Cloning and Subcloning of cDNA Coding for Group Ⅱ Allergen of Dermatophagoides Farinae

Objective: To clone, sequence and subclone the cDNA coding for group Ⅱ allergen of Dermatophagoidesfarinae(Der f2). Methods: The cDNA gene fragment of Der f2 was amplified by RT-PCR. After being purified, the gene fragment was cloned into a vector pMD-18T. The recombinant plasmid pMD-18T-Der f2 was transformed into E. coli JM109. Positive clones were screened and identified by PCR and digested with restriction endonuclease, and the sequence of inserted Der f2 cDNA was also analyzed. Then Der f2 was subcloned into the vector of pET-32a( ). Results: The Der f2 cDNA was specifically amplified from RNA by RTPCR. The recombinant plasmid pMD-18T-Der f2 and pET-32a( ),Der f2 was constructed and digested by SacⅠ and NotⅠ, and the size of gene fragment was 455bp and in accordance with the expected one. Conclusion: The pET-32a( )-Der f2 subclone was constructed successfully.     

头顶一颗珠地下根茎无性繁殖研究

头顶一颗珠为国家二类保护植物，又为名贵药材，其根茎无性繁殖比种子有性繁殖缩短生育期２ａ；从出苗到入药提早１２ｍｏ；平均单株根茎干重增加６％；无性繁殖系数可达１０～１２，在生产上有利用价值     

史记

加拿大著名医师白求恩诞生（1890）；阿斯匹林获准上市（1899）；人类第一次成功使用机械心脏（1952）；世界上第一只试管羔羊诞生（1984）；克林顿禁止人体无性繁殖试验（1997）；中国大陆首例试管婴儿诞生（1988）；     

克隆技术：生命科学研究的机遇与挑战

自从英国科学家用成年绵羊的体细胞克隆出世界首例“多莉”绵羊的消息被海内外的媒体竞相报道后，近一段时间以来，已引起了世界各地生命科学家、政治家和法律学家们的广泛关注，特别是对这一技术是否会用于克隆“人”以及由此所可能产生的社会学和伦理学上的后果，表示出了期盼、忧虑或否定；我国的许多学者也以各种形式提出了面对中国国情的建议，希望政府职能部门能未雨绸级，对我国的克隆技术研究进行行为规范。本文拟对此作一评述，希望这一技术能在我国得到正常发展而不至于走入歧途。一、克隆动物与无性自孩就在人们议论克隆羊的同时…     

Cloning and identification of NS5ATP2 gene and its spliced variant transactivated by hepatitis C virus non-structural protein 5A

AIM: To clone, identify and study new NS5ATP2 gene and its spliced variant transactivated by hepatitis C virus non-structural protein 5A. METHODS: On the basis of subtractive cDNA library of genes transactivated by NS5A protein of hepatitis C virus, the coding sequence of new gene and its spliced variant were obtained by bioinformatics method. Polymerase chain reaction (PCR) was conducted to amplify NS5ATP2 gene. RESULTS: The coding sequence of a new gene and its spliced variant were cloned and identified successfully. CONCLUSION: A new gene has been recognized as the new target transactivated by HCV NS5A protein. These results brought some new clues for studying the biological functions of new genes and pathogenesis of the viral proteins.     

Cloning and characterization of a novel hepatitis B virus core binding protein C12

AIM: To elucidate the biological function of HBV core antigen (HBcAg) on pathogenesis of hepatitis B, a novel gene C12 coding for protein with unknown function interacting with HBcAg in hepatocytes was identified and characterized. METHODS: HBcAg bait plasmid pGBKT7-HBcAg was constructed and transformed into yeast AH109, then the transformed yeast was mated with yeast Y187 containing liver complementary DNA (cDNA) library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-α-gal for screening twice. After extracting and sequencing of plasmid from blue colonies, we isolated a cDNA clone encoding a novel protein designated as C12 that directly interacted with HBcAg. The interaction between HBcAg and C12 was verified again by re-mating. PEGFP-N1-C12 fluorescent protein fusion gene was transfected in 293 and L02 cell, and observed by fluorescent microscope. MTT reduction assay was used to study the action of C12 protein effect on metabolism of mammal cell. Yeast two-hybrid and cDNA microarray were performed to search binding protein and differential expression genes regulated by C12 protein. RESULTS: C12 gene was screened and identified by yeast two-hybrid system 3. The interaction between HBcAg and the novel protein coded by the new gene C12 was further confirmed by re-mating. After 48 h, fluorescence of fusion protein could be observed steadily in the 293 and L02 cell plasma. Under MTT assay, we found that the expression of C12 did not influence the growth of liver cells. Seventeen differential expression genes in HepG2 cells transfected with C12 protein expression plasmid by cDNA microarray, of which 16 genes were upregulated and 1 gene was downregulated by C12 protein. Twenty-one colonies containing 16 different genes coding for C12 protein binding proteins were isolated by yeast two-hybrid, there were 2 new genes with unknown function. CONCLUSION: The novel protein C12 is located in cell plasma, and its overexpression has no significant effect on the metabolism of liver cell. It interacts with many proteins in hepatocytes and may be involved in many processes of gene expression.     

中国不同地理居群半夏遗传多样性分析

半夏Pinellia ternate (Thunb) Breit为天南星科半夏属植物，以块茎入药［1］。我国大部分地区均有分布。但是随着半夏野生资源逐渐减少，民间多以同属其他植物作为代替品入药，加之引种途径各异，长期无性繁殖，导致品种退化。许多地区品种背景资料，种系不清，缺乏统一的分类标准，使得古方中所用道地药材在形态和质量方面出现变化，给半夏的临床用药安全带来很大的隐患。因此，就需要一种可靠的分类鉴定手段。本实验应用AFLP(amplified fragment length polymorphism)分子标记技术，对采自我国30个不同地理居群的半夏遗传多样性进行了分析。     

总序香茶菜的组织培养与快繁研究

目的对总序香茶菜进行组织培养和离体快速繁殖。方法以总序香茶菜带芽茎段为外植体,在附加有不同种类和不同浓度激素的MS培养基上进行培养。结果最佳芽诱导培养基:MS BA0.05～0.5mg/L NAA0.01～0.05mg/L;最佳芽增殖培养基:MS BA0.5mg/L NAA0.05mg/L(或IAA0.01mg/L);最佳生根培养基:MS NAA0.1mg/L 活性炭0.05%;最佳愈伤组织诱导与生长培养基为:MS 2,4-D1.0～2.0mg/L BA0.1～0.2mg/L。结论成功建立了快速无性繁殖系。     

Cloning and expression in Escherichia coli of a new gene of Schistosoma japonicum encoding casein kinase Ⅱ beta subunit

Background Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccinecandidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase Ⅱ beta subunit, of Schistosoma japonicum (S. japonicum) and express it in Escherichia coli (E. coli). Methods The ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized. The full-length cDNA of the new gene was obtained by joining the 3‘ RACE PCR fragment and the EST clone. To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E. coli JM109. The recombinant protein was analyzed by SDS-PAGE and Western-blot. Results A 908 bp cDNA was isolated from S. japonicum and identified to be casein kinase Ⅱ beta subunit gene by sequence analysis. The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8%, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H. sapiens), Xenopus laevi (X. laevi) , Drosophila melanogaster (D.melanogaster), Caenorhabditis elegan (C. elegan), and Schizosaccharomyces pombe (S. promber) respectively. The predicted molecular weight of the protein was 24. 921 kDa. The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391. This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E coil JM109. The recombinant protein could be recognized by the S. japonicum infected rabbit serum. Conclusion The full-length cDNA sequences encoding S. japonicum casein kinase Ⅱ beta subunitwere firstly sequenced, cloned, and expressed in E coil.