日本血吸虫大陆株26kDa谷胱甘肽S—转移酶编码区基因的克隆…

从日本血吸虫大陆株成分离ＲＮＡ，逆转录合成ｃＤＮＡ第一链，根据菲律宾株２６５ｋＤａ谷胱甘肽Ｓ－转移酶（ＧＳＴ）的ｃＤＮＡ序列，设计并合成引物，扩增出２６ｋＤａ的编码区基因，并克隆到ｐＢｌｕｅｓｃｒｉｐｔ质粒，初步酶切鉴定后，从两端对插入片段进行核苷酸序列测定。结果与菲律宾株比较，核苷酸同源性为９９．８％，仅第５８２位碱基不同，菲律宾为Ａ，而大陆株为Ｇ。比较从ｃＤＮＡ推导出的氨基酸序列，两者１００％     

汉坦病毒A9株M基因片段核苷酸序列的测定及分析

采用反转录-ＰＣＲ方法，分节段扩增汉坦病毒Ａ９株Ｍ基因片段ｃＤＮＡ，并克隆入ｐＧＥＭＴ载体中，用双脱氧末端终止法直接测定序列。结果表明，Ａ９株Ｍ片段ｃＤＮＡ由３６１８个碱基组成，编码１１３５个氨基酸，　　核苷酸序列及由其推导的氨基酸序列同７６／１１８株的同源性分别为９４．３３％和９７．８０％，说明汉坦病毒Ａ９株与７６／１１８株在糖蛋白的结构上高度保守。同７６／１１８株比较，在３'末端非编码区变异极大，变异率为２０％，且多了２个核苷酸。Ａ９株Ｍ基因片段核苷酸序列的获得，为利用该ｃＤＮＡ进行基因工程疫苗的研制创造了条件。     

中国广西狂犬病毒野毒株（CGX89－1株）糖蛋白基因核酸序列测定和分析

本文报告了中国广西狂犬病毒野毒株（ＣＧＸ８９－１株）糖蛋白基因ｃＤＮＡ的核苷酸序列及其推导的氨基酸序列。ＣＧＸ８９－１株的糖蛋白基因从起始密码ＡＴＧ到终止密码ＴＡＡ共有１５７５个核苷酸残基，可编码形成５２４个氨基酸残基的多肽链，经修饰后形成具有５０５个氨基酸残基构成的狂犬病毒糖蛋白。ＣＧＸ８９－１株的糖蛋白基因和核苷酸组成分别为：Ａ占２７．１１％，Ｔ占２６．２９％，Ｃ占２１．９７％和Ｇ占２４．６３％。核苷酸序列和巴斯德株（ＰＶ株），国际标准攻击毒株（ＣＶＳ株），中国狂犬病毒疫苗株（３ａＧ株）相比，同源性分别为８４．１％，８３．１％和８４．５％。其推导的氨基酸序列和ＰＶ株、ＣＶＳ株和３ａＧ株相比其同源性分别为９２．４％，８９．７％和８９．５％。ＣＧＸ８９－１株也具有３个Ｎ－糖基化位点，分别位于第３７位、１５７位和３１９位的氨基酸残基上。糖蛋白的膜外区部分重要抗原位点和ＰＶ株、ＣＶＳ株及３ａＧ株具有较高的一致性。     

中国广西狂犬病毒野毒株（CGX89—1株）糖蛋白基因核…

本报告了中国广西狂犬病毒野毒株（ＣＧＸ８９－１株）糖蛋白基因ｃＤＮＡ的核苷酸序列及其推导的氨基酸序列。ＣＧＸ８９－１株的糖蛋白基因从起始密码ＡＴＧ到终止密码ＴＡＡ共有１５７５个核苷酸残基，可编码形成５２４个氨基酸残基的多肽链，经修饰后形成具有５０５个氨基酸残基构成的狂犬病毒糖蛋白。ＣＧＸ８９－１株的糖蛋白基因和核苷酸组成分别为：Ａ占２７．１１％，Ｔ占２６．２９％，Ｃ占２１．９７％和Ｇ占２４．６３％     

肝癌病人血清中庚型肝炎病毒非结构基因（NS）5区cDN …

通过逆转录－套式－聚合酶链反应，从２例肝癌患者血清中扩增出长９９４ｂｐ的庚型肝炎病毒（ＨＧＦ）ｃＤＮＡ序列，聚合酶链反应（ＰＣＲ）产物纯化后以双脱氧末端终止法从双向直接测定其序列。结果，所分离的２株ＨＧＶ ＮＳ５区９９４ｂｐ核苷酸与国外已发表的３株ＨＧＶ序列比较，同源性为８７．２１％￣９３．６７％；２株间的同源性为９３．９２％。根据所测得的ｃＤＮＡ序列推导出其编码的３１３氨基酸序列，在氨基酸水平上     

7型腺病毒疫苗株76.5～87mu核苷酸序列分析

目的获得７型腺病毒疫苗株（Ａｄ７ｖ）７６５－８７ｍｕ核苷酸序列，分析该区段的基因结构和功能。方法从Ａｄ７疫苗株克隆出６８－８７ｍｕ核苷酸片段，应用Ｓａｎｇｅｒ双脱氧法进行核苷酸序列分析。结果Ａｄ７ｖ７６５～８７ｍｕ核苷酸序列长度为３５５７ｂｐ，推测此片段编码Ｅ３区１２１ｋＤ、１９２ｋＤ、２０１ｋＤ、２０５ｋＤ、１０３ｋＤ和１５２ｋＤ蛋白的一部分。所编码的６个蛋白与Ａｄ７原型株（Ａｄ７ｐ）和Ａｄ７ｈ（１９８６年以来南美流行的毒株）核苷酸高度同源（大于９７２％）。１６１ｋＤ蛋白由于缺失一个核苷酸，造成移码突变，编码提前终止。编码７７ｋＤ蛋白的ＯＲＦ由于缺失一段核苷酸而提前终止编码。结论这一结果为阐明Ａｄ７的基因结构与功能并为构建Ａｄ７载体时对Ｅ３区缺失提供依据     

抗bFGF McAb制备,鉴定及轻链可变区基因克隆和序列析

目的：以重组人碱性成纤包生长因子为抗原免疫小鼠,建立多株稳定分泌ｂＦＧＦ单克隆抗体（ＭｃＡｂ）的杂交瘤细胞。半对ｂＦＧＦ单克隆抗体（ＭｃＡｂ）可变区基因进行扩增及序列分析。方法：采用Ｗｅｓｔｅｒｎ ｂｌｏｔ法和免疫沉淀法对抗体特异性和ｌｇ类型进行鉴定,同时,应用分子生物学技术,对Ｖｋ基因的ＤＮＡ片段进行克隆,并对４ａ２Ｖｋ基因进行序列测定。结果：ｂＦＧＦ单克隆抗体可特异性结合人重组ｂＦＧＦ抗原,且     

编码人分化抗原5C5全长cDNA的克隆及功能初探

目的 编码５Ｃ５分化抗原全长ｃＤＮＡ的克隆及功能探索。方法 构建人活化Ｂ细胞株３Ｄ５细胞的λｇｔ１１ ｃＤＮＡ文库，以核苷酸杂交法从ｃＤＮＡ文库中筛选阳性克隆、作核苷酸序列分析，编码蛋白质氨基酸序列的亲疏水分析，Ｎｏｒｔｈｅｒｎ ｂｌｏｔ测５Ｃ５ ｍＲＮＡ转录本长度，用ＲＴ－ＰＣＲ检测５Ｃ５ ｍＲＮＡ在不同细胞株的表达，观察单抗５Ｃ５－Ｇ１对３Ｄ５细胞增殖的影响。结果 从人活化Ｂ细胞株３Ｄ５的λｇ     

登革2型病毒04株5‘和3’末端的序列分析

目的 观察登革２型病毒０４株（Ｄ２－０４）基因组５’和３’末端序列。方法 从Ｄ２－０４感染的Ｃ６／３６细胞中提取总ＲＮＡ,以该ＲＮＡ为模板,利用ＲＡＣＥ法,分别扩增Ｄ２－０４株的５’和３’末端ｃＤＮＡ片段。将其分别与ｐＧＥＭ－Ｔ载体连接得到含有５’端５３５ｂｐ和３’端５０３ｂｐ ｃＤＮＡ的重组质粒,并测定上述ｃＤＮＡ插入片段的序列。将Ｄ２－０４的５’和３’端非编码区的核苷酸序列与其它登芏２型毒株进     

小鼠Ⅲ型17β—羟甾脱氢酶的序列及其表达

１７β－羟甾脱氢酶（１７β－ＨＳＤ）催化１７－酮类固醇与１７β－羟类固醇相互转换。本研究从小鼠睾丸中纯化出小鼠Ⅲ型１７β－ＨＳＤ的ｃＤＮＡ克隆，最长的ｃＤＮＡ核苷酸数为１１３１，包含一个短的５＇非编码序列、９１８个核苷酸编码序列和１５５个核苷酸３＇非编码序列；其起始密码子为ＡＴＧ，终止密码子为ＴＡＧ，开读框架编码为３０５个氨基酸的多肽。该研究为进一步了解１７β－ＨＳＤ的缺乏和性激素代谢的分子机理奠     

Comparison of the cloned genes of the 26- and 28-kilodalton glutathione S-transferases of Schistosoma japonicum and Schistosoma mansoni.

Both Schistosoma japonicum and S. mansoni contain 28- and 26-kDa glutathione S-transferases (GSTs). Despite their immunological cross-reactivity using rabbit antisera, the S. japonicum 28-kDa GST (Sj28) is weakly immunogenic relative to the S. mansoni protein (Sm28) in mouse immunization experiments using GSTs purified from adult worms. The difference in immunogenicity is also observed during schistosome infection in mice. Using surface-labeled living S. japonicum worms, evidence was obtained for a surface location of Sj28 comparable to that reported for the S. mansoni molecule. The nucleotide and deduced amino acid sequences of cDNA clones corresponding to Sj28 and Sm28 were compared. Despite obvious homology (77% identity), differences were found in regions known to contain T epitopes in the S. mansoni protein which may be an explanation for the striking differences in immunogenicity in regard to antibody production in mice. The 26-kDa GSTs of these two parasites (Sj26 and Sm26) are also closely related on the basis of nucleotide and deduced amino acid sequences, there being 82% identity in the putative coding regions. When the amino acid sequences of Sj28 and Sm28 were compared with those of Sj26 and Sm26, the overall sequence identity was approximately 20%. However, a relatively conserved region was identified in otherwise structurally different molecules which may participate in common properties of these enzymes.     

Molecular cloning and tissue distribution of a 26-kilodalton Schistosoma mansoni glutathione S-transferase

A Schistosoma mansoni cDNA library was constructed from the mRNA of adult worms in the expression vector lambda gt11 and screened with a rabbit antiserum raised against the 26-kDa S. mansoni glutathione S-transferase isoforms (Sm GST 26). Two clones were selected and the nucleotide sequences deduced. The predicted amino acid sequence, specified by these cDNAs, shows strong homology with a Schistosoma japonicum 26 kDa glutathione S-transferase and a lower level of homology with mammalian glutathione S-transferase class mu isoenzymes (EC 2.5.1.18). No significant homology score was found with a 28-kDa S. mansoni glutathione S-transferase (Sm GST 28). A study of the tissue distribution of the cloned Sm GST 26 by immunoelectron microscopy shows similarities to Sm GST 28 in that they are present in the tegument and in subtegumentary parenchymal cells. However, a major difference exists in the protonephridial region in which Sm GST 26 is present in the cytoplasmic digitations localized in the apical chamber delineated by the flame cell body, suggesting that Sm GST 26 may be actively excreted by adult worms.     

Further characterisation of the Schistosoma japonicum protein Sj23, a target antigen of an immunodiagnostic monoclonal antibody.

Sj23, the 23-kDa target antigen in Schistosoma japonicum adult worms of the hybridoma monoclonal antibody (mAb) I-134, has been identified and cloned from cDNA libraries, mAb I-134 has been successfully used in immunodiagnostic assays to detect S. japonicum infection in Philippine patients. Sequence analysis has shown that Sj23 is the homologue, with 84% amino acid identity, of Sm23, a 23-kDa molecule from S. mansoni worms previously described from our laboratory. The domain structures of Sj23 and Sm23 are strikingly similar to the human membrane proteins ME491, CD37, CD53 and TAPA-1, which may suggest a functional role for the schistosome molecules in cellular proliferation.     

Immunoglobulin heavy chain cDNA from the teleost Atlantic cod (Gadus morhua L.): nucleotide sequences of secretory and membrane form show an unusual splicing pattern.

Rabbit antibodies to Atlantic cod (Gadus morhua L.) immunoglobulin were affinity purified and used to screen cDNA libraries from spleen and head kidney mRNA. cDNA clones for both the secretory and membrane-bound heavy (H) chain were isolated, the nucleotide and deduced amino acid sequences of which are reported here. Comparisons of the cod secretory H chain amino acid sequence show 24%, 27%, 30% identity to the mu chain of Mus, Xenopus and Ictalurus, respectively. The highest degree of identity was observed in the CH4 domain. The cDNA encoding the transmembrane form shows a novel splicing pattern where the TM1 exon is spliced directly onto the CH3 domain and not to the CH4 domain as in other animal groups. Southern blot analyses with VH and C probes on genomic DNA from cod erythrocytes indicate that there is a unique C gene but several V genes in the cod immunoglobulin H chain locus.     

Molecular cloning and characterization of a novel Schistosoma japonicum "irradiated vaccine-specific" antigen, Sj14-3-3.

A Schistosoma japonicum cDNA coding for a full length S. japonicum 14-3-3 protein was obtained by antibody screening of an adult worm cDNA library using sera taken from mice vaccinated with UV-attenuated cercariae, which are capable of transferring high levels of passive immunity to this parasite. The deduced amino acid sequence consists of 254 amino acids and is highly homologous with 14-3-3 family of proteins from a variety of species (55-69% identity). The recombinant S. japonicun 14-3-3 protein (rSj14-3-3) was expressed and purified in pGEX/E. coli, and in Western blotting was strongly recognised by sera from mice, rats and bovines vaccinated with irradiated S. japonicum cercariae. Analysis of mRNA showed that Sj14-3-3 is expressed in sporocysts and adult worms, but not in cercariae, however mouse antisera against rSj14-3-3 recognised a 29 kDa native antigen in antigen preparations made from eggs, cercariae, schistosomula and adult worms of S. japonicum indicating that this antigen is present in all life-cycle stages. The presence of the native antigen in detergent extracts of intact schistosomula suggests that it is also present in the schistosomular tegument which is the most vulnerable target for immune attack. However, antisera against rSj14-3-3 did not recognise a similar band in S. mansoni or S. haematobium antigens, indicating that, like the UV-attenuated vaccines, this protein induced species-specific immune responses. Southern blot analysis suggested that there may exist more than one gene copy and/or polymorphism for Sj14-3-3. Immunoelectron microscopy confirmed that the native antigen is present throughout the body of adult worms including the tegument, but is less abundant in the muscles. The potential of rSj14-3-3 as a vaccine is now under further investigation.     

PCR amplification and comparison of nucleotide sequences from the groESL heat shock operon of Ehrlichia species.

Degenerate PCR primers derived from conserved regions of the eubacterial groESL heat shock operon were used to amplify groESL sequences of Ehrlichia equi, Ehrlichia phagocytophila, the agent of human granulocytic ehrlichiosis (HGE), Ehrlichia canis, Bartonella henselae, and Rickettsia rickettsii. The groESL nucleotide sequences were less conserved than the previously determined 16S rRNA gene sequences of these bacteria. A phylogenetic tree derived from deduced GroEL amino acid sequences was similar to trees based on 16S rRNA gene sequences. Nucleotide sequences obtained from clinical samples containing E. equi, E. phagocytophila, or the HGE agent were very similar (99.9 to 99.0% identity), and the deduced amino acid sequences were identical. Some divergence was evident between nucleotide sequences amplified from samples originating from the United States (E. equi and the HGE agent) and sequences from the European species, E. phagocytophila. A single pair of PCR primers derived from these sequences was used to detect E. chaffeensis and HGE agent DNA in blood samples from human patients with ehrlichiosis.     

Primary structure of the large (L) RNA segment of nephropathia epidemica virus strain H?lln?s B1 coding for the viral RNA polymerase

The L RNA segment of the nephropathia epidemica virus (NEV) strain H?lln?s B1 was characterized by molecular cloning of the corresponding cDNA and subsequent determination of the DNA nucleotide sequence. The L RNA segment is 6550 nucleotides long with complementarity of 20 bases at the 3' and 5' termini. The viral messenger sense RNA contains one major open reading frame (ORF) with a coding capacity of 2156 amino acid residues encoding a protein with a calculated molecular weight of 246 kDa and an IEP of pH 7.4. Comparison of the deduced amino acid sequences from NEV hantavirus and Bunyamwera virus (BWV) L segment messenger sense RNAs, revealed a high degree of diversity (overall amino acid identity, 17%). However, three clusters of 30-40% amino acid identity were detected. One of these domains, containing an Asp-Asp motif found in many RNA polymerases, also shares amino acid sequence homology with the PB1 polymerase component of influenza type A. These results indicate that the L RNA segment of the NEV codes for the viral RNA-dependent RNA polymerase. The data presented here complete our previous studies on the characterization of the NEV genome by cDNA sequencing of the viral M and S RNA segments.