DNA疫苗的作用机制及新型免疫策略的研究进展

疫苗是预防和控制严重传染病的重要手段，将基因工程用于疫苗生产已经成为生物技术的热点内容之一。治疗性基因工程疫苗是近些年研究的重点，并迅速从治疗传染性疾病的研究扩展到非传染性疾病的研究，如治疗性乙肝疫苗、治疗性肿瘤疫苗等。本文针对这一热点阐述基因工程DNA疫苗的作用机制及新型免疫策略研究的进展。     

禽传染性支气管炎DNA疫苗在鸡体内的分布和安全性

采用本实验室构建的3种禽传染性支气管炎病毒基因的真核表达质粒pIBVS1、pIBVM、pIBVN,按各50 mg/L配制成质量浓度为150 mg/L的DNA疫苗,经腿部肌肉分点注射免疫1周龄SPF雏鸡。分别在免疫后24 h,73、0及60d,采集试验鸡的血液、心、肝、脾、肺、肾、胸腺、性腺（卵巢/睾丸）及注射部位肌肉进行组织总DNA抽提,以组织总DNA为模板进行PCR扩增。另外,在对照组DNA模板中加入不同拷贝数的质粒,确定PCR反应的灵敏性。以纯化后的组织总DNA为模板,PCR法检测质粒DNA整合到鸡细胞染色体基因组上的可能性,评价疫苗的安全性。结果表明,该疫苗在24 h内迅速分布于全身,并能在血液及所有检测的组织器官内持续分布60 d;纯化后的组织基因组DNA经PCR扩增均呈阴性,未发现整合现象,证实该DNA疫苗的安全性好。     

F-2毒素致体外培养大鼠睾丸支持细胞DNA损伤

为探讨F-2毒素对雄性生殖机能的影响,取大鼠睾丸支持细胞进行体外培养,运用单细胞凝胶电泳技术检测51、0、20和40 mg/L的F-2毒素攻毒后24 h支持细胞DNA的损伤情况。结果显示,F-2毒素在一定浓度范围内对体外培养的支持细胞DNA产生损伤作用,且受损程度随着F-2毒素攻毒剂量而升高,具有明显量效关系。除5 mg/L剂量组外,其余各组的细胞受损率、彗星尾长和DNA损伤程度与阴性对照组相比差异极显著（P〈0.01）,表明F-2毒素对体外培养的大鼠睾丸支持细胞有毒性作用,能够损伤细胞DNA。     

Assessment of genetic purity of tomato (Lycopersicon esculentum L.) hybrid using molecular markers

Three DNA molecular marker systems, RAPD, ISSR and SSR, were used to test seed genetic purity of two commercial hybrid tomato (Lycopersicon esculentum L.) cultivars ‘Hezuo 903’ and ‘Sufen No. 8’. Genomic DNA from the two F₁ hybrid cultivars and their corresponding parental lines was screened with 218 RAPD decamer primers, 54 ISSR primers and 49 SSR primers. Among the 321 primers, 4 primers for ‘Hezuo 903’ and 3 for ‘Sufen No. 8’, which could produce both female and male parent-specific markers, were selected for testing the genetic purity. A total of 210 hybrid individuals of each cultivar were analyzed using the identified primers. The combined results of the marker analysis showed that eight of the 210 F₁ plants in ‘Hezuo 903’ and 13 of 210 in ‘Sufen No. 8’ were false hybrids, and the overall genetic purity of the two F₁ hybrid seed lots was 96.2 and 93.8%, respectively. This study showed that RAPD and SSR markers could provide a practical and efficient tool in quality control of the tomato commercial hybrid seeds.     

Analysis of chromosome aberration due to ethidium bromide using AFM

AIM: To study chromosome aberration due to ethidium bromide (EB),a heterocyclic organic compound and an organic fluorescence dye commonly used in biochemical experiment,and to help further understanding the molecular mechanism of tumor or cancer induced by EB and other heterocyclic organic compounds.METHODS: The toxicity action of EB was evaluated from three aspects including DNA,chromosome and embryo stem cells (ESCs) using atomic force microscopy (AFM),and thereinto,the morphology structural difference of ESCs treated with two EB doses was also valuated.RESULTS: The morphological structures of DNA,chromosome and ESCs were dramatically damaged.The average height of DNA decreased 0.5 nm;chromosomal arms were ruptured from centromere location;molecules of cellular membrane congregated and loop-like structure formed,and ES cell masses were collapsed and became dead after large EB doses treatment and mesh-like morphological structure was discernable.CONCLUSION: The toxicity action of EB is strong and destroys the surface structure of DNA and chromosome.EB induces structural aberration of ES cellular membrane and cell death.The results indicate that the action of EB is externalized at gene level and cell level,which is important to study the carcinogenicity of EB.     

Detecting<Emphasis Type="Italic">Orobanche</Emphasis> species by using cpDNA diagnostic markers

Some species of the genusOrobanche are among the most devastating parasitic weeds, causing extensive damage in agricultural fields. Considering the difficult control due to seed longevity in the soil, small seed size, high fecundity and a subterranean phase that allows them to parasitize the host before they emerge and become evident, the development of diagnostic markers is highly recommended. In our study we identified potential molecular diagnostic markers from the plastid genome in order to distinguish among the most importantOrobanche species attacking crops in Andalusia, the southern region of the Iberian Peninsula. The study has consideredO. crenata, O ramosa andO. cumana causing serious losses in legumes, solanaceous crops and sunflower fields, respectively, andO. minor that, although abundant in Andalusia, has to our knowledge not yet been found parasitizing agricultural hosts. We amplified a non-coding region from the plastid genome, studied sequence differences among the amplified fragments and digested those of the same length with selected restriction enzymes. Here, we propose a molecular protocol to distinguish the main parasitic plants in crop fields of southern Spain. Different applications such as identification ofOrobanche seeds in soil or crop seed lots are discussed in order to offer right crop recommendations or to prevent new infestation of parasite-free fields. Recommendations for further development of these diagnostic markers are also considered. http://www.phytoparasitica.org posting Jan. 15, 2007.     

Cloning of DNA fragments specific to the pathotype and race of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

Japanese isolates of Verticillium dahliae, a causal agent of wilt disease in many plants, are classifiable into pathotypes based on their pathogenicity. Because these pathotypes are morphologically indistinguishable, establishing a rapid identification method is very important for the control of this pathogen in Japan. For cloning DNA fragments that are useful for identification and specific detection of V. dahliae pathotypes, we performed random amplified polymorphic DNA (RAPD) analyses using various isolates. One polymerase chain reaction (PCR) product, E10-U48, was specific to isolates pathogenic to sweet pepper. The other product, B68-TV, was specific to race 1 of isolates pathogenic to tomato. The specificity of these sequences was confirmed by genomic Southern hybridization. Further analyses revealed that the region peripheral to B68-TV obtained from the genomic DNA library includes the sequence specific to all isolates pathogenic to tomato (races 1 and 2). Moreover, sequence tagged site (STS) primers designed from B68-TV and its peripheral region showed race-specific and pathotype-specific amplification in a PCR assay. The probes and primers obtained in this study are likely to be useful tools for the identification and specific detection of pathotypes and races of V. dahliae. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession number AB095266.     

马铃薯金线虫和白线虫DNA特异扩增产物的克隆与鉴定

通过采用Fullaondo（1999）研究的特异引物检测方法,对来自英国和比利时的两个马铃薯金线虫（Globodera rostochiensis）种群和英国的一个马铃薯白线虫（G.pallida）种群的DNA进行扩增,其产物以大肠杆菌（Eschetichia coli）为受体菌,经过连接、转化和克隆纯化,筛选获得阳性克隆.经PCR鉴定和测序,英国和比利时的马铃薯金线虫种群的特异性谱带分别是357bp和350bp,而英国的马铃薯白线虫的谱带是782bp,与Fullaondo等人研究报道结果基本相符,从而鉴别出来自不同地理种群的马铃薯金线虫和白线虫.     

中国主栽双孢蘑菇菌株的DNA多态性

本文以中国双孢蘑菇不同主栽产区的9个具代表性的双孢蘑菇菌株为材料,用20个随机引物对其进行RAPD分析。结果表明,20个随机引物中,有15个随机引物的扩增产物DNA片段表现出明显的多态性,其中每个引物对不同菌株扩增出的DNA片段数目各不相同,而且不同引物扩增出的DNA带谱也不同,差异较大。这15个引物对供试菌株总共扩增出168条DNA片段,其中最大的片段可达6.77kb,最小的DNA片段仪有0.31kb。同时,比较了供试菌株两两间的遗传相似性,发现它们之间的变化不大,其相似系数从0.6891到1.0000,而平均相似系数仅为0.8500,表明供试菌株之间的亲缘关系较近,遗传变异不丰富。另外,采用系统聚类法中的类平均法,对供试菌株相似系数两两间进行聚类分析并生成树状图谱,直观准确地揭示了它们之间的差异。当相似系数为77％时,所有供试菌株都被聚为一大类,说明我国主栽双孢蘑菇菌株的遗传基础较狭窄,可能来源于同一品系,这为进一步开展双孢蘑菇菌种的遗传改良提供了理论依据。     

RAPD及其在果树上的应用

就RAPD的特点、操作及其在果树品种鉴定、遗传多样性检测、系谱解析和遗传图谱构建等方面的应用和进展进行了简要的阐述。