用噬菌体多肽库筛选日本血吸虫表膜抗原的模拟表位

为探索可用于诊断的模拟表膜抗原。采用纯化的兔抗表膜抗原IgG作探针 ,免疫筛选噬菌体随机十二肽库 ,经 3轮生物淘洗后 ,随机挑选 30个噬菌体克隆 ,用ELISA检测其与筛选抗体的特异性结合 ,选择两个阳性克隆进行DNA序列测定 ,并用斑点ELISA比较检测正常人和日本血吸虫病患者血清各 10份。结果显示 ,随机挑选的 30个克隆中有 9个噬菌体克隆与筛选的抗体有特异性的结合反应。DNA测序结果显示 ,两个阳性噬菌体克隆 (携带的抗原表位 )所演绎的氨基酸序列与GenBank已知的氨基酸序列无同源性。斑点ELISA结果显示两个抗原表位可被血吸虫病患者血清呈特异性识别     

日本血吸虫尾蚴cDNA文库的免疫学筛选及EST序列的分析

目的为获得日本血吸虫（Schistosoma japonicura，Sj）转化生长因子β1（TGF—β1）基因的部分或全长cDNA序列。同时验证用针对某一蛋白的抗体筛选表达文库是否可以得到相应蛋白对应的基因序列。方法采用兔抗鼠TGF—β1血清，对Sj尾蚴cDNA表达文库进行免疫学筛选，对阳性克隆进行PCR扩增后，将大于500bp的克隆进行复筛，对复筛阳性的克隆进行测序和生物信息学鉴定。结果对大约10^6个噬菌斑进行了初筛。共获得7个阳性克隆；经过PCR扩增后，其中有4个克隆大于500bp，复筛获得3个持续阳性克隆；对测序后的阳性克隆进行生物信息学鉴定，得到的三个克隆均与日本血吸虫辅酶Q10氧化还原酶（SjCHGC）基因具有高的同源性（Bhata分值都大于200）。结论SjCHGC蛋白与兔抗鼠TGF—β1抗体的反应为交叉反应，用抗某一蛋白的抗体筛选表达文库得到相应蛋白的基因序列的方法不一定可行。     

Expression of Schistosoma japonicum antigens in Escherichia coli

A cloned library of DNA complementary to the mRNA of adult Schistosoma japonicum has been prepared and expressed as fusion proteins with Escherichia coli beta-galactosidase. Colonies expressing the S. japonicum cDNA clones were screened both with antibodies from individuals with a history of schistosomiasis and with antibodies obtained from a rabbit immunized with whole adult worms. In both cases colonies were detected which bound antibody, although the frequency of antigen-positive clones was much higher with the rabbit antiserum than with human sera. In both cases the proportion of colonies reacting with antibodies was markedly lower than that published for equivalent screens of Plasmodium falciparum cDNA with sera from individuals with a history of falciparum malaria. Several major S. japonicum antigens were identified by the affinity purification of antibodies using immobilised fusion proteins produced during lytic growth of the recombinant bacteriophage.     

从人T细胞淋巴瘤cDNA文库中筛选含PH结构域编码序列的新基因

目的 从T细胞中获得更多含PH结构域编码序列的新基因。方法 采用低严谨核酸杂交技术，根据T细胞中已知的PH结构域的分子设计探针，对人T细胞淋巴瘤cDNA文库进行筛选。结果 在筛选过程中得到了4个新基因，各cDNA长度依次为：1733bp,2308bp,2130bp和1647bp，分别编码含229，376，211和143个氨基酸残基的多肽。经生物信息学分析，在基因数据库中未发现与此4种cDNA序列类似的基因。该4个基因均已被GenBank收录，登录号分别为：AF334588，AF334589，AF334590和AF334792。结论 这些新基因的获得及进一步研究，可能会为深入探讨T细胞激活机制提供新的线索。     

人类睾丸生精细胞凋亡相关基因TSARG3的克隆

目的：克隆人类睾丸生精细胞凋亡相关基因TSARG3。方法：从已获得的小鼠稳睾和正常睾丸对照中表达量有明显差异的表达序列标签片段（BE644537）入手，构建人同源表达序列标签重叠群，应用基因特异性引物和载体特异性引物，在睾丸cDNA文库的DN或进行巢式PCR扩增、测序，对测序结果进行生物信息学分析。结果：从睾丸cDNA文库中分离出人类睾丸凋亡相关基因的5’末端而获得全长cDNA,命名为TSARG3，GenBank登录号为AF419291（保密期为1年），同时应用相同方法克隆了该基因在小鼠中的同源基因，GenBank登录号为AF419292。结论：获得人类睾丸生精细胞凋亡相关基因TSARG3，该基因可能与人类睾丸生精细胞凋亡有关。     

Selection of the Mimic Epitopes of Antigens from Schistosoma japonicum by Using Phage Display Techniques

Rat was a semi permissive host of Schistosoma. Accordingto the research of Yu XC[1], when rat was infected withSchistosoma japonicum (S.j), the egg granulomas in ratlivers were rarely observed, while majority of eggs wereripe eggs and black dead eggs, and the number of meanliver eggs per gram (LEPG) was 1393.46, apparently lessthan that in permissive host mouse. These results indicatedapparently natural resistance. In our laboratory, enzyme linked immuno electro transfer blot (EITB) …     

人B细胞活化相关基因BC-1514全长cDNA的克隆与原核表达

目的 克隆与B细胞活化相关的新基因及其原核表达。方法 采用差异显示反转录PCR（DDRT-PCR）技术对人扁桃体活化和静止B细胞mRNA的差异表达进行分析。差异显示的片段经过Northern杂交验证后,作为探针进行入活化B细胞cDNA文库的筛选,将所获得的阳性克隆的编码区经PCR扩增后克隆到原核表达载体pGEX-5X-1中,重组质粒经酶切,测序鉴定后转化大肠杆菌BL-21,以IPTG诱导表达融合蛋白。结果 以在活化B细胞高表达的EST32为探针,经3轮筛选人活化B细胞文库获得一个新的全长为1514bp的cDNA克隆（命名为BC-1514）,重组的BC-1514蛋白可在E.coli中以融合蛋白的形式有效表达,其表达量约占细菌总蛋白量的14.3%左右,BC-1514cDNA的GenBank的登录号为AF304442。结论 获得了1条新的与B细胞活化相关的cDNA克隆并在E.coliBL-21中得到了有效表达。     

Construction of cDNA library from NPC tissue and screening of antigenic genes

To construct cDNA library of nasopharyngeal carcinoma (NPC) and obtain the NPC associated or specific antigens from it, we used a powerful new method to identify the antigens eliciting humoral immune response, which is SEREX (serological identification of antigen by recombinant cDNA expression library). Autologous serum of NPC patient was used to screen the reactive clones in the human NPC tissue cDNA library consisted of 3.64×106 recombinants. The 23 exact positive clones were subcloned to monoclonality and the size of cDNA inserts was identified by PCR. Then the nucleotide sequence of cDNA inserts was determined, and the sequence alignments were performed with BLAST software on GenBank database. They represented 16 different antigens. A detailed sequence analysis showed that 10 of 16 genes were high homologous to genes known in GenBank, such as RPL31, S100 A2, MT2A, etc. However, there were also 6 genes with low homology to genes in GenBank. Furthermore, 3 of 6 genes may be novel genes. The associations of these genes to NPC and the roles that they played in the occurrence and development of NPC should be further revealed.     

Molecular characterization of a calcium-binding protein SjCa8 from <Emphasis Type="Italic">Schistosoma japonicum</Emphasis>

A full-length cDNA encoding a cercarial stage-specifically expressed 8-kDa calcium-binding protein (SjCa8) was isolated from Schistosoma japonicum cercarial cDNA library using microarray screen. The putative gene coding for SjCa8 is of 371 bp with an open reading frame of 69 amino acid (aa). The deduced aa sequence showed 83% identity with the Schistosoma mansoni 8-kDa CaBP, 47% identity with Clonorchis sinensis calcium-binding protein, and 38% identity with Fasciola hepatica putative calcium-binding protein. Also, it shares more than 30% identity with the calmodulin of many different species, while the most significant similarity between them lies around the two calcium-binding loop regions. There are two potential sites for phosphorylation and one potential site for N-myristoylation in the sequence. The SjCa8 has also been predicted to contain a single pair of EF-hand Ca(2+)-binding domain. The recombinant SjCa8 (rSjCa8) protein expressed and purified from E. coli has been demonstrated to possess the calcium-binding activity. Immune serum from UV-attenuated S. japonicum cercariae-immunized rabbit detected rSjCa8 by Western blot assay, while the sera from S. japonicum naturally infected rabbit and normal rabbit could not. These findings may contribute to the development of an effective vaccine against schistosomiasis.     

Sera of Schistosoma japonicum-infected patients cross-react with diagnostic 31/32 kD proteins of S. mansoni.

Schistosoma mansoni adult worm antigens were tested for cross-reactions with sera obtained from patients infected with S. japonicum. The sera consistently recognized a doublet of bands, in immunoblots, which had molecular weights of approximately 31 and 32 kilodaltons (kD). This reaction was found to be markedly reduced with sera of patients who had received chemotherapy and who had a low risk of reinfection. Sera obtained from uninfected persons or from patients infected with other parasites never reacted with the antigen doublet. Schistosoma japonicum-infected mice produced antibodies during prepatency which predominantly recognized antigens of this molecular weight range in immunoblots performed with S. mansoni or S. japonicum proteins. Sera from S. mansoni-infected patients with a high specificity for the diagnostic S. mansoni-antigen cross-reacted with a corresponding component also in S. japonicum worms. Immunofluorescence assays performed with sera of schistosomiasis japonica patients confirmed earlier results localizing the diagnostic 31/32 kD antigens in the gut of S. mansoni. These cross-reacting 31/32 kD S. mansoni protein antigens may be applied for the immunodiagnosis of schistosomiasis japonica.     

人肝癌细胞系中肿瘤相关基因MAGE的克隆

目的 克隆人肝癌细胞系HHCC中的肿瘤相关基因MAGE－1的全长cDNA。方法 根据GenBank中MAGE－1基因编码区，设计PCR引物，用RT-PCR手稿,人人肝癌细胞系HHCC中获得MAGE－1全长cDNA，双酶切后连接入质粒pUC19中，转化入大肠杆菌DH5α。挑选阳性克隆提取质粒，进行DNA序列分析，并将测得的核苷酸序列与GenBank中的DNA序列进行BLAST同源性分析。结果 获得MAGE－1全长cDNA,MAGE-6基因463bp片段，以及1个与MAGE－6基因编码区有93％同源性的片段，可能为MAGE家族中的新成员。结论 人肝癌细胞系HHCC可表达多种MAGE基因，可能有效的MAGE基因出现，为研究MAGE基因在HCC中的表达模式及其在HCC免疫治疗中的靶点提供了新的资料。     

Characterization of a large gene family in Schistosoma japonicum that encodes an immunogenic miracidial antigen

Three of eleven clones isolated from a genomic expression library of Schistosoma japonicum DNA using chronically infected human sera also react with chronically infected mouse sera. Characterization of these three clones showed that they contain different members of the same gene family. One clone contains two members of the gene family approximately 2 kb apart and in opposite orientation to each other. DNA sequence homologies between pairs of genes range from 98% to 99.5%. Southern hybridization results indicate there are approximately 40 copies of these genes per haploid genome. Sera from mice immunized with purified fusion protein detected immunoreactive products in the central ganglion and ciliated epidermal cells of miracidia.     

Adult schistosome cDNA libraries as a source of antigens for the study of experimental and human schistosomiasis

Protective immunity has been demonstrated in experimental schistosomiasis and is also believed to occur in man. It can be mediated by antibodies from infected animals or animals immunized with attenuated organisms. Recombinant Escherichia coli synthesizing antigenic polypeptides from the three principal species of schistosome that infect man, Schistosoma mansoni, S. japonicum and S. haematobium, have been constructed. Libraries of adult worm cDNA were prepared from each species in the expression vector lambda gt 11 and directly screened with antibodies from animals experimentally immunized with S. mansoni and S. japonicum and from humans infected with S. haematobium. The S. mansoni clones have been analysed in greatest detail. At least four different types of clones were identified. All the detected recombinant polypeptide antigens were recognised by antibodies from chronically infected mice and most were also recognised by antibodies from mice immunized with attenuated cercariae and anti-surface membrane antibodies. Clones synthesizing species-specific antigens for both S. mansoni and S. japonicum were identified by simultaneous screening of both libraries. At least three types of S. haematobium clones were identified by screening with human infection serum, most of which were species-specific. All the antigens were in the form of fusion peptides with E. coli beta-galactosidase and their expression was induced by isopropylthiogalactopyranoside. Since known protective monoclonal antibodies recognise highly glycosylated membrane proteins which cannot be identified in the form of nascent polypeptides, the direct identification of polypeptide antigens defined by their reactivity, as reported here, is an essential step in producing reagents by recombinant DNA technology, suitable for vaccination and diagnosis.     

Comparison of sequence of cDNA clone with other genomic and cDNA sequences for human C-reactive protein

A clone for C-reactive protein (CRP) has been isolated from a human liver cDNA library; this clone harbors a plasmid, pC81, which has an insert of 1631 bp. When compared to geuomic and cDNA sequences published to date now, pC81 has revealed homologies and differences that might help to clarify the structure of this gene and the presence of allelic variants in man.The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X56214.     

Expression in Escherichia coli of two Schistosoma mansoni genes that encode major antigens recognized by immune mice

Two clones which contain genes encoding Schistosoma mansoni proteins recognized by immune mouse sera were chosen from cDNA lambda gt11 expression vector library by preselecting clones from the library with rabbit antisera against adult worm phosphate-buffered saline (PBS)-soluble antigens. One clone, MAC 182, codes for part of a Mr 70 000 protein; the other clone, MAC 184, codes for a Mr 27 000 protein. The insert sizes of MAC 182 and MAC 184 are 400 bp and 800 bp, respectively. Both clones express S. mansoni beta-galactosidase fusion proteins as products of the construct. Antibodies from either chronically infected mice or mice vaccinated with irradiated cercariae recognize the MAC 182 fusion protein (MAC 182fp) but not the MAC 184 fusion protein (MAC 184fp). Rabbit antibodies prepared against MAC 182fp immunoprecipitate a Mr 70 000 in vitro translation product from adult mRNA and react in Western blot with a corresponding Mr 70 000 protein present in eggs, cercariae and adult worms but absent in schistosomula. Although the MAC 184fp is not recognized directly by chronic infection or vaccinated mouse antibodies, antisera prepared against the purified fusion protein immunoprecipitate a Mr 27 000 in vitro translation product which also reacts with mouse chronic infection sera. The same Mr 27 000 protein appears to be present in eggs, cercariae, schistosomula and adults as determined by Western blots with rabbit antisera against the MAC 184fp. These results suggest that the S. mansoni polypeptide encoded by the MAC 184 gene, when expressed within a fusion protein, fails to present epitopes normally recognized during natural infection. We propose that these epitopes are conformationally determined and are destroyed when the MAC 184 protein is expressed within beta-galactosidase. This abrogation of conformational epitopes may explain the failure of antibodies from chronically infected or vaccinated mice and rabbits to effectively recognize gene products of certain lambda gt11-fusion protein clones.     

应用EST和电子克隆策略研究血吸虫表达基因谱

目的开展血吸虫表达基因谱的研究,寻找新的疫苗候选分子和药物靶标。方法应用表达序列标签（EST）和电子克隆策略。结果获得了552个EST序列和487个电子延伸序列,其中104个EST序列在延伸前未表现同源性,而延伸后表现出有意义的同源性;获得了日本血吸虫基因表达谱的信息以及发现了有潜在药物和疫苗价值的新基因序列。结论本研究为日本血吸虫基因表达谱的研究提供更有效的研究思路。     

Identification and characterization of peptides mimicking the epitopes of metalloprotease of Schistosoma japonicum

In an attempt to isolate and characterize peptides mimicking epitopes of metalloprotease and explore their immunological protection against Schistosoma japonicum （S. japonicum）, polyclonal anti-metalloprotease sera was prepared to screen a 12-mer random peptide library to isolate phages binding specially to antisera IgG. Then, phage ELISA, animal immunization, DNA sequencing, Western blotting and enzymatic activity neutralizing analysis were used to characterize the selected phage clones. All of ten randomly picked clones were shown to be positive. Five peptides of different amino acid sequences deduced from DNA sequences were obtained and two of them （peptides 2 and 3） could induce significant reduction （31.0% and 31.8%, respectively） in worm burden and high reduction （52.6% and 54.9%, respectively） in liver eggs per gram （LEPG）, while, unexpectedly, others （peptides 1, 4 and 5） could not elicit enough protection against infection of S. japonicum. Peptides 2 and 3 could be recognized by S. japonicum infected mouse sera （IMS） and could elicit neutralizing Abs. The results show that peptides 2 and 3 are antigenic and immunogenic. They are true mimics of epitopes of metalloprotease and useful as novel vaccine candidates against S.japonicum. Cellular ＆ Molecular Immunology. 2005;2（3）：219-223.     

用单抗5C5和5C5—G1筛选的613bp cDNA的核苷酸序列分析

用识别人活化Ｂ细胞分化抗原５Ｃ５的单克隆抗体５Ｃ５和５Ｃ５－Ｇ１的混合物从人扁桃体细胞λｇｔ１１ ｃＤＮＡ文库筛选到３个阳性克隆。其ｃＤＮＡ插入到质粒ｐＵＣ１８，经双链双脱氧测序法作核苷酸序列分析，知其中一个ｃＤＮＡ长６１３ｂｐ，另二个长４６７ｂｐ，后者与前者的前４６７ｂｐ完全重叠。６１３ｂｐ ｃＤＮＡ中有一个开放阅读框架，从１０３ｂｐ到４２９ｂｐ，共３２７ｂｐ。Ｎｏｒｔｈｅｒｎ印迹分析显示，此６