Immunogenicity Analysis of Prokaryotic Expression Products of Kaposi＇s Sarcoma Associated Herpesvirus orf65

To purify the protein encoding the small capsid protein (SCP) of KSHV and analyze its immunogenicity, the carboxyl terminus of orf65 of Kaposi's sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E. coli containing pQE-80L-orf65 was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and the fusion protein was purified by chromatography. The expressed protein and its purified product were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and showed that 9 kDa was the expected size of the purified orf65 protein. The antiserum was produced in rabbit which was immunized by purified orf65 protein. An ELISA assay was established to analyze the immunogenicity of the purified orf65 protein. The ELISA analysis demonstrated that orf65 protein has strong immune activity, and the immune activity of polyclonal antibody against orf65 was more than 4 fold higher than that in the serum of the non-immunized rabbit. These results demonstrate that purified orf65 protein has very strong immunogenicity and can be used in screening KSHV infection in the general population using ELISA.     

猪繁殖与呼吸综合征病毒orf7和orf5双基因的原核表达研究

根据PRRSV已知序列设计两对引物,采用RT-PCR方法分别扩增orr7和orf5基因片段.利用EcoRI、SpeⅠ和Hind Ⅲ位点将orf7和orf5基因片段依次克隆到pMD-18T载体,构建成重组质粒pMD18NE,并比较所克隆基因序列的同源性.将串联的orf7和orf5基因亚克隆到原核表达载体pGEX-KG,构建重组原核融合表达质粒pGEX-KGNE,并转化BL21,SDS-PAGE和Western-blot分析表明orf7和orf5基因与GST获得了融合表达,并且表达的融合蛋白GST-NE具有免疫学反应活性,这为猪繁殖与呼吸综合征血清学诊断方法的建立及疫苗研究打下基础.     

猪繁殖与呼吸综合征病毒orf7和orf5双基因的原核表达研究

根据PRRSV已知序列设计两对引物,采用RT-PCR方法分别扩增orf7和orf5基因片段。利用EcoRI、SpeI和HindIIl位点将orf7和orf5基因片段依次克隆到pMD-18T载体,构建成重组质粒pMDl8NE,并比较所克隆基因序列的同源性。将串联的orf7和orf5基因亚克隆到原核表达载体pGEX．KG,构建重组原核融合表达质粒pGEX．KGNE,并转化BL21,SDS．PAGE和Western．blot分析表明：orf7和orf5基因与GST获得了融合表达,并且表达的融合蛋白GST-NE具有免疫学反应活性,这为猪繁殖与呼吸综合征血清学诊断方法的建立及疫苗研究打下基础。     

Thermodynamic and spectroscopic investigations of TMPyP4 association with guanine- and cytosine-rich DNA and RNA repeats of C9orf72

Background

An expansion of the hexanucleotide repeat (GGGGCC)n·(GGCCCC)n in the C9orf72 promoter has been shown to be the cause of Amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). The C9orf72 repeat can form four-stranded structures; the cationic porphyrin (TMPyP4) binds and distorts these structures.

单引物方法克隆盐角草orf25基因

采用单引物RT-PCR扩增的方法,从新疆野生植物盐角草（Salicornia europaea）中克隆获得了1.7kb的cDNA片段,经过测序和序列分析,发现基因片段包含了orf25基因完整的读码框架。采用序列同源性分析方法,结果显示新疆盐角草orf25基因与甜菜线粒体同源性高达98%,与烟草线粒体同源性达到95%,与小麦同源性为92%,与玉米同源性为88%,表明orf25基因在植物中是高度保守的一种基因,同时说明野生植物盐角草中也存在与农作物相似的雄性不育相关基因,orf25基因编码的功能性蛋白可能在影响植物雄性不育改良作物品种方面具有重要的意义。     

C20orf54抑制食管癌细胞增殖并促进其凋亡

目的观察过表达C20orf54对食管癌细胞周期和凋亡的影响,探讨C20orf54基因在食管癌发生、发展中的作用。方法应用免疫组织化学染色方法检测C20orf54蛋白在食管癌组织和癌旁正常组织中的表达;应用MTT法和流式细胞术检测过表达C20orf54对食管癌细胞系EC9706细胞增殖能力、细胞凋亡与细胞周期的影响。结果 C20orf54在食管癌组织中免疫反应性显著低于癌旁正常组织。稳定转染表达C20orf54的EC9706细胞生长明显受抑制,且G0/G1期细胞积聚,周期阻滞,细胞凋亡增多。结论 C20orf54基因可能参与食管癌的生长调控,食管癌的发生与C20orf54基因的表达有关。     

A large open reading frame (orf1995 ) in the chloroplast DNA of Chlamydomonas reinhardtii encodes an essential protein

An open reading frame potentially encoding a protein of 1995 amino acids (orf1995) has been found in the chloroplast genome of the green alga Chlamydomonas reinhardtii. Besides having a short hydrophobic N-terminal domain with five putative transmembrane helices, the predicted orf1995 product is highly basic. orf1995 might be a homologue of the ycf1 gene in land plants, whose function has not yet been determined. Mutants of C. reinhardtii transformed with a disruption of orf1995 remain heteroplasmic for the wild-type and disrupted alleles of this gene, indicating that the orf1995 product is essential for cell survival.     

粤蓝链霉菌榴菌素生物合成基因orf20的功能

【目的】在大肠杆菌中,转录因子SoxR作为胞内氧化还原感应器,参与抗氧化胁迫的全局性调控。粤蓝链霉菌榴菌素生物合成基因簇内存在一个类soxR基因orf20,但其生理功能仍不清楚。【方法】将orf20基因在大肠杆菌中进行表达,分析携带重组质粒的大肠杆菌对百草枯抗性的变化。同时通过修改后的PCR-targeting方法构建粤蓝链霉菌orf20删除的突变株,分析突变株的表型变化和对百草枯抗性水平的变化。【结果】重组ORF20在羧基端含有组氨酸标签,并在大肠杆菌中获得可溶性表达,携带重组质粒pET28b-orf20的大肠杆菌对百草枯的抗性水平显著提高。粤蓝链霉菌orf20删除突变株仍具有产孢能力,生长特性没有改变,对百草枯的抗性水平也没有变化,但榴菌素的产量大幅提高,是野生株的3.3倍。【结论】在大肠杆菌中,orf20基因的编码产物能够被百草枯激活,替代SoxR参与抗氧化胁迫的调控。在粤蓝链霉菌中,orf20基因不参与抗氧化胁迫,而对榴菌素的产生有负调控效应。     

Accelerated molecular evolution of insect orthologues ofERG28/C14orf1: A link with ecdysteroid metabolism?

We have analysed the evolution ofERG28/C14orf1, a gene coding for a protein involved in sterol biosynthesis. While primary sequence of the protein is well conserved in all organisms able to synthesize sterolsde novo, strong divergence is noticed in insects, which are cholesterol auxotrophs. In spite of this virtual acceleration, our analysis suggests that the insect orthologues are evolving today at rates similar to those of the remaining members of the family. A plausible way to explain this acceleration and subsequent stabilization is that Erg28 plays a role in at least two different pathways. Discontinuation of the cholesterogenesis pathway in insects allowed the protein to evolve as much as the function in the other pathway was not compromised.     

水稻细胞质雄性不育相关基因orf290功能域的初步研究

[目的]探讨线粒体基因orf290主要功能结构域与水稻配子体细胞质雄性不育的关系。[方法]利用生物信息学方法,分析ORF290功能结构域,构建其不同功能结构域的表达载体,完成遗传转化,采用碘-碘化钾染色法观察阳性植株的花粉育性及小穗育性,统计分析ORF290功能结构域与育性的关系。[结果]与保持系相比,各功能结构域的转基因植株农艺性状的差异不明显,花粉育性及自然结实率均有所降低,35S::orf290(T_0代)、35S::Rf1b 5'-orf290(T_1代)、35S::Rf1b 5'-orf216(T_0代)和35S::orf216(T_0代)花粉育性(结实率)分别为:50. 7%(46. 5%)、33%(42. 15%)、38. 7%(37. 9%)和60. 8%(50%)。[结论]结果表明orf290是细胞质雄性不育相关基因,且ORF290功能结构域位于C端,N端具有线粒体定位信号。     

叶用芥菜细胞质雄性不育相关基因orf 220的分子特性

采用榨菜细胞质雄性系为不育源,通过变种间杂交和回交的方法,转育叶用芥菜细胞质雄性不育种质。根据Brassica nap型细胞质雄性不育相关基因off222设计兼并引物,利用特异引物PCR方法,分别在不育源、F1、BC2以及BC3世代中扩增出一条600～700bp大小的特异条带,而在叶用芥菜保持系中无此条带,具细胞质基因遗传特性。进一步序列分析表明,此特异条带大小为663bp,具有起始密码子和终止密码子,编码220个氨基酸,定名为off220。同时,off220推导的氨基酸序列存在两个跨膜区,氨基端与Oenothera berteriana中的COXⅢ(线粒体细胞色素氧化酶复合体亚基)、萝卜中的ATP8(线粒体ATP酶复合体亚基)以及向日葵中的ORFB(ATP酶复合体α亚基相关)蛋白氨基端高度同源。RT-PCR方法分析表明,off220基因的表达为组成性表达,无器官特异性。     

新基因C10orf97参与压力超负荷性心肌肥厚

目的检测新基因C10orf97是否参与压力超负荷型心肌肥厚病程。方法通过缩窄大鼠胸主动脉横支构建压力超负荷诱导的心肌肥厚模型,在缩窄手术后的连续时间点应用血流动力学检测评价心室重构和心功能,应用实时荧光定量PCR法检测心肌肥厚标志基因心房利钠肽和C10orf97的mRNA表达。结果主动脉缩窄手术后,大鼠心脏显著肥厚,心脏体重比逐渐增加,心功能先受损后代偿性增强。心房利钠肽表达显著上调,在缩窄后第15天升高为假手术组40倍。C10orf97基因的表达在缩窄后第2天即显著上调为假手术组的2倍,在第4天降低,随后逐渐上升,第15天时表达量升高为假手术组的3倍。结论C10orf97基因参与了压力超负荷引起的心肌肥厚病程。     

沉默c2orf68基因对结直肠癌细胞增殖的影响

目的:通过RNA干扰技术抑制人结直肠癌SW480、SW620及LS174T细胞株中c2orf68基因的表达,进一步观察其生物学特性的改变,以阐明c2orf68基因对结直肠癌细胞增殖的影响。方法:利用siRNA在线设计软件设计合成干扰片段,并将2个干扰片段转入所试细胞株中。应用RT-PCR法检测细胞mRNA的表达;利用MTT实验观察细胞增殖情况;应用流式细胞技术检测细胞凋亡。结果:转染siRNA片段后,SW480、SW620及LS174T细胞株的mRNA表达都下降,其抑制率分别为:50.05%、44.79%;49.69%、30.00%;50.15%、45.79%。随后,选取干扰效率高的siRNA1干扰片段做后续实验。MTT实验发现,siRNA转染LS174T细胞后,细胞的增殖能力明显下降,细胞的增殖抑制率为21.2%(P0.05)。同时,采用流式细胞仪检测细胞的凋亡,实验表明,实验组细胞凋亡率为18.13%(P0.05)。结论:成功筛选了抑制结直肠癌c2orf68基因的特异性干扰片段,该片段可以下调人结直肠癌细胞株中c2orf68的mRNA,抑制人结直肠癌细胞的生长,促进细胞凋亡,说明该基因可能与结直肠癌的细胞增殖有关,进一步调控相应生物学特性的改变,引起结直肠癌的发生。     

Identification of Integrin α3 as a New Substrate of the Adenovirus E4orf6/E1B 55-Kilodalton E3 Ubiquitin Ligase Complex

The human adenovirus E4orf6 and E1B55K proteins promote viral replication by targeting several cellular proteins for degradation. The E4orf6 product has been shown by our group and others to form an E3 ubiquitin ligase complex that contains elongins B and C and cullin family member Cul5. E1B55K associates with this complex, where it is believed to function primarily to introduce bound substrates for degradation via proteasomes. In addition to p53, its first known substrate, the E4orf6/E1B 55-kDa complex (E4orf6/E1B55K) was shown to promote the degradation of Mre11 and DNA ligase IV; however, additional substrates are believed to exist. This notion is strengthened by the fact that none of these substrates seems likely to be associated with additional functions shown to be mediated by the E4orf6-associated E3 ubiquitin ligase complex, including export of late viral mRNAs and blockage of export of the bulk cellular mRNAs from the nucleus. In an attempt to identify new E4orf6/E1B55K substrates, we undertook a proteomic screen using human p53-null, non-small-cell lung carcinoma H1299 cells expressing either E4orf6 protein alone or in combination with E1B55K through infection by appropriate adenovirus vectors. One cellular protein that appeared to be degraded by E1B55K in combination with the E4orf6 protein was a species of molecular mass ∼130 kDa that was identified as the integrin α3 subunit (i.e., very late activation antigen 3 alpha subunit). Preliminary analyses suggested that degradation of α3 may play a role in promoting release and spread of progeny virions.Viruses are well known to promote replication by inhibiting or enhancing endogenous cellular machinery or, in some cases, by reprogramming key cellular pathways. Human adenoviruses have developed effective ways to modulate the immune response, apoptosis, double-strand break repair, mRNA export, and translation to optimize virus replication and the spreading of progeny virions. The expression of adenovirus E1A proteins stabilizes p53 and induces apoptosis (8, 33); however, this effect is reversed in infected cells by the action of two early products: the E1B 55-kDa (E1B55K) and E4orf6 proteins (35, 36). We and others have shown that these proteins act through the formation of an E3 ubiquitin ligase complex analogous to the SCF and VBC complexes but which contains, in addition to elongins B and C and the RING protein Rbx1, the cullin family member Cul5 (18, 41, 43). This E4orf6-mediated E3 ligase complex blocks p53-induced apoptosis (35, 36) by promoting the ubiquitination of p53, followed by its degradation by proteasomes (41, 43). E4orf6 protein mediates the assembly of the complex by its interaction with elongin C through its three BC boxes (11, 41, 43). E1B55K, which appears to associate with the E4orf6 protein only when present in the ligase complex (4), is thought to function as a substrate recognition factor that brings substrates to the complex because, although both E4orf6 and E1B55K bind p53 independently, interaction of E1B55K with p53 is essential for the efficient degradation of p53 (41, 48). In addition to protecting infected cells from early lysis via p53-induced apoptosis, the E4orf6/E1B55K ligase complex performs other functions essential for virus replication. Two other substrates of the complex have been identified: a member of the MRN DNA repair complex, Mre11, and the central component of the nonhomologous end-joining DNA repair system, DNA ligase IV (2, 56). Degradation of both of these proteins prevents viral genome concatenation, which interferes with the packaging of viral DNA into virions (2, 56). E1B55K binds to p53, Mre11, and DNA ligase IV and has been demonstrated to colocalize with p53 and Mre11 in perinuclear cytoplasmic bodies termed aggresomes (1, 2, 32). More recently, we and others have obtained results that suggest that the E4orf6-associated E3 ligase complex regulates viral and cellular mRNA export (5, 66). The Cul5-based ligase activity was shown to be essential for selective viral mRNA export and the block of cellular mRNA export from the nucleus (66), thus contributing to the shutoff of cellular protein synthesis initiated by L4-100K (20). The actual substrates of the complex responsible for regulating mRNA export are currently unknown.As discussed in detail below, our efforts to identify substrates of the E4orf6/E1B55K complex led us to consider a member of the integrin family as a potential substrate. Integrins are members of a family of surface receptors that function in several ways through the formation of cell-extracellular matrices and cell-cell interactions (reviewed in references 21, 26, and 63). Integrins are typically composed of two transmembrane glycoproteins forming heterodimers of α and β subunits each of approximately 80 to 150 kDa. There are at least 18 α subunits and 8 β subunits in mammals that can dimerize in limited combinations to form more than 20 functionally distinct integrins with different ligand specificities. Integrin heterodimers function as transmembrane receptors that link external factors to intracellular signaling pathways. In addition to roles in cell adhesion, these communication events are implicated in a large range of cellular processes, including proliferation, differentiation, translation, migration, and apoptosis. Some of these processes depend on the intracellular trafficking pathways of the integrins (reviewed in references 9, 24, 40, and 44), including the long-loop recycling pathway in which integrins present in clathrin-coated endosomes move first to the perinuclear recycling center, where some accumulate, including the β1 integrin subunit (31), before returning to the plasma membrane. The integrin α3β1 is a member of the β1 integrin subfamily in which the α3 subunit (VLA-3a) is coupled to the β1 subunit to form the very late activation antigen (VLA-3 or CD49c) (21, 59, 60). α3β1 is expressed in a wide range of tissues in which it binds a variety of extracellular matrix substrates, including fibronectin, collagen, thrombospondin 1, and laminins 1, 5, 8, 10, and 11 (13). These associations allow the integrin α3β1 to fill its primary role in cell adhesion. α3β1 also participates in intercellular adhesion through several protein-protein interactions (10, 27, 53, 55, 58), making it a major contributor in the regulation of cellular adhesion.Human adenovirus type 5 (Ad5) particles interact with cell surface receptors to facilitate internalization into target cells. In the high-affinity interacting model (reviewed in reference 29), the viral fiber knob polypeptide binds the coxsackie adenovirus receptor (CAR) protein on the surface of cells as the primary cell binding event (primary receptor). The penton base polypeptide then binds a cell surface integrin (secondary receptor), leading to entry of the capsid into the cell by a process termed receptor-mediated endocytosis or clathrin-mediated endocytosis. Several types of integrins have been identified as being used by Ad5 to mediate virus internalization: αMβ1, αMβ2, αVβ1, αVβ3, αVβ5, and α5β1 (22, 30, 49, 65). Salone et al. have shown that α3β1 serves as an alternative cellular receptor for adenovirus serotype 5 (49). It promotes entry of the virus into cells, transduction of DNA, and mediates adenovirus infection in both CAR-positive and CAR-negative cell lines. Thus, in addition to functions related to cell adhesion, integrin α3β1 plays an important role in the adenovirus infection cycle.To identify new targets for degradation by the E4orf6/E1B55K ubiquitin ligase, we used a proteomic screen covering most cellular proteins to look for any polypeptide that exhibited a significant decrease in amount following the coexpression from appropriate adenovirus vectors of the E4orf6 protein and E1B55K. This screen revealed several interesting candidates, including integrin α3, a species of 130 kDa that also was found to be reduced in wild-type (wt) virus infection. The degradation of α3 was seen to be dependent on the Cul5-based ligase complex driven by E4orf6 and E1B55K. We also found evidence that the E4orf6/E1B55K ligase complex appears to be involved in cell detachment from the extracellular matrix, a function that could play a role in virus spread.     

Alterations of RNA Editing for the Mitochondrial ATP9 Gene in a New orf220-type Cytoplasmic Male-sterile Line of Stem Mustard （Brassica juncea var. tumida）

RNA editing for the mitochondrlal ATP9 gene of encoding regions has been observed in both cytoplasmic malesterile and maintainer lines of stem mustard, where its editing capacity varied spatially and temporally in the cytoplasmic male sterility （CMS） line. There were four RNA editing sites for the mitochondrial ATP9 gene according to Its normal editing sites in mustard, of which three sites occurred as C-to-U changes and one as a U-to-C change. As a result, the hydrophobicity of deduced ATP9 protein was reduced due to the conversions at its 17th, 45th and 64th positions. Meanwhile, the conservation of deduced ATP9 protein was enhanced by changes at the 56th position. Loss of a specific editing site for ATP9 was observed in juvenile roots, senile roots, senile leaves and floret buds of the CMS line. Comparatively, complete RNA editing for ATP9 gene was retained in juvenile roots, juvenile leaves and floret buds of its maintainer line; however, the loss of a specific editing site for ATP9 gene occurred at senile roots and senile leaves in its maintainer line. These observations allow us to produce a hypothesis that the dysfunction of a specific mitochondrial gene arising from RNA editing could probably be a factor triggering CMS and organ senescence through unknown cross-talk pathways during development.     

重组C8orf32蛋白的表达、纯化及抗体制备

C8orf32是一种功能未知基因,其mRNA含量在乳腺癌组织中显著高于正常乳腺组织。将其开放式阅读框插入pGEX-6P1原核表达载体T7启动子控制下的GST编码基因下游,构建了C8orf32蛋白表达质粒pGEX-6P-C8。表达质粒转化入大肠杆菌BL21(DE3)菌株,经IPTG诱导,成功表达了GST- C8orf32融合蛋白。经带有GST标签的位点特异性蛋白酶切割除去GST-C8orf32融合蛋白的GST标签,获得了N端带有8个多余氨基酸残基的C8orf32蛋白,蛋白纯度为95%左右。N端氨基酸序列分析表明该蛋白N端氨基酸序列正确,质谱鉴定进一步证明所表达C8orf32蛋白的正确性。用制备的C8orf32蛋白免疫新西兰白兔,获得了能够正确识别C8orf32蛋白的抗血清。该蛋白及其抗体的成功制备,为进一步研究C8orf32蛋白的结构功能和体内分布打下了基础。