaminopimelic acid (DAP) and will undergo lysis in environments deprived of DAP. This has allowed the development of a balanced-lethal system for the expression of heterologous antigens in vaccine strains using vectors containing the wild-type asd gene from Streptococcus mutans and asd mutant Salmonella hosts [Nakayama et al., Biotechnology 6 (1988) 693-697]. We have cloned the asd gene from S. typhimurium, characterized the gene product and used this gene to construct Asd+ expression cloning vectors. In addition we have constructed an asd cassette and a transposon derived from Tn5 that allow the rapid modification of other vectors for use with delta asd vaccine strains of S. typhimurium adding versatility to the Asd+ vector/delta asd host system of plasmid maintenance.     

人乳头瘤病毒HPV16 E7蛋白基因在沙门氏菌疫苗菌株X4072中的表达

采用ＰＣＲ方法,从ＨＰＶ１６型质粒中分离出ＨＰＶ１６Ｅ７基因片段,用ＥｃｏＲＩ和Ｓａｌｌ双酶解后,定向插入到表达质粒ｐＹＡ３３３２（ａｓｄ＋）的Ｐｔｒｃ启动子下游,构建成ｐＹＡ３３３２－ＨＰＶ１６－Ｅ７重组表达质粒。在大肠杆菌Ｘ６２１２上筛选出重组质粒后,通过中间菌株Ｘ３７３０的转化过渡,将重组质粒导入鼠伤寒沙门氏减毒疫苗菌株Ｘ４０７２（ａｓｄ－）,构建成宿主和载体之间具有平衡致死结构的ＨＰＶ１６     

宋内Ⅰ相抗原和霍乱CT-B共表达的免疫保护效果观察

将编码宋内氏痢疾菌(Shigella sonnei)I相O抗原的基因和霍乱弧菌(Vibrio choler-ae)的CT-B基因克隆至带asd基因的质粒PYA248,得重组质粒PMGL105。将该重组质粒转入asd基因缺失的减毒伤寒沙门氏菌X4072,构成了一个不带抗药性基因的载体-宿主平衡致死系统。一系列实验表明,该重组菌X4072(PMGL105)能稳定地表达宋内I相O抗原和霍乱弧菌的CT-B抗原。小鼠免疫保护实验表明,该重组菌对有毒的宋内氏I相痢疾杆菌及霍乱弧菌的攻击均具有良好保护作用。     

果蝇心脏发育起调控作用的候选基因的筛选

果蝇的早期心脏发育与脊椎动物的早期发育模式具有惊人的相似,所以果蝇成为研究脊椎动物心脏发育的模式动物,通过对其心脏发育基因的研究,可加速揭示人体心脏的发育机理。为进一步筛选并克隆出新的心脏发育基因,本实验采用经化学诱变的平衡致死系的果蝇,进行心脏特异性抗体染色,观察到10个致死系表现出心脏突变表型,并将已确定遗传学部位的6个品系缩小到更小区域。 Screening of the Genes in Controlling HeartDevelopment of Drosophila YANG Yue-jun,WU Xiu-shan,LI Min College of life sciences,Hunan Normal University,Changsha 410081,China Abstract:It is becoming increasingly evident that remarkable similaries of heart development are revealed in Drosophila and vertebrate,Therefore Drosophila can be used as a prototype to explore the vertebrate.This can in accelerate to revealing of the machanisms of human heart development.In order to screen and clone new genes that control the heart development,we have established the balanced-lethal lines by chemical mutagen and performed the heart-specific antibody.Ten of lines showed mutant phenotype,of which 6 were determined the smaller genetic sites for gene location. Key words：Drosophila; heart develop; genes     

Enhanced protection against nasopharyngeal carriage of <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> elicited by oral multiantigen DNA vaccines delivered in attenuated <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

Developing carrier systems and choosing appropriate antigens are essential steps in improving the safety and efficacy of Streptococcus pneumoniae DNA vaccines, which have enhanced the mucosal protection against nasopharyngeal colonization. In this study, we reconstructed a Salmonella-based balanced-lethal host- eukaryotic vector system, which was used as carrier to orally deliver the Streptococcus pneumoniae multiantigen DNA vaccines encoding psaA (pneumococcal surface adhesion A) and pspA′ (N-terminal of pneumococcal surface protein A) genes. The results showed that the multiantigen DNA vaccines using the new vector system as carrier afforded better protection than the vaccination with injected intramuscularly (i.m.) against Streptococcus pneumoniae D39 colonization infection in BALB/c mice models. This finding has associated with a high level of sIgA in the nasal mucosa as well as systemic IgG antibodies and a shift toward a Th1-mediated immune response. These studies have demonstrated the feasibility and advantage of using the new Salmonella-based balanced-lethal host-eukaryotic vector system as carrier to deliver S. pneumoniae DNA vaccines.     

Construction of a trivalent candidate vaccine against Shigella species with DNA recombination

In this work asd gene of Shigella flexneri 2a strain T32 was replaced by Vibrio cholerae toxin B subunit (ctxB) gene with DNA recombination in vivo and in vitro. The resulting derivative of T32, designed as FWL01, could stably express CtxB, but its growth in LB medium depended on the presence of diaminopimelic acid (DAP). Then form I plasmid of Shigella sonnei strain S7 was labeled with strain T32 asd gene and mobilized into FWL01. Thus a trivalent candidate oral vaccine strain, designed as FSW01, was constructed. In this candidate strain, a balanced-lethal system was constituted between the host strain and the form I plasmid expressing S, sonnei O antigen. Therefore the candidate strain can express stably not only its own O antigen but also CtxB and O antigen of S. sonnei in the absence of any antibiotic. Experiments showed that FSW01 did not invade HeLa cells or cause keratoconjunctivitis in guinea pigs. However, rabbits immunized FSW01 can elicit significant immune responses. In mice and rhesus monkey