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1.
Identification of a human immunodominant B-cell epitope within the GRA1 antigen of Toxoplasma gondii by phage display of cDNA libraries. 总被引:16,自引:0,他引:16
Elisa Beghetto Andrea Pucci Olga Minenkova Andrea Spadoni Luca Bruno Wilma Buffolano Dominique Soldati Franco Felici Nicola Gargano 《International journal for parasitology》2001,31(14):1659-1668
Excreted secreted antigens of the protozoan parasite Toxoplasma gondii play a key role in stimulating the host immune system during acute and chronic infection. With the aim of identifying the immunodominant epitopes of T. gondii antigens involved in the human B-cell response against the parasite, we employed a novel immunological approach. A library of cDNA fragments from T. gondii tachyzoites was displayed as fusion proteins to the amino-terminus of lambda bacteriophage capsid protein D. The lambdaD-tachyzoite library was then affinity-selected by using a panel of sera of pregnant women, all infected with the parasite. Some of the clones identified through this procedure matched the sequence of the dense granule GRA1 protein (p24), allowing us to identify its antigenic regions. In particular, the analysis of human antibody response against the recombinant GRA1 antigen fragments revealed the existence of an immunodominant epitope (epi-24 peptide). 相似文献
2.
Svensson J Andersson C Reseland JE Lyngstadaas P Bülow L 《Protein expression and purification》2006,48(1):134-141
Amelogenin is a dental enamel matrix protein involved in formation of dental enamel. In this study, we have expressed two different recombinant murine amelogenins in Escherichia coli: the untagged rM179, and the histidine tagged rp(H)M180, identical to rM179 except that it carries the additional N-terminal sequence MRGSHHHHHHGS. The effects of the histidine tag on expression levels, and on growth properties of the amelogenin expressing cells were studied. Purification of a crude protein extract containing rp(H)M180 was also carried out using IMAC and reverse-phase HPLC. The results of this study showed clearly that both growth properties and amelogenin expression levels were improved for E. coli cells expressing the histidine tagged amelogenin rp(H)M180, compared to cells expressing the untagged amelogenin rM179. The positive effect of the histidine tag on amelogenin expression is proposed to be due to the hydrophilic nature of the histidine tag, generating a more hydrophilic amelogenin, which is more compatible with the host cell. Human osteoblasts treated with the purified rp(H)M180 showed increased levels of secreted osteocalcin, compared to untreated cells. This response was similar to cells treated with enamel matrix derivate, mainly composed by amelogenin, suggesting that the recombinant protein is biologically active. Thus, the histidine tag favors expression and purification of biologically active recombinant amelogenin. 相似文献
3.
Elisa Beghetto Andrea Pucci Olga Minenkova Andrea Spadoni Luca Bruno Wilma Buffolano Dominique Soldati Franco Felici Nicola Gargano 《International journal for parasitology》2001,31(14)
Excreted secreted antigens of the protozoan parasite Toxoplasma gondii play a key role in stimulating the host immune system during acute and chronic infection. With the aim of identifying the immunodominant epitopes of T. gondii antigens involved in the human B-cell response against the parasite, we employed a novel immunological approach. A library of cDNA fragments from T. gondii tachyzoites was displayed as fusion proteins to the amino-terminus of lambda bacteriophage capsid protein D. The lambdaD-tachyzoite library was then affinity-selected by using a panel of sera of pregnant women, all infected with the parasite. Some of the clones identified through this procedure matched the sequence of the dense granule GRA1 protein (p24), allowing us to identify its antigenic regions. In particular, the analysis of human antibody response against the recombinant GRA1 antigen fragments revealed the existence of an immunodominant epitope (epi-24 peptide). 相似文献
4.
重组HIV-1壳体蛋白在转基因枸杞根系中的分泌表达 总被引:7,自引:1,他引:7
P24壳体蛋白(capsid, CA)是HIV¬-1早期感染的一个重要标志.用含有植物表达载体pCAMBIA 1305.2- MA4-CA(包含GRP信号肽和MA4-CA融合基因)的农杆菌菌株侵染枸杞,将转有MA4-CA融合基因的转化株诱导生根,并进行毛状根的培养; western blot证实根系及培养液中的MA4-CA融合蛋白以二聚体的形式存在,分子量为50 kDa;免疫组织化学显示,CA定位在细胞浆、细胞壁和细胞间隙中,充分证实了利用GRP信号肽可以引导重组蛋白分泌表达。建立枸杞中HIV-1壳体蛋白的根分泌表达系统,为研究植物HIV-1 CA-病毒样颗粒(VLPs)疫苗奠定基础。 相似文献
5.
H J Ehrlich S R Jaskunas B W Grinnell S B Yan N U Bang 《The Journal of biological chemistry》1989,264(24):14298-14304
Human protein C, like other serine proteases, is normally secreted as an inactive zymogen. It is converted to its active form extracellularly by limited proteolysis with the thrombin-thrombomodulin complex. This activation results from the removal of a 12-residue activation peptide from the NH2 terminus of the heavy (COOH-terminal) chain. We report here a successful strategy for the activation of human protein C during post-translational cellular processing, resulting in the secretion of activated protein C from transfected mammalian cells. Deletion of the nucleotides encoding the activation peptide resulted in the expression of a protease with less than 5% of the expected activity. However, the replacement of the activation peptide with an 8-residue sequence (Pro-Arg-Pro-Ser-Arg-Lys-Arg-Arg) involved in the proteolytic processing of the human insulin receptor precursor resulted in the direct expression of fully activated protein C. The mutant protein was shown to be correctly processed by NH2-terminal sequence analysis. This strategy for successful expression of an activated form of protein C may apply to the expression of active forms of other proteases which are naturally expressed as zymogens. 相似文献
6.
Küttner G Giessmann E Wessner H Scholz C Reinhardt D Winkler K Marx U Höhne W 《BioTechniques》2004,36(5):864-870
The peptide tag GATPQDLNTML, corresponding to amino acids 46-56 of the human immunodeficiency virus type 1 (HIV-1) capsid protein p24, is the linear epitope of the murine monoclonal antibody CB4-1. This antibody shows high affinity (KD = 1.8 x 10(-8) M) to the free epitope peptide in solution. The original p24 peptide tag and mutant derivatives were fused to the C terminus of a single-chain antibody (scFv) and characterized with respect to sensitivity in Western blot analyses and behavior in purification procedures using affinity chromatography. The p24 tag also proved to be a suitable alternative to the (Gly4Ser)3 linker commonly used to connect single-chain antibody variable regions derived from a heavy (VH) and light chain (VL). Binding of CB4-1 antibody to the p24 tag was not hampered when the tag was located internally in the protein sequence, and the specific antigen affinity of the scFv was only slightly reduced. All scFv variants were solubly expressed in Escherichia coli and could be purified from the periplasm. Our results highlight the p24 tag as a useful tool for purifying and detecting recombinantly expressed scFvs. 相似文献
7.
Vauloup-Fellous C Pène V Garaud-Aunis J Harper F Bardin S Suire Y Pichard E Schmitt A Sogni P Pierron G Briand P Rosenberg AR 《The Journal of biological chemistry》2006,281(38):27679-27692
The capsid of hepatitis C virus (HCV) particles is considered to be composed of the mature form (p21) of core protein. Maturation to p21 involves cleavage of the transmembrane domain of the precursor form (p23) of core protein by signal peptide peptidase (SPP), a cellular protease embedded in the endoplasmic reticulum membrane. Here we have addressed whether SPP-catalyzed maturation to p21 is a prerequisite for HCV particle morphogenesis in the endoplasmic reticulum. HCV structural proteins were expressed by using recombinant Semliki Forest virus replicon in mammalian cells or recombinant baculovirus in insect cells, because these systems have been shown to allow the visualization of HCV budding events and the isolation of HCV-like particles, respectively. Inhibition of SPP-catalyzed cleavage of core protein by either an SPP inhibitor or HCV core mutations not only did not prevent but instead tended to facilitate the observation of viral buds and the recovery of virus-like particles. Remarkably, although maturation to p21 was only partially inhibited by mutations in insect cells, p23 was the only form of core protein found in HCV-like particles. Finally, newly developed assays demonstrated that p23 capsids are more stable than p21 capsids. These results show that SPP-catalyzed cleavage of core protein is dispensable for HCV budding but decreases the stability of the viral capsid. We propose a model in which p23 is the form of HCV core protein committed to virus assembly, and cleavage by SPP occurs during and/or after virus budding to predispose the capsid to subsequent disassembly in a new cell. 相似文献
8.
Viral incorporation of cyclophilin A (CyPA) during the assembly of human immunodeficiency virus type-1 (HIV-1) is crucial for efficient viral replication. CyPA binds to the previously identified Gly-Pro90 site of the capsid protein p24, but its role remained unclear. Here we report two new interaction sites between cyclophilins and p24. Both are located in the C-terminal domain of p24 around Gly-Pro157 and Gly-Pro224. Peptides corresponding to these regions showed higher affinities (Kd approximately 0.3 microM) for both CyPA and cyclophilin B than the best peptide derived from the Gly-Pro90 site ( approximately 8 microM) and thus revealed new sequence motifs flanking Gly-Pro that are important for tight interaction of peptide ligands with cyclophilins. Between CyPA and an immature (unprocessed) form of p24, a Kd of approximately 8 microM was measured, which corresponded with the Kd of the best of the Gly-Pro90 peptides, indicating an association via this site. Processing of immature p24 by the viral protease, yielding mature p24, elicited a conformational change in its C-terminal domain that was signaled by the covalently attached fluorescence label acrylodan. Consequently, CyPA and cyclophilin B bound with much higher affinities ( approximately 0.6 and 0.25 microM) to the new, i.e. maturation-generated sites. Since this domain is essential for p24 oligomerization and capsid cone formation, CyPA bound to the new sites might impair the regularity of the capsid cone and thus facilitate in vivo core disassembly after host infection. 相似文献
9.
Jean L Hackett F Martin SR Blackman MJ 《The Journal of biological chemistry》2003,278(31):28572-28579
Erythrocyte invasion by the malaria merozoite is prevented by serine protease inhibitors. Various aspects of the biology of Plasmodium falciparum subtilisin-like protease-1 (PfSUB-1), including the timing of its expression and its apical location in the merozoite, suggest that this enzyme is involved in invasion. Recombinant PfSUB-1 expressed in a baculovirus system is secreted in the p54 form, noncovalently bound to its cognate propeptide, p31. To understand the role of p31 in PfSUB-1 maturation, we examined interactions between p31 and both recombinant and native enzymes. CD analyses revealed that recombinant p31 (rp31) possesses significant secondary structure on its own, comparable with that of folded propeptides of some bacterial subtilisins. Kinetic studies demonstrated that rp31 is a fast binding, high affinity inhibitor of PfSUB-1. Inhibition of two bacterial subtilisins by rp31 was much less effective, with inhibition constants 49-60-fold higher than that for PfSUB-1. Single (at the P4 or P1 position) or double (at P4 and P1 positions) point mutations of residues within the C-terminal region of rp31 had little effect on its inhibitory activity, and truncation of 11 residues from the rp31 C terminus substantially reduced, but did not abolish, inhibition. None of these modifications prevented binding to the PfSUB-1 catalytic domain or rendered the propeptide susceptible to proteolytic digestion by PfSUB-1. These studies provide new insights into the function of the propeptide in PfSUB-1 activation and shed light on the structural requirements for interaction with the catalytic domain. 相似文献
10.
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12.
Processing of the intracellular form of the west Nile virus capsid protein by the viral NS2B-NS3 protease: an in vitro study. 总被引:8,自引:7,他引:1
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According to the existing model of flavivirus polyprotein processing, one of the cleavages in the amino-terminal part of the flavivirus polyprotein by host cell signalases results in formation of prM (precursor to one of the structural proteins, M) and the membrane-bound intracellular form of the viral capsid protein (Cint) retaining the prM signal sequence at its carboxy terminus. This hydrophobic anchor is subsequently removed by the viral protease, resulting in formation of the mature viral capsid protein found in virions (Cvir). We have prepared in vitro expression cassettes coding for both forms of the capsid protein, for the prM protein, for the C-prM precursor, and for the viral protease components of West Nile flavivirus and characterized their translation products. Using Cint and Cvir translation products as molecular markers, we have observed processing of the intracellular form of the West Nile capsid protein by the viral protease in vitro both upon cotranslation of the C-prM precursor and the viral protease-encoding cassette and by incubation of C-prM translation products with a detergent-solubilized extract of cells infected with a recombinant vaccinia virus expressing the active viral protease. The cleavage of Cint by the viral protease at the predicted dibasic site was verified by introduction of point mutations into the cleavage site and an adjacent region. These studies provide the first direct demonstration of processing of the intracellular form of the flavivirus capsid protein by the viral protease. 相似文献
13.
Two-Helper RNA System for Production of Recombinant Semliki Forest Virus Particles 总被引:11,自引:6,他引:5
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Alphavirus expression systems based on suicidal virus particles carrying recombinant replicons have proven to be a very efficient way to deliver genes for heterologous protein expression. However, present strategies for production of such particles have biosafety limitations due to the generation, by RNA recombination, of replication-proficient viruses (RPVs). Here we describe a new packaging system for Semliki Forest virus (SFV) based on a the use of a two-helper system in which the capsid and spike proteins of the C-p62-6K-E1 polyprotein are expressed from two independent RNA molecules. The capsid gene contains a translational enhancer and therefore that sequence was also engineered in front of the spike sequence p62-6K-E1. A sequence coding for the foot-and-mouth disease virus 2A autoprotease was inserted in frame between the capsid translational enhancer and the spike genes. This allows production of the spike proteins at high levels with cotranslational removal of the enhancer sequence and normal biosynthesis of the spike complex. The autoprotease activity of the capsid protein was abolished by mutation, further increasing the biosafety of the system. Cotransfection of cells with both helper RNAs and an SFV vector replicon carrying the LacZ gene led to production of recombinant particles with titers of up to 8 × 108 particles per 106 cells. Extensive analysis failed to demonstrate the presence of any RPVs, emphasizing the high biosafety of the system based on two-helper RNAs. 相似文献
14.
A N Vzorov Iu Iu Tentsov V B Grigor'ev S A Shulenin M M Garaev O P Bogdan A G Bukrinskaia 《Molekuliarnaia biologiia》1990,24(6):1666-1674
Monkey kidney cells CV-1 were infected with recombinant vaccinia virus carrying HIV-1 gag gene with a deletion of 230 nucleotide pairs from the 3'-terminus. The main gene product detected in the lysates of infected cells was the gag precursor rp50. The protein was accumulated on the cell membranes suggesting that it had a myristylated N-terminus, and was cleaved by a recombinant virus specific protease with the formation of two proteins, p17 and p24 corresponding in molecular masses to mature gag proteins. Virus-like particles similar to immature HIV virions were budding from the surface of infected cells. They look like the ring of optically dense material covered with a lipid bilayer, of the same size (100-120 nm) and of the same density in a sucrose gradient (1.16-1.18 g/ml) as HIV-1 virions. The particles contained rp50 and cellular heterogeneous RNA. Thus, the unprocessed gag precursor with deleted 77 amino acid residues from the C-terminus is able to form virus-like particles in the absence of env proteins and virus-specific RNA, and these particles are budding from the cell surface. The question about the use of extracellular Gag-particles for AIDS diagnostic work and construction of vaccines is discussed. 相似文献
15.
共表达口蹄疫病毒衣壳前体蛋白P1-2A基因和蛋白酶3C基因牛肾细胞株的筛选及稳定性 总被引:2,自引:0,他引:2
[目的]筛选稳定表达口蹄疫病毒衣壳蛋白的牛肾细胞(Madin-Darby bovinekidney,MDBK)株.[方法]采用聚合酶链式反应(Polymerase chain reaction,PCR)方法从重组质粒pMD-P1-2A和pMD-3C中分别扩增口蹄疫病毒衣壳前体蛋白P1-2A基因和蛋白酶3C基因,将两基因依次插入逆转录病毒载体pBABE-puro.重组逆转录病毒载体pBABE-puro/P1-2A-3C和pVSV-G质粒载体用脂质体介导共转染GP2-293包装细胞.产生的重组逆转录病毒感染MDBK细胞后使用嘌呤霉素筛选抗性细胞.利用克隆环套取法得到单克隆细胞.经间接免疫荧光和酶联免疫吸附测定(Enzyme-linkedimmunosorbent assay,ELISA)方法检测MDBK细胞中衣壳蛋白的表达,并在电镜下观察口蹄疫病毒空衣壳.[结果]成功筛选到稳定表达口蹄疫病毒衣壳蛋白的MDBK细胞株,衣壳前体蛋白P1-2A在蛋白酶3C裂解作用下正确组装成空衣壳.[结论]该研究为口蹄疫亚单位疫苗的研制提供了实验材料. 相似文献
16.
Xiaoliang Liu Akemi Ota Michiko Watanabe Shigeharu Ueda Atsushi Saitoh Hideo Shinagawa Atsuo Nakata Takashi Kurimura Xiaoui Wang Yu Zhao Kiyoshi Kondo Jiro Seki Shinichi Miyake Nobuo Sakato Hajime Fujio 《Microbiology and immunology》1995,39(10):775-785
We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (KA = 1.9 × 105?1.4 × 108 M?1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (KA = 2.0 × 105?1.8 × 107 M?1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains. 相似文献
17.
Govinda S Kutlow T Bentivegna CS 《Journal of biochemical and molecular toxicology》2000,14(4):195-203
A putative ribosomal protein (rp) mRNA in Chironomus riparius has been found using differential display (DD). Its sequence has 84.8% identity with mosquito rp L8, Aedes albopictus, and is approximately 0.9 kb. Studies were undertaken in order to evaluate rp as a control for environmentally relevant genes. Responses of Drosophila heat shock 70 gene (hsp70) were used to establish heat shock temperatures and cadmium (Cd) concentrations for Chironomus experiments and to validate DD. Expression of hsp70 was induced over control by 28 degrees C at 30 minutes and 1 mM Cd at 24 hours (p< or =0.05). For Chironomus, DD, Northern blot, and nuclease sensitivity were used to measure responses to two stressors: heat shock for 30 minutes and Cd for 24 or 48 hours. Differential display and nuclease sensitivity assays found expression of rp mRNA at 37 degrees C and 16 mM Cd to be similar to controls. Northern blots indicated statistically significant effects for heat shock (p = 0.046) but not Cd (p = 0.406). However, mRNA levels at 37 degrees C were increased only 1.72-fold over controls. A concentration of 24 nM actinomycin D suppressed rp expression as measured by nuclease sensitivity assays. Stressors should not affect rp mRNA levels below their LC-50s. 相似文献
18.
Mussels Mytilus coruscus can adhere to various solid surface in the presence of moisture. Mussel foot protein-3 (mfp-3) has been suggested as the main adhesive protein in the plaques closest to the adhesion interface and been the focus of substantial biomaterials development research within the last decade. The byssal plaques of M. coruscus were accumulated and variants of a family known as mcofp3 (Mytilus coruscus foot protein 3) were purified from acetic acid/urea extracts of plaques, with their N-terminal sequences determined thereafter. The cDNA sequence coding for the mcofp3 precursor was obtained from M. coruscus foot cDNA library. These precursors contain a putative signal peptide of 24 residues, a mature peptide sequence of 41-56 amino acids rich in Tyr, Gly, Pro, and Asn. The recombinant mcofp3 fused with a hexa-histidine affinity ligand was successfully expressed through an Escherichia coli expression system, and the recombinant mcofp3 was purified using affinity chromatography followed by reverse phase high performance liquid chromatography (HPLC). The DOPA content and adhesive properties of purified recombinant mcofp3 with or without tyrosinase modification were compared with the native mcofp3. These assays showed that recombinant mcofp3 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments. 相似文献
19.
Using the cis-acting human cytomegalovirus (HCMV) packaging elements (pac 1 and pac 2) as DNA probes, specific DNA-protein complexes were detected by electrophoretic mobility shift assay (EMSA) in both HCMV-infected cell nuclear extracts and recombinant baculovirus-infected cell extracts containing the HCMV p130 (pUL56) protein. DNA-binding proteins, which were common in uninfected and infected cell extracts, were also detected. Mutational analysis showed that only the AT-rich core sequences in these cis-acting motifs, 5′-TAAAAA-3′ (pac 1) and 5′-TTTTAT-3′ (pac 2), were required for specific DNA-protein complex formation. The specificity of the DNA-protein complexes was confirmed by EMSA competition. Furthermore, a specific endonuclease activity was found to be associated with lysates of baculovirus-infected cells expressing recombinant p130 (rp130). This nuclease activity was time dependent, related to the amount of rp130 in the assay, and ATP independent. Nuclease activity remained associated with rp130 after partial purification by sucrose gradient centrifugation, suggesting that this activity is a property of HCMV p130. We propose a possible involvement of p130 in HCMV DNA packaging.Human cytomegalovirus (HCMV), one of eight human herpesviruses, can cause serious illness in neonates as well as in immunocompromised adults (2). For example, transplant and AIDS patients may develop life-threatening diseases as a consequence of primary infection or reactivation of latent infection. Present therapeutic approaches are limited, and new strategies that may result from a better understanding of the molecular events involved in viral maturation are needed.The HCMV virion consists of an envelope, an amorphous tegument, and an icosahedral nucleocapsid, which is assembled in the nuclei of infected cells. The precise molecular events of HCMV capsid assembly and subsequent DNA packaging are not well understood. It is generally accepted that viral DNA is packaged into a procapsid consisting of major capsid protein (UL86), minor capsid protein (UL85), minor capsid protein-binding protein (UL46), smallest capsid protein (UL47/48), assembly protein (UL80.5), and proteinase precursor protein (UL80a) (8). The assembly protein is removed during DNA insertion. It is unclear how the concatenated viral DNA contacts empty capsids and is cleaved and packaged into the capsid.Recent studies with herpes simplex virus type 1 (HSV-1) mutants that were temperature sensitive suggest that cleavage of the concatenated DNA does not occur in the absence of packaging (1). One possible model would be the involvement of cleavage packaging protein(s) which could facilitate incorporation of DNA into the procapsid by attaching to a specific motif within the viral genome. With HSV-1, the UL36 gene product (ICP1) and a smaller protein (possibly encoded by UL37) are part of a complex that recognizes the HSV-specific a sequence and are required for cleavage and packaging of viral DNA from concatemers (6, 7). In addition, the HSV-1 ICP 18.5 (UL28) gene product and the pseudorabies virus (PrV) homolog (16) were also reported to play an important role in DNA packaging (1, 14). Addison et al. (1) demonstrated that empty capsids were observed under conditions nonpermissive for the expression of the HSV-1 ICP 18.5 gene product. The HSV-1 ICP 18.5 mutants failed to cleave concatenated viral DNA in noncomplementing cells, suggesting that cleavage and packaging require ICP 18.5. Similar results were reported by Mettenleiter et al. (14) for PrV mutant protein. These observations suggest that the HSV-1 UL36, UL37, and UL28 gene products are involved in cleavage and packaging of concatenated viral DNA.In a recent study, we identified and partially characterized the gene product of HCMV UL56 (4). The HCMV UL56 gene product of 130 kDa is the homolog of the HSV-1 UL28 gene product. It is therefore postulated that UL56 possesses properties comparable to those of HSV-1 UL28, implying an involvement in cleavage and packaging of DNA. The HCMV genomic a sequence is a short sequence located at both termini of the genome and repeated in an inverted orientation at the L-S junction. The a sequence plays a key role in replication as a cis-acting signal for cleavage and packaging of progeny viral DNA and circularization of the viral genome. The HCMV a sequence contains two conserved motifs, pac 1 and pac 2, which are required for cleavage and packaging of the viral DNA (18). Both sequence motifs are located on one side of the cleavage site. The pac 1 and pac 2 motifs have an AT-rich core flanked by a GC-rich sequence. During the initial step of viral DNA packaging, a capsid-associated protein may bind to the pac sequences and may be involved in cleavage of the viral DNA concatemer.In this study, electrophoretic mobility shift assays (EMSAs) were performed with DNA probes spanning the region of these cis-acting elements. These studies demonstrate that specific proteins from HCMV-infected nuclear extracts or baculovirus-UL56-infected cell extracts bind to the pac motifs. Using affinity-purified monospecific antibodies, we show that p130 is present in specific DNA-protein complexes containing the pac motifs of the viral genome. Furthermore, evidence is presented for a sequence-specific endonuclease activity of recombinant HCMV p130, using circular plasmid DNA bearing the a sequence as a substrate. 相似文献
20.
构建并表达HIV-1 CAP2NC蛋白,探索其体外自组装条件。通过PCR技术扩增HIV-1(NL4-3毒株)CAP2NC基因片段,并将其连接到原核表达载体pTO-T7,获得重组质粒pTO-T7-CAP2NC,然后转化至大肠杆菌BL21(DE3)菌株,经疏水层析纯化后获得重组蛋白CAP2NC。SDS-PAGE结果表明,重组蛋白CAP2NC可在大肠杆菌可溶高效表达,经纯化后纯度约为95%。ELISA检测表明重组蛋白CAP2NC可被HIV-1衣壳蛋白特异性单克隆抗体识别,具有较好反应活性。重组蛋白透析后在非原性SDS-PAGE中呈现为多种聚体形式。分子筛排阻层析分析CAP2NC蛋白透析后可进行组装,负染电镜进一步观察显示CAP2NC蛋白在RNA存在条件下,可形成空心管状颗粒,其形态结构与HIV-1病毒衣壳体外自组装形成的类似。上述结果表明HIV-1 CAP2NC蛋白具有体外自组装的性质,为进一步在体外研究非成熟病毒样颗粒结构奠定基础。 相似文献