植物病毒表达载体pCIYVV／CP／W的构建及GFP表达

[目的]研究了植物病毒表达载体pCIYVV／CP／W的构建及用pClYVV／CP／W表达绿色荧光蛋白（GFP）,为植物反应器生产有用蛋白、开发有效的植物病毒载体提供参考。[方法]用三叶草黄脉病毒侵染性全长cDNA克隆pCIYVV基因组的NIb／CP基因之间的一段多克隆位点和两侧含有病毒蛋白酶NIa切割识别序列的多聚脱氧核糖核苷酸接头,构建pCIYVV／CP／W载体,并将绿色荧光蛋白基因插入pCIYVV／CP／W中构建pCIYVV／CP／W／GFP载体,用反转录PCR对重组病毒克隆转录情况进行检测,并用Western杂交对重组病毒克隆表达的目的基因产物进行测定。[结果]pCIYVV／CP／W／GFP接种的蚕豆幼苗表现出与野生型CIYVV相同的症状,发病率达100％,表明重组病毒克隆pCIYVV／CP／W／GFP．有侵染性,外源基因的插入没有破坏载体pCIYVV／CP／W的开放阅读框;以感染叶总RNA为模板,对pCIYVV／CP／W／GFP表达GFP外源基因的稳定性进行了检测,从F0（重组病毒质粒最初转录出的病毒）一直检测到Fd子代病毒。检测结果表明,外源基因在L子代病毒基因组中稳定存在;以从感染叶中提取的总蛋白为抗原,以GFP抗体为第1抗体,以碱性磷酸酶标记抗体为第2抗体,用Western杂交对重组病毒克隆pCVYVV／CP／W／GFP表达外源蛋白进行了定性检测。从F0一直检测到F4子代病毒。检测结果表明,重组病毒克隆pCIYVV／CP／W／GFP至少到F4子代病毒能稳定表达GFP。     

A second nuclear protein is encoded by Epstein-Barr virus in latent infection

A region of the Epstein-Barr virus (EBV) genome that is important in inducing cell proliferation includes a single long open reading frame. Part of this open reading frame has been fused to the lacZ gene and expressed in Escherichia coli. Antisera to the fusion protein identify a protein in the nuclei of latently infected growth-transformed lymphocytes and in Burkitt tumor cells grown in vitro. This nuclear protein is encoded by a different virus-gene than that which encodes the previously described EBV nuclear antigen, EBNA.     

A new HTLV-III/LAV protein encoded by a gene found in cytopathic retroviruses

The DNA of the HTLV-III/LAV group of retroviruses contains certain additional open reading frames that are not found in typical avian or mammalian retroviruses. The role of these sequences in encoding for gene products that may be related to pathogenesis remains to be resolved. An open reading frame whose 5' end overlaps with the pol gene, but is unrelated to the env gene, has been observed in HTLV-III/LAV and visna virus, both cytopathic mammalian retroviruses. Evidence presented here shows that this open reading frame is a bona fide coding sequence of HTLV-III/LAV and that its product, a protein with a molecular weight of 23,000, induces antibody production in the natural course of infection.     

Expression in Escherichia coli of open reading frame gene segments of HTLV-III

Human T-cell lymphotropic virus type III (HTLV-III), the causative agent of the acquired immune deficiency syndrome (AIDS), was recently isolated and its genomic structure analyzed by DNA cloning methods. In the studies reported here a combined cloning and expression system was used to identify HTLV-III encoded peptides that react immunologically with antibodies in sera from AIDS patients. Cloned HTLV-III DNA was sheared into approximately 500-base-pair fragments and inserted into an "open reading frame" expression vector, pMR100. The inserted DNA was expressed in Escherichia coli transformants as a polypeptide fused to the lambda CI protein at its amino terminus and to beta-galactosidase at its carboxyl terminus. Sera from AIDS patients containing antibodies to HTLV-III were then used to screen for immunoreactive fusion proteins. Twenty clones, each specifying a fusion protein strongly reactive with AIDS serum, were identified. DNA sequence analysis indicated that the HTLV-III fragments were derived from the open reading frame DNA segments corresponding to the gag and pol gene coding regions and also the large open reading frame region (env-lor) located near the 3' end of the viral genome.     

黑曲霉木聚糖酶基因（xynA）在大肠杆菌中的表达及酶学分析

从黑曲霉(Aspergillus niger)F19中克隆得到木聚糖酶基因xynA.将不带原基因信号肽编码序列的xynA基因段以正确的阅读框架克隆到大肠杆菌(Escherichia coli)表达载体pET-28a(+)上,并转化E.co-li BL21,获得重组工程菌BLX1.经过IPTG诱导,xynA获得特异性表达,其表达产物以包涵体和胞内可溶性蛋白2种形式存在.经过SDS-PAGE分析,重组蛋白分子质量约为24 ku.酶学分析表明,最适反应温度为50℃,最适反应pH为5.0,在酸性条件下具有较好的稳定性.     

Identification of HTLV-III/LAV sor gene product and detection of antibodies in human sera

The nucleotide sequence of the genome of HTLV-III, the infectious agent etiologically associated with the acquired immune deficiency syndrome, predicts a small open reading frame, termed sor, located between the pol and env genes. A DNA segment containing 82 percent of the sor region was inserted into a prokaryotic expression vector, pJL6, to determine whether sor encodes a viral protein and to gain some insight into its possible function. The bacterially synthesized sor protein reacted with sera from individuals infected with HTLV-III, indicating that sor is expressed as a protein product or products that are immunogenic in vivo. Antibodies to the purified, bacterially synthesized sor protein were found to react specifically with the same protein and also with a protein of molecular weight 23,000 (23K) in HTLV-III-infected H9 cell extracts. The 23K protein comigrated with a protein immunoprecipitated by the serum of a hemophiliac patient with antibodies to HTLV-III, suggesting that this protein is probably the sor gene product.     

小鼠性腺特异表达基因(GSE)的克隆及初步研究

从小鼠的睾丸中克隆了GSE(gonad-specific expression gene)基因,并对该基因进行序列分析和Southern、Northern杂交分析.核苷酸序列分析表明,GSE基因的ORF长度为745 bp,编码247个氨基酸,预测蛋白质分子量为27.6 kDa;Southern杂交证明GSE可能是单拷贝的基因;Northern杂交显示,GSE基因在小鼠体细胞组织(心脏、肝脏、肾脏、大脑、骨骼肌和脾脏)中没有检测到其表达,在睾丸中表达量很高,在卵巢中表达量较低.从推断出的氨基酸序列表明,GSE蛋白在细胞中可能是一个不带有信号肽的可溶性蛋白.     

黄曲条跳甲RCN基因的克隆与表达

【目的】克隆黄曲条跳甲钙离子结合蛋白（reticulocalbin, RCN）,并分析其序列特征和表达谱。【方法】结合转录组测序及荧光定量PCR技术,鉴定和分析黄曲条跳甲钙离子结合蛋白基因的序列特征、功能及表达谱。【结果】获得的黄曲条跳甲RCN基因的cDNA序列全长为1 197 bp,开放阅读框为984 bp,共编码327个氨基酸残基。其蛋白分子含有5个亮氨酸拉链结构域（EF-hand）,与钙离子结合的模体可能为DX（N/D）X（D/N）XXXXXXE。cDNA序列的系统发育分析表明,黄曲条跳甲的RCN与赤拟谷盗的亲缘关系最近。荧光定量PCR分析表明,RCN在黄曲条跳甲雌雄成虫的不同部位都有表达,具有一定的广谱性,其中在头部和中肠的表达量相对较高;触角中雌虫的相对表达量是雄虫的1.9倍,而在生殖系统中,雄虫的相对表达量是雌虫的2.1倍。【结论】成功鉴定了黄曲条跳甲的一种钙离子结合蛋白基因,初步分析了该基因与钙离子结合的模体序列及在虫体不同部位转录水平的表达情况。     

An overlapping protein-coding region in influenza A virus segment 3 modulates the host response

Influenza A virus (IAV) infection leads to variable and imperfectly understood pathogenicity. We report that segment 3 of the virus contains a second open reading frame ("X-ORF"), accessed via ribosomal frameshifting. The frameshift product, termed PA-X, comprises the endonuclease domain of the viral PA protein with a C-terminal domain encoded by the X-ORF and functions to repress cellular gene expression. PA-X also modulates IAV virulence in a mouse infection model, acting to decrease pathogenicity. Loss of PA-X expression leads to changes in the kinetics of the global host response, which notably includes increases in inflammatory, apoptotic, and T lymphocyte-signaling pathways. Thus, we have identified a previously unknown IAV protein that modulates the host response to infection, a finding with important implications for understanding IAV pathogenesis.     

Homology of cytokine synthesis inhibitory factor (IL-10) to the Epstein-Barr virus gene BCRFI

Complementary DNA clones encoding mouse cytokine synthesis inhibitory factor (CSIF; interleukin-10), which inhibits cytokine synthesis by TH1 helper T cells, were isolated and expressed. The predicted protein sequence shows extensive homology with an uncharacterized open reading frame, BCRFI, in the Epstein-Barr virus genome, suggesting the possibility that this herpes virus exploits the biological activity of a captured cytokine gene to enhance its survival in the host.     

登革病毒E蛋白基因表达载体的构建

利用高效表达载体pET系统，构建广东地区登革流行株E蛋白基因的高效表达载体，表达载体经酶切及测序鉴定表明载体构建正确。为探讨E蛋白在登革病毒致病机制中的作用。以及针对本地登革病毒高效、特异的单克隆抗体和高效价的特异高免血清的制备奠定了基础。     

Identification of the protein encoded by the E6 transforming gene of bovine papillomavirus

Papillomaviruses (PV) contain several conserved genes that may encode nonstructural proteins; however, none of these predicted gene products have been identified. Papillomavirus E6 genes are retained and expressed as RNA in PV-associated human and animal carcinomas and cell lines. This suggests that the E6 gene product may be important in the maintenance of the malignant phenotype. The E6 open reading frame of the bovine papillomavirus (BPV) genome has been identified as one of two BPV genes that can independently transform mouse cells in vitro. A polypeptide encoded by this region of BPV was produced in a bacterial expression vector and used to raise antisera. The antisera specifically immunoprecipitated the predicted 15.5-kilodalton BPV E6 protein from cells transformed by the E6 gene. The E6 protein was identified in both the nuclear and membrane fractions of these transformed cells.     

鸭瘟病毒河南株gI基因的克隆与测序

参考GenBank中鸭瘟病毒gI基因序列设计并合成引物,以鸭瘟病毒河南分离株DNA为模板进行PCR扩增,将扩增片段克隆至PGEM-T载体上,得到含gI基因重组质粒。经酶切鉴定,并对重组阳性质粒进行序列测定。结果表明,获得的片段含鸭瘟病毒gI基因,全长1 089 bp,与已报道的其他疱疹病毒gI基因具有较高的同源性,编码432个氨基酸,蛋白分子质量为39.7ku、等电点(PI)为6.06。     

鸟传染性支气管炎病毒类木瓜蛋白酶基因的克隆和表达

[目的]为探究鸟传染性支气管炎病毒(IBV)的感染和复制过程及其防治提供信息和途径。[方法]以鸟IBV的cDNA为模板,利用PCR技术克隆IBV类木瓜蛋白酶基因。将回收片段与pGEX-6p-1载体连接并转入大肠杆菌DH5α中。在IPTG诱导下将克隆的重组质粒在大肠杆菌BL21中进行表达,经纯化获得目标蛋白。[结果]克隆的类木瓜蛋白酶基因全长为924 bp,编码308个氨基酸的完整开放读码框。它与Genbank中IBV M41毒株类木瓜蛋白酶基因的序列完全一致,证实该基因为IBV的类木瓜蛋白酶基因。在IPTG诱导下,BL21菌株成功表达目标基因和GST融合蛋白,融合蛋白约为60 kD。经纯化和酶切后所得蛋白产物的分子量约为34 kD。[结论]该基因的成功克隆和表达为有关蛋白结构与功能的研究奠定了基础。     

平邑甜茶非共生血红蛋白基因GLB1的克隆、表达及转化研究

根据GenBank上苹果GLB1基因的EST序列设计引物,利用PCR技术克隆到平邑甜茶GLB1同源基因MhGLB1（GenBank注册号：GQ423619）,该基因包含一个477bp的开放阅读框,编码158个氨基酸,分子量为17．8ku。成功构建了35S：：MhGLB1正义表达载体,并对“S以12粉”番茄进行农杆菌介导的遗传转化。荧光定量PCR结果显示,MhGLB1在根、茎、叶中均有所表达,但在根中的表达量最高;NO3-和SNP处理能够诱导根中MhGLB1的表达。     

犬瘟热病毒H基因原核表达质粒的构建

为H蛋白进一步表达作为诊断用抗原、建立特异的犬瘟热病毒(CDV)抗体检测方法及CDV的预防奠定基础,以已构建含有犬瘟热病毒H基因的pMD18-H质粒为模板,利用合成的特异性引物进行PCR扩增,得到1824bp的目的DNA片段,将所得H基因克隆至经相同双酶切处理后的pET32a(+)原核表达载体中,获得重组质粒pET32...     

Isolation and characterization of a novel protein (X-ORF product) from SIV and HIV-2

A protein designated p14 was purified from a simian immunodeficiency virus (SIVMne) and was shown by amino acid sequence analysis to be nearly identical to the predicted translational product of a unique open reading frame (X-ORF) in the nucleotide sequences of SIVmac and human immunodeficiency virus type 2 (HIV-2). Thus the X-ORF is proven to be a new retroviral gene. The p14 is present in SIVMne in molar amounts equivalent to those of the gag proteins. This is the first example of a retrovirus that contains a substantial quantity of a viral protein that is not a product of the gag, pro, pol, or env genes. SIV p14 and its homolog in HIV-2 may function as nucleic acid binding proteins since purified p14 binds to single-stranded nucleic acids in vitro. Antisera to the purified protein detected p14 in SIVMne, SIVmac, and a homologous protein (16 kilodaltons) in HIV-2 but did not react with HIV-1. Diagnostic procedures based on this novel protein will distinguish between HIV-1 and HIV-2.     

紫大麦草中DREB类转录因子基因Hvi917DREB2的克隆、原核表达及序列分析

DREB类转录因子是一类与植物逆境胁迫响应相关的重要蛋白,本研究利用同源克隆方法和RACE技术从紫大麦草(Hordeum violaceum)中分离到1个DREB类转录因子基因,命名为Hvi917 DREB2。序列分析表明Hvi917 DREB2基因含有1个792bp的开放阅读框,编码264个氨基酸残基,含有1个保守的AP2结构域,属于AP2大家族。该基因编码的氨基酸序列与Genebank中短芒大麦草AP2蛋白HbDREB2、DREB1(登录号分别为:AAU29412.1和AER42620.1)具有99%的氨基酸序列一致性。将该基因构建成原核表达载体,经IPTG诱导表达了1个分子量为34.5kD的蛋白。     

鲤春病毒血症病毒基质蛋白基因的原核表达及多克隆抗体的制备

根据鲤春病毒血症病毒(SVCV)基因组序列(GenBank:NC_002803),人工合成M基因开放阅读框序列,克隆至原核表达载体pET-32a(+),转化工程菌株BL21 (DE3),在0.1 mmol·L-1 IPTG、37℃下诱导5h,获得高效表达的M蛋白,但其主要以包涵体的形式存在,经超声破碎、包涵体纯化后,再...     

O型口蹄疫病毒VP1基因的克隆与表达

以O型口蹄疫病毒(FMDV)流行毒株为研究对象,通过RT-PCR扩增出VP1基因,克隆至表达载体pET-32a中,分析比较不同地区O型口蹄疫病毒VP1基因序列,为构建VP1重组基因工程苗奠定基础．经测序表明,目的基因VP1已以正确的阅读框架整合至表达质粒中,应用大肠杆菌BL21(DE3)为宿主菌,通过IPTG诱导方法,表达包含VP1基因产物的融合蛋白,经SDS-PAGE和Western—blotting分析,表明表达蛋白表达量高,反应原性良好．