YEAST two-hybrid and itc studies of alpha and beta spectrin interaction at the tetramerization site

Yeast two-hybrid (Y2H) and isothermal titration calorimetry (ITC) methods were used to further study the mutational effect of non-erythroid alpha spectrin (αII) at position 22 in tetramer formation with beta spectrin (βII). Four mutants, αII-V22D, V22F, V22M and V22W, were studied. For the Y2H system, we used plasmids pGBKT7, consisting of the cDNA of the first 359 residues at the N-terminal region of αII, and pGADT7, consisting of the cDNA of residues 1697–2145 at the C-terminal region of βII. Strain AH109 yeast cells were used for colony growth assays and strain Y187 was used for β-galactosidase activity assays. Y2H results showed that the C-terminal region of βII interacts with the N-terminal region of αII, either the wild type, or those with V22F, V22M or V22W mutations. The V22D mutant did not interact with βII. For ITC studies, we used recombinant proteins of the αII N-terminal fragment and of the erythroid beta spectrin (βI) C-terminal fragment; results showed that the K_d values for V22F were similar to those for the wild-type (about 7 nM), whereas the K_d values were about 35 nM for V22M and about 90 nM for V22W. We were not able to detect any binding for V22D with ITC methods. This study clearly demonstrates that the single mutation at position 22 of αII, a region critical to the function of nonerythroid α spectrin, may lead to a reduced level of spectrin tetramers and abnormal spectrin-based membrane skeleton. These abnormalities could cause abnormal neural activities in cells.     

不同亚型流感病毒NS1蛋白的细胞定位差异分析

NS1蛋白是流感病毒编码的一种小分子多功能蛋白,可在病毒的复制过程中抑制宿主细胞的抗病毒免疫应答。为研究不同亚型流感病毒的NS1蛋白在细胞内的定位差异,分别用H1N1亚型WSN、PR8和CA04毒株,H9N2亚型SD毒株及H7N9亚型AH01毒株感染A549、MDCK细胞系以及构建的可表达不同亚型流感病毒NS1蛋白的p CMV-Myc-NS1质粒转染293T细胞,用激光共聚焦显微镜观察发现不同亚型流感病毒在不同细胞系和时间点的定位差异,感染后24 h时WSN和PR8毒株的NS1主要定位于细胞质中,而CA04和SD毒株主要定位于细胞核内。另外,观察过表达的WSN、SD和AH01毒株NS1的细胞定位,转染后24 h时WSN毒株NS1定位于细胞质中,而SD和AH01毒株主要定位于细胞核中。经氨基酸序列比对,对WSN毒株NS1蛋白进行关键氨基酸点突变,结果显示单一位点的改变未导致NS1蛋白细胞定位的改变,其细胞定位的差异不是由单一位点决定的。综上所述,分析不同亚型中的NS1的定位差异,这对进一步了解NS1蛋白同宿主细胞不同区域的蛋白的相互作用、流感病毒的调节机制以及病毒感染细胞中天然免疫反应具有一定的指导意义。     

A型流感病毒NS1与突触后密度蛋白-95的相互作用

目的 A型流感病毒NS1蛋白是一种多功能的致病因子,能够与被感染细胞中的多种蛋白相互结合,影响并干扰宿主细胞内的信号转导、蛋白质合成及抗病毒反应。突触后密度蛋白(Postsynaptic density protein95,PSD-95)主要存在于神经元及SH-SY-5Y等神经来源的细胞株中。假设NS1能够与PSD-95结合,则更有利于了解A型流感病毒对神经元及相关细胞的作用机制。方法通过酵母双杂交,GST-pull down及免疫荧光技术分别从体外和体内两方面检测NS1与PSD-95的相互作用。结果酵母双杂交表明,仅转染PGAD-NS51/PGBK-PSD-95的QDO有菌落生长,且α-半乳糖苷酶活性显著高于阳性对照;而转染PGAD-NS32/PGBK-PSD-95的QDO无菌落生长;GST-pull down表明仅NS51与PSD-95孵育后,能够被Western-blot检测到;免疫荧光表明NS51与PSD-95可能存在共定位,而NS32与PSD-95则不存在共定位。结论 H5N1(A/chicken/Guangdong/1/2005)的NS1能够与PSD-95结合;反之,H3N2(A/Shantou/602/06)的NS1则不能。     

A型流感病毒NS1蛋白羧基端PL结构域影响NS1在细胞核内的定位

A型流感病毒NS1蛋白羧基端4个氨基酸可以与PDZ结构域(the domain of PSD95,Dig and ZO-1)相结合,称为PL结构域(PDZ ligand domain)．对不同亚型或毒株的流感病毒而言,其NS1蛋白PL结构域的组成存在比较大的差异．有研究发现这种差异能够影响NS1与宿主细胞蛋白的相互作用进而影响病毒的致病力．为进一步探讨PL结构域对NS1蛋白生物学特性的影响,首先构建出4种不同亚型流感病毒(H1N1、H3N2、H5N1、H9N2)来源的NS1绿色荧光蛋白表达质粒．在此基础上,对野生型H3N2病毒NS1表达质粒进行人工改造,将其PL结构域缺失或者替换为其他亚型流感病毒的PL结构域,制备出4种重组NS1蛋白表达质粒．通过比较上述不同NS1蛋白在HeLa细胞中的定位情况发现,只有野生型H3N2病毒的NS1蛋白可以定位于核仁当中,而野生型H1N1、H5N1、H9N2病毒的NS1蛋白以及PL结构域缺失或替代的H3N2病毒NS1蛋白都不能定位于核仁．而通过比较上述NS1蛋白在流感病毒易感的MDCK细胞中的定位,进一步发现所有这些蛋白均不定位于核仁．上述结果表明：PL结构域的不同可以明显影响NS1蛋白在HeLa细胞核内的定位和分布,这有可能造成其生物学功能的差异．同时,NS1蛋白在细胞核内的定位还与宿主细胞的来源有着密切关系．     

Human trachea primary epithelial cells express both sialyl(α2-3)Gal receptor for human parainfluenza virus type 1 and avian influenza viruses, and sialyl(α2-6)Gal receptor for human influenza viruses

We reported previously that the dominant receptors of influenza A and B viruses, and human and murine respiroviruses, were sialylglycoproteins and gangliosides containing monosialo-lactosamine type I-and II-residues, such as sialic acid-α2-3(6)-Galβ1-3(4)-GlcNAcβ1-. In addition, the Siaα2-3Gal linkage was predominantly recognized by avian and horse influenza viruses, and human parainfluenza virus type 1 (hPIV-1), whereas the Siaα2-6Gal linkage was mainly recognized by human influenza viruses (Paulson JC in “The Receptors' [Conn M Ed] 2, 131–219 (1985); Suzuki Y, Prog Lipid Res 33, 429–57 (1994); Ito T, J Virol 73, 6743–51 (2000); Suzuki Y, J Virol 74, 11825–31 (2000); Suzuki T, J. Virol 75, 4604–4613 (2001); Suzuki Y, Biol. Pharm. Bull. 28, 399–408 (2005)). To clarify the distribution of influenza virus receptors on the human bronchial epithelium cell surface, we investigated a primary culture of normal human bronchial epithelial (NHBE) cells using two types of lectin (MAA and SNA), which recognize sialyl linkages (α2-3 and α2-6), using fluorescence-activated cell-sorting analysis. The results showed that both α2-3- and α2-6-linked Sias were expressed on the surface of primary human bronchial epithelial cells. The cells infected by hPIV-1 bound to MAA, confirming that cells targeted by hPIV-1 have α2-3-linked oligosaccharides. We also compared the ability of hPIV-1 and human influenza A virus to infect primary human bronchial epithelial cells pre-treated with Siaα2-3Gal-specific sialidase from Salmonella typhimurium. No difference was observed in the number of sialidase pre-treated and non-treated cells infected with human influenza A virus, which binds to Siaα2-6Gal-linked oligosaccharides. By contrast, the number of cells infected with hPIV-1 decreased significantly upon sialidase treatment. Thus, cultured NHBE cells showed both α2-3-linked Sias recognized by hPIV-1 and avian influenza virus receptors, and α2-6-linked Sias recognized by human influenza virus receptors.     

Vaccination of pigs against swine influenza viruses by using an NS1-truncated modified live-virus vaccine

Swine influenza viruses (SIV) naturally infect pigs and can be transmitted to humans. In the pig, genetic reassortment to create novel influenza subtypes by mixing avian, human, and swine influenza viruses is possible. An SIV vaccine inducing cross-protective immunity between different subtypes and strains circulating in pigs is highly desirable. Previously, we have shown that an H3N2 SIV (A/swine/Texas/4199-2/98 [TX98]) containing a deleted NS1 gene expressing a truncated NS1 protein of 126 amino acids, NS1black triangle126, was attenuated in swine. In this study, 4-week-old pigs were vaccinated with the TX98 NS1black triangle126 modified live virus (MLV). Ten days after boosting, pigs were challenged with wild-type homologous H3N2 or heterosubtypic H1N1 SIV and sacrificed 5 days later. The MLV was highly attenuated and completely protected against challenge with the homologous virus. Vaccinated pigs challenged with the heterosubtypic H1N1 virus demonstrated macroscopic lung lesions similar to those of the unvaccinated H1N1 control pigs. Remarkably, vaccinated pigs challenged with the H1N1 SIV had significantly lower microscopic lung lesions and less virus shedding from the respiratory tract than did unvaccinated, H1N1-challenged pigs. All vaccinated pigs developed significant levels of hemagglutination inhibition and enzyme-linked immunosorbent assay titers in serum and mucosal immunoglobulin A antibodies against H3N2 SIV antigens. Vaccinated pigs were seronegative for NS1, indicating the potential use of the TX98 NS1black triangle126 MLV as a vaccine to differentiate infected from vaccinated animals.     

Heterologous interactions between NS1 proteins from different influenza A virus subtypes/strains

Non-structural protein 1 (NS1) of the influenza virus plays a crucial role in modulating the host immune response and facilitating virus replication. The formation of a homodimer or an oligomer is necessary for NS1 to exert its function efficiently. In the present study, the NS1 protein from the A/Shantou/602/06(H3N2) virus (herein abbreviated as NS32) was found to interact with NS1 from A/Shantou/169/06(H1N1), A/Chicken/Guangdong/1/05(H5N1) and A/Quail/Hong Kong/G1/97(H9N2) (abbreviated as NS11, NS51 and NS92, respectively) viruses, although NS32 shares 17.4%?C20.9% sequence diversity with NS11, NS51 and NS92. This indicates that the heterologous interactions between NS1 proteins from different influenza A virus subtypes/ strains may be a common event during co-infection.     

β-Galactosidase production by the psychrotolerant yeast Guehomyces pullulans 17-1 isolated from sea sediment in Antarctica and lactose hydrolysis

A psychrotolerant yeast Guehomyces pullulans 17-1 was isolated from sea sediment in Antarctica. It was found that it could yield both extracellular and cell-bound β-galactosidase. After optimization of the medium and cultivation conditions, it was found that the yeast strain produced over 17.2 U mL⁻¹ of β-galactosidase activity within 120 h at the flask level while the yeast strain produced over 25.3 U mL⁻¹ of β-galactosidase activity within 144 h during the 2-L fermentation. This is the highest β-galactosidase activity produced by the wild type yeast strains reported so far. However, the optimal pH and temperature for the crude β-galactosidase were 4.0 and 50 °C, respectively. Lactose could be converted into glucose and galactose and a large amount of reducing sugar could be released from milk under catalysis of the yeast culture.     

Subtyping animal influenza virus with general multiplex RT-PCR and Liquichip high throughput (GMPLex)

This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of H1, H3, H5, H7, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR (GM RT-PCR). It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus (IV) rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features: high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10⁻⁵(= 280ELD₅₀) forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10⁻⁴(=2800 ELD₅₀). The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.Key words: Influenza Virus, General multiplex RT-PCR, Iuminex assay, Subtyping, HA and NA genes     

A rapid method for liquid β-galactosidase reporter assay in Saccharomyces cerevisiae

Although β-galactosidase assay is widely used for various studies in yeast a quantitative estimation of low enzyme activities with standard reactives remains hampered. It requires long reaction time and large amounts of cells. To overcome existing limitations we developed protocol, which incorporates realization reaction in miniaturized format, cell lysis in reaction buffer and simplification the normalization of β-galactosidase activity. These features allow faster reaction kinetics, accurate and simple quantification of low enzyme activities. To perform studies in vivo conditions we constructed a reporter plasmids based on the low copy yeast vector Ycp50. We adapted our assay on the yeast protein Rpn4 which is highly unstable with a half-life of only 2 min. We demonstrated that detection of Rpn4–LacZ fusion is achieved in 40 min in our method, whereas in standard assay it requires 4–5 h. Moreover, we implemented our approach for promoter dissection investigation. Thus, we present rapid, convenient and less labor-intensive method for assessment β-galactosidase activity.     

High throughput flow cytometry based yeast two-hybrid array approach for large-scale analysis of protein-protein interactions

The analysis of protein-protein interactions is a key focus of proteomics efforts. The yeast two-hybrid system (Y2H) has been the most commonly used method in genome-wide searches for protein interaction partners. However, the throughput of the current yeast two-hybrid array approach is hampered by the involvement of the time-consuming LacZ assay and/or the incompatibility of liquid handling automation due to the requirement for selection of colonies/diploids on agar plates. To facilitate large-scale Y2H assays, we report a novel array approach by coupling a GFP reporter based Y2H system with high throughput flow cytometry that enables the processing of a 96-well plate in as little as 3 min. In this approach, the yEGFP reporter has been established in both AH109 (MATa) and Y187 (MATα) reporter cells. It not only allows the generation of two copies of GFP reporter genes in diploid cells, but also allows the convenient determination of self-activators generated from both bait and prey constructs by flow cytometry. We demonstrate a Y2H array assay procedure that is carried out completely in liquid media in 96-well plates by mating bait and prey cells in liquid YPD media, selecting the diploids containing positive interaction pairs in selective media and analyzing the GFP reporter directly by flow cytometry. We have evaluated this flow cytometry based array procedure by showing that the interaction of the positive control pair P53/T is able to be reproducibly detected at 72 hr postmating compared with the negative control pairs. We conclude that our flow cytometry based yeast two-hybrid approach is robust, convenient, quantitative, and is amenable to large-scale analysis using liquid-handling automation.     

β-Mannanolytic system of Aureobasidium pullulans

A xylanolytic yeast strain Aureobasidium pullulans NRRL Y 2311-1, was found to produce all enzymes required for complete degradation of galactomannan and galactoglucomannan. The enzymes differed in function and cellular localization: endo-β-1,4-mannanase was secreted into the culture fluid, β-mannosidase was strictly intracellular, and α-galactosidase and β-glucosidase were found both extracellularly and intracellularly. Among these enzyme components, only extracellular β-mannanase and intracellular β-mannosidase were inducible. The production of β-mannanase and β-mannosidase was 10- to 100-fold higher in galactomannan medium than in medium with one of the other carbon sources. β-mannanase and β-mannosidase were coinduced in glucose-grown cells by galactomannan, galactoglucomannan, and β-1,4-manno-oligosaccharides. The natural inducer of extracellular β-mannanase and intracellular β-mannosidase appeared to be β-1,4-mannobiose. Synthesis of both enzymes was completely repressed by glucose, mannose, or galactose. The synthetic glycoside methyl β-d-mannopyranoside served as a nonmetabolizable inducer of both β-mannosidase and β-mannanase. Received: 24 June 1996 / Accepted: 26 September 1996     

Avian influenza virus A/HK/483/97(H5N1) NS1 protein induces apoptosis in human airway epithelial cells

Avian H5N1 influenza virus causes a remarkably severe disease in humans, with an overall case fatality rate of greater than 50%. Human influenza A viruses induce apoptosis in infected cells, which can lead to organ dysfunction. To verify the role of H5N1-encoded NS1 in inducing apoptosis, the NS1 gene was cloned and expressed in human airway epithelial cells (NCI-H292 cells). The apoptotic events posttransfection were examined by a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end-labeling assay, flow cytometric measurement of propidium iodide, annexin V staining, and Western blot analyses with antibodies specific for proapoptotic and antiapoptotic proteins. We demonstrated that the expression of H5N1 NS1 protein in NCI-H292 cells was sufficient to induce apoptotic cell death. Western blot analyses also showed that there was prominent cleavage of poly(ADP-ribose) polymerase and activation of caspase-3, caspase-7, and caspase-8 during the NS1-induced apoptosis. The results of caspase inhibitor assays further confirmed the involvement of caspase-dependent pathways in the NS1-induced apoptosis. Interestingly, the ability of H5N1 NS1 protein to induce apoptosis was much enhanced in cells pretreated with Fas ligand (the time posttransfection required to reach >30% apoptosis was reduced from 24 to 6 h). Furthermore, 24 h posttransfection, an increase in Fas ligand mRNA expression of about 5.6-fold was detected in cells transfected with H5N1 NS1. In conclusion, we demonstrated that the NS1 protein encoded by avian influenza A virus H5N1 induced apoptosis in human lung epithelial cells, mainly via the caspase-dependent pathway, which encourages further investigation into the potential for the NS1 protein to be a novel therapeutic target.     

A型流感病毒NS1基因密码子去优化改造引起病毒毒力减弱

根据A型流感病毒密码子使用偏嗜性,选取稀有密码子对A/Puerto Rico/8/34(H1N1)病毒NS1基因内部110个氨基酸区域进行密码子同义突变改造,并全基因合成NS基因,利用反向遗传操作技术拯救出含有密码子去优化NS1基因的重组病毒(deoNS)。体外细胞噬斑形成实验和病毒生长曲线证明该病毒在MDCK细胞内的感染和复制能力比野生型病毒低约1000倍;BALB/c小鼠体内致病力实验证明deoNS病毒不能引起小鼠发病和死亡,该病毒在小鼠肺内的复制滴度比野生型病毒低100～1000倍。本研究探索了通过基因组密码子去优化改造途径降低A型流感病毒毒力的可行性,首次证明流感病毒NS1基因密码子去优化同义突变可以降低病毒毒力,为流感减毒活疫苗的研究提供了新的思路。     

Non structural protein of avian influenza A (H11N1) virus is a weaker suppressor of immune responses but capable of inducing apoptosis in host cells

ABSTRACT: BACKGROUND: The Non-Structural (NS1) protein of Influenza A viruses is an extensively studied multifunctional protein which is commonly considered as key viral component to fight against host immune responses. Even though there has been a lot of studies on the involvement of NS1 protein in host immune responses there are still ambiguities regarding its role in apoptosis in infected cells. Interactions of NS1 protein with host factors, role of NS1 protein in regulating cellular responses and apoptosis are quiet complicated and further studies are still needed to understand it completely. RESULTS: NS1 genes of influenza A/Chicken/India/WBNIV2664/2008 (H5N1) and A/Aquatic bird/India/NIV-17095/2007(H11N1) were cloned and expressed in Human embryonic kidney (293T) cells. Microarray based approach to study the host cellular responses to NS1 protein of the two influenza A viruses of different pathogenicity showed significant differences in the host gene expression profile. NS1 protein of H5N1 resulted in suppression of IFN-beta mediated innate immune responses in 293T cells, leading to down-regulation of the components of JAK-STAT pathway like STAT1 which further suppressed the expression of pro-inflammatory cytokines like CXCL10 and CCL5. The degree of suppression of host immune genes was found considerable with NS1 protein of H11N1 but was not as prominent as with H5N1-NS1. TUNEL assay analyses were found to be positive in both the NS1 transfected cells indicating both H5N1 as well as H11N1 NS1 proteins were able to induce apoptosis in transfected cells. CONCLUSIONS: We propose that NS1 protein of both H5N1 and H11N1 subtypes of influenza viruses are capable of influencing host immune responses and possess necessary functionality to support apoptosis in host cells. H11N1, a low pathogenic virus without any proven evidence to infect mammals, contains a highly potential NS1 gene which might contribute to greater virus virulence in different gene combinations.     

Website for avian flu information and bioinformatics

Highly pathogenic influenza A virus H5N1 has spread out worldwide and raised the public concerns. This increased the output of influenza virus sequence data as well as the research publication and other reports. In order to fight against H5N1 avian flu in a comprehensive way, we designed and started to set up the Website for Avian Flu Information () from 2004. Other than the influenza virus database available, the website is aiming to integrate diversified information for both researchers and the public. From 2004 to 2009, we collected information from all aspects, i.e. reports of outbreaks, scientific publications and editorials, policies for prevention, medicines and vaccines, clinic and diagnosis. Except for publications, all information is in Chinese. Till April 15, 2009, the cumulative news entries had been over 2000 and research papers were approaching 5000. By using the curated data from Influenza Virus Resource, we have set up an influenza virus sequence database and a bioinformatic platform, providing the basic functions for the sequence analysis of influenza virus. We will focus on the collection of experimental data and results as well as the integration of the data from the geological information system and avian influenza epidemiology. Contributed equally to this work Special Project of Informatization of Chinese Academy of Sciences in “the Eleventh Five-Year Plan”, E-Science Application of Research on Resources, Disease Monitoring and Risk Assessment of Important Wild Birds in Qinghai Lake Region (Grant No. INFO-115-D02), China International Science and Technology Cooperation of the Ministry of Science and Technology, China (MOST) (Grant Nos. 2006DFB32010 and 2007DFC30240), MOST Grant 2005CB523001 (Program 973), National Key Technologies Research & Development Program (Grant 2006BAD06A01), and a grant from NIH (Grant No. 3 U19 AI051915-05S1), the China Post-doctor fellowship (Grant No. 20070420542).     

Using yeast two-hybrid system to detect interactions of ATP synthase subunits from Spinacia oleracea

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding α, β, γ, δ and ε subunits ofSpinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding β-galactosidase was detected. Of all the combinations, that of γ and ε subunit genes showed the highest level of reporter gene expression, while those of α and β, a and ε, β and ε and β and δ induced stable and significant reporter gene expression. The combination of δ and ε as well as that of δ and γ induced weak and unstable reporter gene expression. However, combinations of α and γ, β and γ and α and δ did not induce reporter gene expression. These results suggested that specific and strong interactions between γ and ε, α and β, α and ε, β and ε and β and δ subunits, and weak and transient interactions between δ and ε and δ and γ subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into the structural change of ATP synthase during catalysis.     

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,利用免疫共沉淀技术验证二者之间的相互作用.结果 pGBKT7-PML-C诱饵蛋白质粒和pACT2-GINS2靶蛋白质粒共转化AH109酵母菌后,可见蓝色阳性克隆生长;pCMV-HA-PML-C及pCMV-Myc-GINS2真核表达载体构建成功,共转染293细胞,抗HA多克隆抗体沉淀与HA-PML-C相互作用的蛋白复合物后,用抗Myc单克隆抗体进行Western印迹检测,可以检测到Myc-GINS2蛋白.结论 利用酵母双杂交和免疫共沉淀技术在胞内验证了PML-C与GINS2间存在相互作用.     

Using yeast two-hybrid system to detect interactions of ATP synthase subunits fromSpinacia oleracea

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding α, β, γ, δ and ε subunits ofSpinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding β-galactosidase was detected. Of all the combinations, that of γ and ε subunit genes showed the highest level of reporter gene expression, while those of α and β, a and ε, β and ε and β and δ induced stable and significant reporter gene expression. The combination of δ and ε as well as that of δ and γ induced weak and unstable reporter gene expression. However, combinations of α and γ, β and γ and α and δ did not induce reporter gene expression. These results suggested that specific and strong interactions between γ and ε, α and β, α and ε, β and ε and β and δ subunits, and weak and transient interactions between δ and ε and δ and γ subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into the structural change of ATP synthase during catalysis.     

Cross Protection against Influenza A Virus by Yeast-Expressed Heterologous Tandem Repeat M2 Extracellular Proteins

The influenza M2 ectodomain (M2e) is well conserved across human influenza A subtypes, but there are few residue changes among avian and swine origin influenza A viruses. We expressed a tandem repeat construct of heterologous M2e sequences (M2e5x) derived from human, swine, and avian origin influenza A viruses using the yeast expression system. Intramuscular immunization of mice with AS04-adjuvanted M2e5x protein vaccines was effective in inducing M2e-specific antibodies reactive to M2e peptide and native M2 proteins on the infected cells with human, swine, or avian influenza virus, mucosal and systemic memory cellular immune responses, and cross-protection against H3N2 virus. Importantly, M2e5x immune sera were found to confer protection against different subtypes of H1N1 and H5N1 influenza A viruses in naïve mice. Also, M2e5x-immune complexes of virus-infected cells stimulated macrophages to secrete cytokines via Fc receptors, indicating a possible mechanism of protection. The present study provides evidence that M2e5x proteins produced in yeast cells could be developed as a potential universal influenza vaccine.