青鳉(Oryzias latipes)smad3的克隆与表达分析

鱼类性染色体的原始性导致性别决定基因的多样性.高等动物中的单个基因,在鱼类中因为基因组复制而产生多个同源基因,呈遗传多样性.TGF-β家族在硬骨鱼性别决定和性别分化的过程中发挥关键作用.smad3是性腺体细胞衍生因子(gsdf)的下游基因,可将TGF-β信号从细胞表面传递至细胞核.本研究从青鳉精巢和卵巢组织cDNA中克...     

鱼类Fc受体研究

Fc受体是免疫细胞表面一种重要受体分子,通过与免疫球蛋白Fc段结合触发多种生物学功能,是联系体液免疫和细胞免疫的桥梁。部分硬骨鱼中已经发现了Fc受体,在斑马鱼、斑点又尾鲴和鲤鱼中都克隆到了Fc受体的γ亚基,在鲨鱼和大西洋鲑中证明有能够与免疫球蛋白结合的Fc受体存在,并在斑点叉尾鲴、河豚和虹鳟中存在着类似α亚基的Fc受体。对鱼类Fc受体的发现和研究必将为了解鱼类的免疫机制及免疫进化提供重要的资料。     

神经导向因子在肿瘤中的作用及其机制

神经导向因子Netrins作为分泌蛋白与膜结合蛋白,在神经细胞迁移、分化及凋亡等生物学过程中发挥重要的作用。自发现以来,Netrins被认为负责指导中枢神经系统组织形态的发育,随后发现它也广泛参与一些非神经组织的黏附、迁移和分化等生理过程,如血管生成、淋巴管形成和炎症等。最近的研究表明,Netrins也参与调控肿瘤的发生与发展,在结直肠癌、胰腺导管腺癌等多种肿瘤组织中发挥重要的调控作用。由于Netrins的功能多样性且通过结合不同受体在肿瘤组织中发挥不同的生物学效应,其在肿瘤中的具体作用机制尚不清楚。文中结合课题组目前的实验研究进展,阐述了近年来关于Netrins的研究,特别是在肿瘤领域取得的最新研究结果,总结了其作用机制,并对其研究及应用前景进行了展望。     

鱼类细胞系在病毒学研究中的应用进展

鱼类细胞培养尽管起步较晚,但截至目前已有近280余株不同的鱼类细胞系相继建立起来,在生理学、病毒学、毒理学、肿瘤及基因工程等多个领域发挥重要作用。主要对鱼类细胞系在病毒学研究中的最新应用,并结合作者自身的研究,尤其侧重于鱼类病毒分离、重要功能基因鉴定、抗病毒免疫和病毒致病机理研究等方面的进展作一概述。     

Alu元件研究进展

Alu序列是灵长类动物特有的且最重要的短散在元件,在基因组中约有100万份拷贝,占基因组11%以上。这一基因家族曾被认为是垃圾序列,但随着对Alu序列研究的深入,Alu在基因调控表达网络中的功能逐渐被发现,Alu与人类的疾病密切相关,并且可能影响人类衰老,在人类多样性中也发挥重要作用。     

鱼类特异的基因组复制及鱼类多样性(英文)

过去的研究认为在脊椎动物的进化历程中曾发生了两次基因组复制.而最近的系统发生学和比较基因组学研究提出辐鳍鱼还发生了第3次基因组复制,即鱼类特异的基因组复制(The fish-specific genome dupli-cation).目前,基因组复制是生物进化研究中的热点问题.硬骨鱼是世界上现存鱼类中最多的一类,由多于现存脊椎动物半数的物种组成,在形态和生理适应类型上表现了明显的差异.硬骨鱼在进化上的成功和惊人的生物多样性可能与它们的基因组复杂性有关.     

鱼类特异的基因组复制

辐鳍鱼类是脊椎动物中种类最多、分布最广的类群,其基因组大小不等。过去的观点认为,在脊椎动物进化历程中曾发生了两次基因组复制。近期的系统基因组学研究资料进一步提出,在大约350百万年,辐鳍鱼还发生了第三次基因组复制,即鱼类特异的基因组复制(fish-specificgenomeduplication,FSGD),且发生的时间正处在“物种极度丰富”的硬骨鱼谱系(真骨总目)和“物种贫乏”的谱系(辐鳍鱼纲基部的类群)出现分歧的时间点,表明FSGD与硬骨鱼物种和生物多样性的增加有关。进一步开展鱼类比较基因组学和功能基因组学研究将进一步验证FSGD这一假说。     

海洋鱼类肠道微生物研究进展及应用前景

肠道微生物在动物体内数量庞大、结构复杂,在宿主生长发育过程中发挥着重要作用.淡水鱼类肠道微生物的研究主要集中于其对鱼类生长代谢、营养吸收及免疫调节等方面的影响,海洋鱼类则研究相对较少,且多集中于肠道微生物多样性及其变动影响因素分析.本文在归纳总结鱼类肠道微生物功能及其研究方法的基础上,着重分析肠道微生物在海洋鱼类生长发...     

爬行动物MHC基因研究进展

主要组织相容性复合体(MHC)是有颌脊椎动物中发现的编码免疫球蛋白受体的高度多态的基因群,因其在免疫系统中的重要作用而备受关注。脊椎动物不同支系间MHC的结构和演化差异较大。尽管MHC基因特征在哺乳类、鸟类、两栖类和鱼类中已被较好地描述,但对爬行动物MHC的了解仍较少。鉴于爬行动物对于理解MHC基因的演化占据很重要的系统发育位置,研究其MHC具有重要意义。本文就近年来爬行动物MHC的分子结构、多态性维持机制、功能和主要应用的研究现状进行了系统地回顾和总结,并展望了其研究前景。     

B细胞免疫球蛋白与T细胞抗原受体基因的种系结构及其体细胞重排(续)

T细胞受体基因 以~(125)Ⅰ标记的抗原测试T细胞,发现T细胞能与抗原结合,在放射活性很高时被杀灭,余下的T细胞就不再能结合此种抗原。这提示T细胞也具有抗原受体,且有克隆多样性。但对TCR的本质多年来捉摸不定,推测也是一种Ig样分子,故称之为IgT或Igx。1983年同时有几个实验室制备了与TCR反应的克隆特异性抗体。1984年以来相继克隆了TCR基因的     

Immunoglobulin light chains in medaka (Oryzias latipes)

The gene segments encoding antibodies have been studied in many capacities and represent some of the best-characterized gene families in traditional animal disease models (mice and humans). To date, multiple immunoglobulin light chain (IgL) isotypes have been found in vertebrates and it is unclear as to which isotypes might be more primordial in nature. Sequence data emerging from an array of fish genome projects is a valuable resource for discerning complex multigene assemblages in this critical branch point of vertebrate phylogeny. Herein, we have analyzed the genomic organization of medaka (Oryzias latipes) IgL gene segments based on recently released genome data. The medaka IgL locus located on chromosome 11 contains at least three clusters of IgL gene segments comprised of multiple gene assemblages of the kappa light chain isotype. These data suggest that medaka IgL gene segments may undergo both intra- and inter-cluster rearrangements as a means to generate additional diversity. Alignments of expressed sequence tags to concordant gene segments which revealed each of the three IgL clusters are expressed. Collectively, these data provide a genomic framework for IgL genes in medaka and indicate that Ig diversity in this species is achieved from at least three distinct chromosomal regions.     

Vaccination and immune responses against atypical Aeromonas salmonicida in spotted wolffish (Anarhichas minor Olafsen) juveniles

To study the effect of early vaccination, wolffish juveniles of size 50 and 90 mm, respectively, were vaccinated with an oil-adjuvanted atypical A. salmonicida bacterin. Vaccination resulted in significant protection after challenge with the homologous bacterial strain and specific antibody responses were demonstrated against whole bacteria as well as purified A-layer protein and LPS by ELISA and Western blotting but individual variation in immune responses was apparent. The A-protein was the most immunogenic bacterial component. In addition, higher numbers of immunoglobulin producing cells were detected by in situ hybridisation in kidney and spleen of vaccinated fish compared to non-vaccinated fish. Plasma cells were also present in gut and gills in equal numbers irrespective of treatment. No plasma cells were found in the skin. Finally, the frequencies of expressed V(H)families and C(L)isotypes of wolffish immunoglobulins were shown by PCR. The relative expression of the three variable regions of the Ig heavy chain and the three isotypes of the Ig light chain in the spotted wolffish spleen seemed to be unaffected by immunisation with a complex antigen like the A. salmonicida bacterin.     

Characterization of parvalbumin isotypes in white muscle from the barbel and expression during development

Parvalbumin isotypes PA II, PA III, PA IVa, and PA IVb were isolated by chromatography from trunk white muscle of barbel and physicochemically characterized. Electrospray ionization mass spectroscopy revealed that PA II has a lower molecular weight than the other isotypes and that PA IVa and PA IVb each consist of two subforms. Isotype distribution was studied by polyacrylamide gel electrophoresis. In adult fish, the total parvalbumin titre decreased and the isotype distribution varied from the anterior to the posterior myotomes. In the course of barbel development, the total parvalbumin titre increased rapidly as fish standard length increased from 1·3 to 5 cm; then sloped down gently as the length increased to 60 cm. At least six parvalbumin isotypes were identified, three of which are different forms (a, b, and c) of PA II. These three forms were present together at the larval stage, but PA IIc and chiefly PA IIb appeared as early isotypes, contrary to PA IIa which was present until the adult period. Later PA IVb accounted for up to 90% of the total parvalbumin content; PA III and PA IVa are minor adult isotypes. Temporal and spatial variations in the total parvalbumin titre and in the differential expression of barbel parvalbumin isotypes very likely reflected the functional requirements of the fish axial musculature according to fish size and myotome location. Physiologically, the larval isotypes could promote faster relaxation of fast fibres than the adult isotypes, and hence favour shorter contraction times.     

Immunological study of muscle parvalbumin isotypes in three African catfish during development

Eleven parvalbumin isotypes expressed during the development of clariids Heterobranchus longifilis and Clarias gariepinus and claroteid Chrysichthys auratus were purified and electrophoresed on sodium-dodecyl-sulfate polyacrylamide gels. Immunochemical cross-reactions among these proteins were investigated by immunoblotting, using purified antibodies raised against three isotypes chosen at different stages of fish development. Antibodies raised against H. longifilis PA I (larval-juvenile isotype) and against C. gariepinus PA IIIa (juvenile-adult isotype) cross-reacted to a rather similar extent despite a weaker cross-reaction of anti-PA IIIa with larval-juvenile isotypes. On the other hand, antibodies raised against H. longifilis PA IV (an exclusively adult isotype) recognized markedly only H. longifilis PA IV and C. gariepinus PA IIIb. These two adult isotypes most likely belong to the alpha lineage, and all the others to the beta lineage. These results show that parvalbumin isotypes synthesized at different stages of fish growth differ structurally, and that the most marked difference is between larval-juvenile and adult clariid isotypes.     

Isotyping of human C4 complement using differences in the functional activity of C4A and C4B isotypes

The difference in the functional activity of the isotypes A and B of component C4 of human complement was used to determine their ratio and to detect the inherited deficiency of the isotypes. ELISA methods were developed for the quantitative assay of component C4 (conventional sandwich method) and its functional activity. When determining the functional activity, the classic pathway of the complement and therefore of component C4 was activated by activators sorbed on ELISA microplates (immunoglobulin IgG3 or liposaccharide of the Shigella sonnei cell walls, which activates the complement by binding component C1). The nascent fragment C4b is covalently bound to the target activator; C4Ab binds better to the target protein (immunoglobulin), and C4Bb to the target carbohydrate (liposaccharide). Therefore, when immunoglobulin is a target activator, isotype C4A is bound and determined; and when the complement is activated by liposaccharide, isotype C4B is determined. The ratio of the activities determined by the two methods indicates a deficiency in the individual isotypes of component C4 or its absence. The rabbit polyclonal monospecific antibodies against the human component C4 and the conjugates of these antibodies with horseradish peroxidase were used in the methods described.     

Design and characterization of a plasmid vector system capable of rapid generation of antibodies of multiple isotypes and specificities

We present a detailed method for constructing a vector system composed of plasmids encoding immunoglobulin genes that have been constructed in such a fashion so as to allow for the generation and secretion of antibodies of multiple specificities and isotypes via a rapid and easy cloning-and-ligation scheme. Restriction sites within each plasmid allow for the removal of variable domains, constant domains, leader sequences, or the entire immunoglobulin gene. Degenerate primers are used to clone variable regions from hybridoma cDNAs, allowing for the creation of antibodies with varying binding specificities. Sequence-specific primers are used to clone antibody constant domains, allowing for the creation of antibodies of multiple isotypes from a variety of lineages. A high-efficiency leader sequence has been inserted into the start of each gene to improve secretion. Antibodies constructed through this system are efficiently secreted by transfected cells, and are fully functional in antigen-specific binding assays.     

Isotyping of component C4 of human complement using differences in the functional activity of isotypes C4A and C4B

The difference in the functional activity of the isotypes A and B of component C4 of human complement was used to determine their ratio and to detect the inherited deficiency of the isotypes. ELISA methods were developed for the quantitative assay of component C4 (conventional sandwich method) and its functional activity. When determining the functional activity, the classic pathway of the complement and therefore of component C4 was activated on activators sorbed on ELISA microplates: immunoglobulin IgG3 or liposaccharide of theShigella sonnei cell walls, which activates the complement by binding component C1. The nascent fragment C4b is covalently bound to the target activator; C4Ab binds better to the target protein (immunoglobulin), and C4Bb to the target carbohydrate (liposaccharide). Therefore, when immunoglobulin is a target activator, isotype C4A is bound and determined; and when the complement is activated with liposaccharide, isotype C4B is determined. The radio of the activities determined by the two methods indicates the deficiency in the individual isotypes of component C4 or its absence. The rabbit polyclonal monospecific antibodies against the human component C4 and the conjugates of these antibodies with horseradish peroxidase were used in the methods described.     

Generation and Characterization of Antibodies against Asian Elephant (Elephas maximus) IgG,IgM, and IgA

Asian elephant (Elephas maximus) immunity is poorly characterized and understood. This gap in knowledge is particularly concerning as Asian elephants are an endangered species threatened by a newly discovered herpesvirus known as elephant endotheliotropic herpesvirus (EEHV), which is the leading cause of death for captive Asian elephants born after 1980 in North America. While reliable diagnostic assays have been developed to detect EEHV DNA, serological assays to evaluate elephant anti-EEHV antibody responses are lacking and will be needed for surveillance and epidemiological studies and also for evaluating potential treatments or vaccines against lethal EEHV infection. Previous studies have shown that Asian elephants produce IgG in serum, but they failed to detect IgM and IgA, further hampering development of informative serological assays for this species. To begin to address this issue, we determined the constant region genomic sequence of Asian elephant IgM and obtained some limited protein sequence information for putative serum IgA. The information was used to generate or identify specific commercial antisera reactive against IgM and IgA isotypes. In addition, we generated a monoclonal antibody against Asian elephant IgG. These three reagents were used to demonstrate that all three immunoglobulin isotypes are found in Asian elephant serum and milk and to detect antibody responses following tetanus toxoid booster vaccination or antibodies against a putative EEHV structural protein. The results indicate that these new reagents will be useful for developing sensitive and specific assays to detect and characterize elephant antibody responses for any pathogen or vaccine, including EEHV.