红花黄酮醇合酶基因的全长cDNA克隆及植物表达载体的构建

黄酮醇合酶(flavonol synthase,FLS)是黄酮化合物代谢途径中的关键酶之一。本文在红花转录组测序结果中获得中间序列的基础上,采用RT-PCR和RACE技术,从我国传统中药材红花花瓣中克隆到黄酮醇合酶基因的全长c DNA。该基因全长1 201 bp,开放阅读框1 011 bp,编码336个氨基酸。系统进化分析表明,红花FLS基因编码氨基酸与同属菊科植物氨基酸具有一定的同源性,其中与金光菊的亲缘关系最近。通过分子生物学方法,成功构建p BASTA-FLS植物表达载体。为后续研究该基因的生物功能及黄酮化合物合成机制奠定了基础。     

Chinese 1基因型弓形虫棒状体蛋白 ROP16的基因克隆表达及生物信息学分析

目的：构建弓形虫Chinese 1基因型TgCtWh3（以后均简称Wh3）株棒状体蛋白（ rhoptry protein ,ROPs）16的原核和真核重组表达质粒及其3D结构。方法参照ROP16序列分别设计引物,采用PCR从弓形虫Chinese 1基因型Wh3株基因组DNA中扩增出编码ROP16的基因片段,克隆至pMD18-T载体;经PCR及测序分析鉴定;阳性克隆的质粒分别亚克隆至原核表达载体pET-28a和真核表达载体pEGFP-C2,分别转化大肠杆菌BL21和DH5α,PCR和酶切鉴定转化菌落插入的序列;将构建的原核表达菌株经IPTG诱导,SDS-PAGE和免疫印迹分析融合蛋白的表达;将构建的真核重组质粒经脂质体转染293T细胞,观察其在细胞中表达;采用生物信息学方法分析构建了蛋白的3 D结构。结果各组均PCR扩增出约2．1 kb ROP16基因的特异片段,序列检测结果均正确;分别亚克隆到原核表达载体pET-28a和真核表达载体pEGFP-C2中,成功构建了Chinese 1基因型弓形虫棒状体蛋白ROP16的原核表达质粒和真核表达质粒;原核表达质粒在大肠杆菌中表达了ROP16的融合蛋白;真核表达质粒在293T细胞中成功表达,成功构建出ROP16基因的3D结构图。结论以pET-28a和pEGFP-C2为载体,分别成功构建并表达了ROP16的原核和真核重组质粒,并构建出其3D结构图。     

Complete sequence of a mariner transposable element from the predatory mite Metaseiulus occidentalis isolated by an inverse PCR approach

Degenerate primers designed and synthesized based on two conserved regions of the mariner transposase open reading frame were used to amplify a 454 bp DNA fragment from M. occidentalis. Two inverse primers were then synthesized and used to amplify flanking genomic DNA fragments from M. occidentalis by a ligation-mediated inverse PCR. The complete mariner element (Moc 1) was 1284 bp long, including the imperfect 28 bp inverted terminal repeat sequences, and shared 59% similarity to an active 1286 bp long D. mauritiana mariner element (Mos 1). Insertions, deletions and substitutions were observed in the Moc 1 sequence at several positions. No intact open reading frame was detected and the Moc 1 element is considered inactive. Stringent Southern blot hybridizations revealed at least twelve copies of mariner sequences similar to Moc 1 in the colonies tested.     

细胞核抗原Sm B′的基因克隆和原核表达

目的为建立抗Sm自身抗体检测方法,自行克隆、表达和鉴定细胞核抗原Sm B′。方法应用逆转录聚合酶链反应(RT-PCR)技术,从HL-60细胞株中克隆Sm B′全长基因,将PCR产物直接进行TA克隆、鉴定及测序,再定向克隆至pGEx-5T载体中,转入大肠杆菌BL-21,阳性克隆鉴定后在异丙基-β-D-硫代半乳糖苷(IPTG)诱导下表达,产物行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法(western blot)鉴定。结果PCR产物约为700 bp,与预期657 bp接近,测序结果与GenBank核酸数据库报道完全一致。pGEX-5T-Sm B′重组阳性克隆酶切鉴定正确,SDS-PAGE和western blot结果显示融合蛋白分子量为51 000,具有与抗Sm B′抗体的反应性。结论成功克隆表达核抗原Sm B′,为建立抗Sm自身抗体检测方法奠定了基础。     

A cDNA clone encoding a peptide highly specific for hepatitis C infection

A random primed lambda gtll-cDNA library was constructed from donors plasma presumably infected by blood-borne non-A, non-B hepatitis (hepatitis C:HC) agent and immunoscreened with serum pooled from patients with acute or chronic HC. Twelve lambda gtll-cDNA clones encoding antigens associated with HC infection in Japan as well as in the USA were isolated. Of these one clone consisting of 114 nucleotides and showing a discrete band on an immunoblot analysis, was extensively studied. The clone is not derived from the host DNA encoding one polypeptide specific and highly sensitive for serum from patients with HC and has no homology to the nucleotide sequences of known human viruses including hepatitis A, B and D viruses, Ebstein-Barr virus, coxsackievirus, immunodeficiency virus type 1 or Japanese encephalitis virus. These results suggest that this clone is derived from the genome of HC agent.     

Tissue‐specific expression and silencing phenotypes of mitochondrial phosphate carrier paralogues in several insect species

The mitochondrial phosphate carrier gene (PiC) encodes a membrane protein that mediates the supply of inorganic phosphate from the cytosol into the mitochondrial matrix. This substrate‐specific transport system plays an important role in efficient ATP synthesis. Mammals appear to have only one PiC with two alternative splicing variants whose functional differences remain unclear. The present study is the first to characterize the multiple genes that encode PiC in insects. Bombyx mori was found to have two PiC paralogues, one ubiquitous and one testis‐specific, the latter seeming to be present only in Lepidoptera. Drosophila melanogaster was found to harbour two PiC paralogues, whereas Liriomyza chinensis, another dipteran, has three PiC paralogues. Two PiCs were found to be present in Plautia stali, and silencing either of these genes affected the normal development of P. stali nymphs, although their expression patterns differed amongst tissues. Schistocerca gregaria and Locusta migratoria have two PiC each, with different expression patterns. Tribolium castaneum was found to have only one PiC, which appears to play an essential role in larval development. Thus, although the inorganic phosphate transport system appears to be conserved across eukaryotes, PiC has become specialized in the different tissues of different insect species.     

Leukotriene B4 12-hydroxydehydrogenase/15-ketoprostaglandinΔ13-reductase (LTB4 12-HD/PGR) responsible for the reduction of a double-bond of the α,β-unsaturated ketone of an aryl propionic acid non-steroidal anti-inflammatory agent CS-670

CS-670 is a non-steroidal anti-inflammatory agent with an α,β-unsaturated ketone structure. It exerts its pharmacological activity after being transformed to the active metabolite (2S,1′R,2′S)-trans-alcohol. Two consecutive reductions are needed for the formation of the active metabolite, reduction of the double-bond of the α,β-unsaturated ketone moiety, followed by reduction of the resulting saturated ketone. The objective of the current study was to identify the enzyme responsible for reduction of the double-bond. An enzyme purified from rat liver cytosol as a single band on sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was analysed by a Mascot database search of nano-LC tandem mass spectrometry (MS/MS) data and the enzyme was identified as 2-alkenal reductase (EC 1.3.1.74), which is known as an β-nicotinamide adenine dinucleotide phosphate (NADPH)-dependent alkenal/one oxidoreductase and has a role for leukotriene B₄ 12-hydroxydehydrogenase/15-ketoprostaglandinΔ13-reductase (LTB₄ 12-HD/PGR). The identification was confirmed by cloning LTB₄ 12-HD/PGR cDNA from rat liver, expressing it in Escherichia coli, and characterizing the properties of the enzyme. The identity was further supported by the subcellular localization in cytosol, a cofactor requirement for NADPH, substrate specificity, and substantial inhibition by 15-ketoPGF_2α, benzylideneacetophenone, indomethacin, and quercitrin. In addition to catalysing the biological reduction of eicosanoids, including prostaglandins, leukotrienes, and lipoxins, LTB₄ 12-HD/PGR was also determined to function as a xenobiotic-metabolizing enzyme.     

Clinical potential of the HA-1 peptide,a minor histocompatibility antigen

Minor histocompatibility (H) antigens are the targets of host versus graft (HVG), graft versus host (GVH) and graft versus leukaemia (GVL) immune responses following transplantation of organs or tissues between donor/recipient pairs matched for transplantation antigens encoded by the major histocompatibility complex (MHC: HLA in humans). There is a particular clinical problem in predicting and treating GVH disease, which occurs in a significant proportion of bone marrow transplant (BMT) patients, even those with HLA-identical sibling donors. However, many of these recipients receive BMT as part of the treatment for leukaemia and there is a correlation in them between harmful GVH and potentially therapeutic GVL, implying the same target antigens. The molecular identity of minor H antigens is therefore a key issue. This patent describes the recent identification of one of the human minor H antigen (HA-1) and proposes methods for using the nonameric peptide identified, VLHDDLLEA, or analogues of it, to modulate HVG and GVH responses, to promote GVL and, with knowledge of the polymorphism of the encoding gene, to type BMT recipients and their potential donors for presence of the antigen.     

Variations of Phospholipases A2 in the Geographic Venom Samples of Pitvipers

The geographic variations of phospholipases A₂ (PLAs) in the venom of four medically important pit vipers were investigated. We have studied the PLAs by HPLC‐purification, cDNA cloning and sequencing, mass characterization, and functional classification. We found that: 1) Anti‐platelet acidic PLA isoforms in the venoms of Calloselasma rhodostoma from five southeastern Asian countries, and those of the Crotalus v. viridis from seven American States are differentially expressed depending on locality. The variations could be attributed to their distinct specificities towards the platelets of different prey, and to possible adaptation for playing other functional roles. In contrast, structures of the myonecrotic and the edema‐inducing basic PLAs in both venoms were relatively conserved. 2) A special type of the acidic anti‐platelet PLA is present in the venom of some Protobothrops species. Its expression level is diminished in the snake of the southern or the tropical ranges. 3) The venom of Bamboo tree vipers (Trimeresurus stejnegeri) in Taiwan and China showed extraordinary geographic variations in their acidic and basic PLAs. The high RNA‐polymorphism of their venom proteins may have been derived from interbreeding between several ancestral pit viper species. In addition, migration, isolation of different populations and rapid evolution of the venom proteins to adapt for diversified diets may have resulted in further variations in this venom species.