柯萨奇病毒A16型2 B基因的酵母双杂交诱饵载体的构建及其特性鉴定

目的：构建柯萨奇病毒A16的2B基因的酵母诱饵表达载体,用于筛选与2B相互作用的蛋白。方法 PCR扩增2B全序列,克隆入pGBKT7?BD,将构建好的 pGBKT7?2B转化到酵母细胞 Y2HGold;用Western印迹分析诱饵蛋白的表达,检测诱饵蛋白有无毒性和自激活效应。结果成功克隆了pGBKT7?2B并转化至Y2 HGold中,转化细胞Y2 HGold [pGBKT7?2B]可以表达诱饵蛋白2B,2B蛋白对转化细胞无细胞毒性和自激活效应。结论成功构建了酵母诱饵表达载体pGBKT7?2B,可用于酵母双杂交筛选与2B蛋白相互作用的宿主靶蛋白。     

GnRH缺陷疾病致病基因 RNF216互作蛋白的筛选

目的:通过酵母双杂交实验筛选与环指蛋白216(ring finger protein 216, RNF216)相互作用的蛋白,进一步阐明RNF216在GnRH缺陷疾病中的作用。方法:构建pGBKT7- RNF216重组表达载体,将其转化到Y2HGold酵母中,与人cDNA文库进行杂交,筛选与RNF216相互...     

N40:一种新近发现的由流感病毒PB1节段编码的蛋白

N40蛋白是一种新发现的甲型流感病毒表达产物,该蛋白由流感病毒基因组PBl节段编码,是流感病毒PBl蛋白的变体,同PB1蛋白相比在N末端有40个氨基酸残基的缺失.N40蛋白和PB1节段编码的另外两种产物(PB1蛋白、PB1-F2蛋白)在表达水平上相互关联.2009年甲型H1N1亚型流感病毒的基阗组中存在N40蛋白的开放阅读框.N40蛋白的生物学意义以及同流感病毒毒力的关系尚有待于深入研究.     

N40:一种新近发现的由流感病毒PB1节段编码的蛋白

N40蛋白是一种新发现的甲型流感病毒表达产物,该蛋白由流感病毒基因组PBl节段编码,是流感病毒PBl蛋白的变体,同PB1蛋白相比在N末端有40个氨基酸残基的缺失.N40蛋白和PB1节段编码的另外两种产物(PB1蛋白、PB1-F2蛋白)在表达水平上相互关联.2009年甲型H1N1亚型流感病毒的基阗组中存在N40蛋白的开放阅读框.N40蛋白的生物学意义以及同流感病毒毒力的关系尚有待于深入研究.     

N40:一种新近发现的由流感病毒PB1节段编码的蛋白

N40蛋白是一种新发现的甲型流感病毒表达产物,该蛋白由流感病毒基因组PBl节段编码,是流感病毒PBl蛋白的变体,同PB1蛋白相比在N末端有40个氨基酸残基的缺失.N40蛋白和PB1节段编码的另外两种产物(PB1蛋白、PB1-F2蛋白)在表达水平上相互关联.2009年甲型H1N1亚型流感病毒的基阗组中存在N40蛋白的开放阅读框.N40蛋白的生物学意义以及同流感病毒毒力的关系尚有待于深入研究.     

应用酵母双杂交系统筛选与乙型流感病毒BM2相互作用的蛋白质

目的　筛选与乙型流感病毒BM2蛋白相互作用的蛋白质。方法　应用酵母双杂交系统,以BM2 (2 6 10 9)插入载体pGBKT7作为诱铒,在四缺培养基上筛选人KidneyMATCHMAKERcDNA文库,寻找与BM2蛋白相互作用的宿主蛋白。结果　筛选得到6个阳性AD 文库质粒,并用酵母双杂交实验验证了阳性AD 文库质粒与BM2的相互作用。将阳性AD 文库质粒测序并对测序结果做BLAST分析,发现它们分别是N 乙酰神经氨酸酶丙酮酸裂解酶,Angiopoietin 3、锌指蛋白2 5 1、核糖体蛋白S2 0、蛋白精氨酸N 甲基转移酶1(PRMT)、转录因子样1(TCFL1)。结论　BM2能与这些转录翻译功能相关的蛋白质相互作用,表明BM2蛋白在病毒生活周期中发挥重要作用。     

酵母双杂交筛选与单剪接型2.2 kb乙型肝炎病毒剪接特异性新蛋白相互作用的肝细胞蛋白

目的 筛选与单剪接型2.2 kb乙型肝炎病毒(HBV)剪接特异性新蛋白相互作用的肝细胞蛋白.方法 PCR扩增单剪接型2.2 kb HBV剪接特异性新基因TPss并克隆于诱饵载体pGBKT7,在证实TPss蛋白不具有自激活作用的前提下,以酵母双杂交系统筛查与TPss蛋白相互作用的肝细胞蛋白,进而通过哺乳动物细胞双杂交实验验证候选肝细胞蛋白与TPss蛋白在Huh7和HepG2肝细胞中的相互作用.结果 构建酵母双杂交诱饵载体pGBKT7-TPss,Western blot显示其在酵母中表达TPss蛋白.酵母双杂交筛选及哺乳动物细胞双杂交证实TPss蛋白可与4种肝细胞蛋白相互作用,即组织蛋白酶B、微粒体环氧化物水解酶、组织蛋白酶D与纤维蛋白原γ链.结论 TPss可与多种肝细胞蛋白相互作用.     

IQSEC1通过病毒蛋白PB1调控甲型流感病毒的增殖

目的探讨IQSEC1是否通过病毒蛋白PB1调控甲型流感病毒的增殖。方法首先克隆甲型流感病毒[A/Shanghai/02/2013(H7N9)]的8个基因;其次,通过免疫共沉淀检测IQ模体Sec7结构域蛋白1(IQSEC1)与聚合酶PB1(PB1)存在相互作用;此外,通过过表达或者敲低IQSEC1的方法检测IQSEC1对PB1核定位的影响;最后,过表达或者敲低IQSEC1后检测Influenza A virus[A/Shanghai/02/2013(H7N9)]。结果病毒感染条件下,外源IQSEC1和PB1存在相互作用。当过表达IQSEC1时,细胞中IQSEC1的表达量上升,相应的PB1在细胞核中的定位减少;当用敲低IQSEC1时,细胞中IQSEC1的表达量下降,相应的PB1在细胞核中的定位上升。过表达IQSEC1后,甲型流感病毒的增殖水平下降(P0.05)。敲低IQSEC1后,甲型流感病毒的增殖水平上升(P0.05)。结论 IQSEC1通过减少甲型流感病毒蛋白PB1的核定位抑制甲型流感病毒的增殖。     

PML-C与GINS2蛋白相互作用的胞内验证

目的 通过胞内实验验证PML-C与GINS2蛋白之间的相互作用.方法 将诱饵蛋白质粒pGBKT7-PML-C和文库蛋白质粒pACT2-GINS2共转化AH109酵母菌,通过一对一的酵母双杂交技术验证两者在活细胞内的相互作用;构建pCMV-HA-PML-C及pCMV-Myc-GINS2真核表达载体并共转染人胚肾293细胞...     

酵母双杂交技术筛选NECL1胞内区的结合蛋白

目的 筛选人源膜表面黏附分子NECL1蛋白胞内区相互作用蛋白。 方法 构建含人NECL1蛋白胞内区氨基酸编码序列的诱饵质粒pGBKT7-NECL1C,对人胎脑cDNA文库进行酵母双杂交筛选。用GST pull down实验进行体外蛋白相互作用的验证。结果 酵母双杂交阳性克隆测序后显示共存在9段不同序列（存在重复克隆）。比对氨基酸序列得到5个可能相互作用蛋白。通过GST pull down 实验验证了其中两个蛋白与NECL1胞内区的相互作用。结论 应用酵母双杂交系统,获得了一些候选的NECL1胞内区相互作用蛋白。     

PB1-F2 gene in influenza a viruses of different hemagglutinin subtype

The second ORF frame (+1) of PB1 polymerase gene of Influenza A virus (IAV) encodes the PB1-F2 protein. The length of PB1-F2 encoded by the A/Puerto Rico/8/34 (H1N1) (PR8) virus is 87 aa. The analysis of nucleotide sequences of PB1 gene of 626 IAV isolates available in GenBank and Influenza Sequence Database revealed that this gene has mostly the capacity to encode a putative protein of 90 aa. The predicted extra three amino acids in the 90-aa PB1-F2 are to a great extent conservative. Some IAV isolates, particularly human, avian and swine with hemagglutinin (HA) of H1 subtype can potentially encode a C-terminally truncated PB1-F2 of various lengths. The C-terminally truncated PB1-F2 in H1 isolates is lacking the region responsible for mitochondrial targeting and apoptosis. About 50% of avian isolates of H9 subtype possess an ORF for truncated PB1-F2. Eighteen aa, 10 at the N-terminus and 8 at the C terminus are strictly conservative in all 148 human isolates.     

N-terminal region of the PB1-F2 protein is responsible for increased expression of influenza A viral protein PB1

Influenza A virus (IAV) PB1-F2 protein is encoded by an alternative reading frame (+1) within the PB1 gene. PB1-F2 has been shown to contribute to the pathogenesis of influenza virus infection as well as to the secondary bacterial infection. More recently has been shown that PB1-F2 protein may regulate a viral RNA (vRNA) polymerase activity by the interaction with PB1 protein. We proved that PB1-F2 protein increased the level of expression of PB1 protein and vRNA in the infected cells. Moreover, we demonstrated that a higher level of vRNA expression resulted in the increase of expression of multiple viral proteins, including NP, M1, and NS1. Finally, we used plasmids expressing N-terminal (1-50 aa) or C-terminal (51-87 aa) region of the PB1-F2 molecule for transfection of MDCK cells co-infected with influenza A/Puerto Rico/8/34 (H1N1) virus deficient in the PB1-F2 protein expression (PR8ΔPB1-F2). These experiments clearly showed that N-terminal region of PB1-F2 protein was responsible for the increase in PB1 protein expression. C-terminal region of PB1-F2 protein had no effect. Thus, we have identified the important function for N-terminal region of PB1-F2 protein.     

Molecular signatures of virulence in the PB1-F2 proteins of H5N1 influenza viruses

The PB1-F2 protein of influenza A viruses contributes to pathogenesis in animal models. Specific molecular signatures of virulence within PB1-F2 have been mapped for some functions. The 66S polymorphism may modulate interferon activity, and four C-terminal amino acids, 62L, 75R, 79R, and 82L, contribute to cytokine release and inflammatory responses in specific virus backgrounds. All available PB1-F2 sequences from H5N1 subtype influenza A viruses were analyzed. The majority (82.5%) of H5N1 sequences available in the Influenza Research Database code for PB1-F2 proteins with 4 or more of these virulence associated amino acids. Most of these are avian sequences from highly pathogenic strains isolated in Asia or Africa. The 66S polymorphism was uncommon (5.3% of sequences) but was found in association with the other 4 inflammatory amino acids in select highly pathogenic strains in Asia. These analyses suggest that if an H5N1 virus were to emerge as a pandemic strain, the PB1-F2 protein will be a contributor to pathogenesis. Research on the pathogenic effect of these signatures in an H5N1 background should be undertaken. Surveillance efforts should include sequencing of the PB1 gene segment and analysis for these molecular signatures to allow for the potential prioritization of resources during pandemic planning.     

INFLUENZA VIRUS INDUCTION OF APOPTOSIS BY INTRINSIC AND EXTRINSIC MECHANISMS

It is now firmly established that apoptosis is an important mechanism of influenza virus-induced cell death both in vivo and in vitro. Data are predominately from experiments with influenza A virus and in vitro experimental systems. Multiple influenza virus factors have been identified that can activate intrinsic or extrinsic apoptotic induction pathways. Currently there is no evidence for influenza virus directly accessing the apoptosis execution factors. The best-studied influenza virus inducers of apoptosis are dsRNA, NS1, NA, and a newly described gene product PB1-F2. PB1-F2 is the only influenza virus factor to date identified to act intrinsically by localization and interaction with the mitochondrial-dependent apoptotic pathway. Both dsRNA and NA have been shown to act via an extrinsic mechanism involving proapoptotic host-defense molecules: PKR by induction of Fas-Fas ligand and NA by activation of TGF- &#103. PKR is capable of controlling several important cell-signaling pathways and therefore may have multiple effects; a predominant one is increased interferon (IFN) production and activity. NS1 has been shown to be both proapoptotic and antiapoptotic. Use of influenza virus NS1 deletion mutants has provided evidence for NS1 interference with apoptosis, IFN induction, and related cell-signaling pathways. Influenza virus also has important exocrine paracrine effects, which are likely mediated via TNF family ligands and oxygen, free radicals capable of inducing apoptosis. Little is known about activation of inhibitors of apoptosis such as inhibitory apoptotic proteins. Whether all these factors always have a role in influenza virus-induced apoptosis is unknown. The kinetics of synthesis of influenza virus factors affecting apoptosis during the replication cycle may be an important aspect of apoptosis induction.     

Influenza virus induction of apoptosis by intrinsic and extrinsic mechanisms

It is now firmly established that apoptosis is an important mechanism of influenza virus-induced cell death both in vivo and in vitro. Data are predominantly from experiments with influenza A virus and in vitro experimental systems. Multiple influenza virus factors have been identified that can activate intrinsic or extrinsic apoptotic induction pathways. Currently there is no evidence for influenza virus directly accessing the apoptosis execution factors. The best-studied influenza virus inducers of apoptosis are dsRNA, NS1, NA, and a newly described gene product PB1-F2. PB1-F2 is the only influenza virus factor to date identified to act intrinsically by localization and interaction with the mitochondrial-dependent apoptotic pathway. Both dsRNA and NA have been shown to act via an extrinsic mechanism involving proapoptotic host-defense molecules: PKR by induction of Fas-Fas ligand and NA by activation of TGF-beta. PKR is capable of controlling several important cell-signaling pathways and therefore may have multiple effects; a predominant one is increased interferon (IFN) production and activity. NS1 has been shown to be both proapoptotic and antiapoptotic. Use of influenza virus NS1 deletion mutants has provided evidence for NS1 interference with apoptosis, IFN induction, and related cell-signaling pathways. Influenza virus also has important exocrine paracrine effects, which are likely mediated via TNF family ligands and oxygen, free radicals capable of inducing apoptosis. Little is known about activation of inhibitors of apoptosis such as inhibitory apoptotic proteins. Whether all these factors always have a role in influenza virus-induced apoptosis is unknown. The kinetics of synthesis of influenza virus factors affecting apoptosis during the replication cycle may be an important aspect of apoptosis induction.     

Influenza a virus PB1-F2 protein

PB1-F2 protein (PB1-F2) is encoded by the alternative (+1) ORF in the PB1 gene of influenza A viruses (IAVs). This protein has a number of unique features, namely its absence from some animal IAV isolates, variable expression in individual infected cells, rapid proteasome-dependent degradation, mitochondrial localization, and apoptotic or pro-apoptotic properties. Localization of PB1-F2 to mitochondria is mediated via C-terminal basic amphipathic alpha-helix. PB1-F2 affects apoptosis and may contribute to the pathogenicity and lethality of IAVs. Sequence analysis showed that, in addition to the strains with an ORF for full-length PB1-F2, there are some with an ORF for different truncated forms of PB1-F2. Several other viruses encode proteins with structure and function similar to PB1-F2 of IAVs.     

人肝再生增强因子相互作用蛋白基因的克隆

目的：寻找与人肝再生增强因子(hALR)相互作用的蛋白质．探讨其在肝再生过程中的分子生物学机制。方法：采用酵母双杂交系统，以hALR作诱饵蛋白筛选预转化的人肝cDNA文库，对阳性克隆进行生物信息学分析。结果：筛选出6组与hALR具有特异性相互作用的蛋白基因，分别是：血清白蛋白、金属硫蛋白、Na／K-ATPase、硒蛋白P和两个未知功能基因的cDNA序列。结论：初步克隆了与hALR相互作用蛋白基因，为以后深入研究这些蛋白质与hALR之间的相互作用，进一步揭示hALR的作用机制奠定了基础。     

Synthesis and cellular location of the ten influenza polypeptides individually expressed by recombinant vaccinia viruses

A complete set of recombinant vaccinia viruses that express each of the influenza virus polypeptides has been constructed. PB1, PB2, PA, HA, NP, M1, and NS1 genes were derived from influenza virus A/PR/8/34, NA from influenza virus A/Cam/46, and M2 and NS2 genes from influenza virus A/Udorn/72. Cells infected with these recombinant viruses synthesize influenza polypeptides that are precipitable with specific antisera and that have electrophoretic mobilities similar to the corresponding influenza virus polypeptides. Indirect immunofluorescence studies have shown that HA, NA, and MS2 proteins migrate to the cell surface; PB2, PB1, PA, NP, and NS1 proteins migrate to the cell nucleus; and M1 and NS2 are distributed throughout the cell, although NS2 accumulates preferentially in nuclei. These transport processes occurred independently of other influenza polypeptides and are therefore attributable to the intrinsic properties of the influenza polypeptides themselves.     

Current knowledge on PB1-F2 of influenza A viruses

Almost 10 years ago, an eleventh protein of influenza A viruses was discovered in a search for CD8+ T-cell epitopes. This protein was named PB1-F2 since it is encoded in the +1 reading frame of the PB1 gene segment. Various studies have shown that PB1-F2 has a pleiotropic effect: (1) The protein can induce apoptosis in a cell type-dependent manner, (2) PB1-F2 is able to promote inflammation, and (3) finally it up-regulates viral polymerase activity by its interaction with the PB1 subunit. These properties could contribute to an enhanced pathogenicity. However, the underlying mechanism is not fully understood yet. New data suggest that some effects of PB1-F2 are strain-specific and host-specific.