A potyvirus-based gene vector allows producing active human S-COMT and animal GFP, but not human sorcin, in vector-infected plants

Potato virus A (PVA), a potyvirus with a (+)ssRNA genome translated to a large polyprotein, was engineered and used as a gene vector for expression of heterologous proteins in plants. Foreign genes including jellyfish GFP (Aequorea victoria) encoding the green fluorescent protein (GFP, 27 kDa) and the genes of human origin (Homo sapiens) encoding a soluble resistance-related calcium-binding protein (sorcin, 22 kDa) and the catechol-O-methyltransferase (S-COMT; 25 kDa) were cloned between the cistrons for the viral replicase and coat protein (CP). The inserts caused no adverse effects on viral infectivity and virulence, and the inserted sequences remained intact in progeny viruses in the systemically infected leaves. The heterologous proteins were released from the viral polyprotein following cleavage by the main viral proteinase, NIa, at engineered proteolytic processing sites flanking the insert. Active GFP, as indicated by green fluorescence, and S-COMT with high levels of enzymatic activity were produced. In contrast, no sorcin was detected despite the expected equimolar amounts of the foreign and viral proteins being expressed as a polyprotein. These data reveal inherent differences between heterologous proteins in their suitability for production in plants.     

Development of a new tobamovirus-based viral vector for protein expression in plants

Plants are becoming an interesting alternative system for the heterologous production of pharmaceutical proteins, providing a more scalable, cost-effective, and biologically safer option than the current expression systems. The development of plant virus expression vectors has allowed rapid and high-level transient expression of recombinant genes, and, in turn, provided an attractive plant-based production platform. Here we report the development of vectors based on the tobamovirus Pepper mild mottle virus (PMMoV) to be used in transient expression of foreign genes. In this PMMoV vector, a middle part of the viral coat protein gene was replaced by the green fluorescent protein (GFP) gene, and this recombinant genome was assembled in a binary vector suitable for plant agroinoculation. The accumulation of GFP was evaluated by observation of green fluorescent signals under UV light and by western blotting. Furthermore, by using this vector, the multiepitope gene for chikungunya virus was successfully expressed and confirmed by western blotting. This PMMoV-based vector represents an alternative system for a high-level production of heterologous protein in plants.

     

Analysis in human immunodeficiency virus type 1 vectors of cis-acting sequences that affect gene transfer into human lymphocytes.

Human immunodeficiency virus type 1 (HIV-1) can be used to generate recombinant viral vectors for delivery of heterologous genes to human CD4-positive lymphocytes. To define the cis-acting sequences required for efficient gene transfer, a number of HIV-1 vectors containing a previously identified packaging signal, long terminal repeats, and additional gag, pol, and env viral sequences were designed. By providing the viral proteins in trans, recombinant viruses were generated and analyzed for their abilities to transfer genes into human T lymphocytes. Inclusion of up to 653 nucleotides derived from the 5' end of the gag gene in the vector improved the efficiency of gene transfer, but inclusion of additional gag or pol sequences did not further improve this efficiency. The increased efficiency of gene transfer associated with the inclusion of 5' gag sequences in the vector arose, at least in part, from an increase in the packaging of vector RNA. The presence of the Rev-responsive element (RRE) increased the efficiency of transfer of vectors containing significant lengths of gag sequence, as expected from the Rev requirement for nucleus-to-cytoplasm transport of unspliced vector RNA containing intact packaging signals. However, the presence of a RRE did not affect the transfer efficiency of smaller vectors lacking significant lengths of gag sequences, arguing against a specific role for the RRE in packaging or vector transfer. These results contribute to an understanding of the minimal cis-acting sequences that operate in the context of HIV-1 vectors for delivering genes into human lymphocytes.     

Transient expression of<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> ascorbate peroxidase 3 in<Emphasis Type="Italic"> Nicotiana benthamiana</Emphasis> plants infected with recombinant potato virus X

We have explored the transient over-expression of Arabidopsis thaliana ascorbate peroxidase 3 (APX3) in Nicotiana benthamiana using a viral vector based on the potato virus X (PVX). Plants infected with a PVX:APX3 hybrid had a similar progression of viral particles compared to control plants infected with a PVX:GFP hybrid, indicating that infection was not affected by the over-expression of heterologous APX3. Our results also showed that in PVX:APX3-infected plants, the hybrid virus directed a high level of APX3 expression and the recombinant protein was functional, as inferred from the higher APX activity compared to mock and PVX:GFP hybrid-infected plants. The PVX recombinant expression system used is a simple and quick method for transient expression of heterologous APXs, which are expected to suffer specific processing in plant cells.     

SV40PolyA顺式活化基因元件中不完整茎环结构的发现和序列研究

研究室的前期工作发现,Alu串连序列插入pEGFP-C1质粒的GFP基因下游,瞬时转染HeLa细胞抑制GFP基因表达,2F2R(来自SV40PolyA反序5′端的第2个60 bp)插入GFP和Alu串连序列之间可以解除Alu序列对GFP基因的抑制作用。文章通过删减2F2R发现,45R(2F2R 5′端的45 bp)、30R和22R可以活化基因,且二串连体活化基因作用高于单体。Secloop(2F2R近中部的22 bp)和Poly4(2F2R 3′端的30 bp)不能活化基因。30R与Poly4用9碱基连接形成30R-Poly4,其活化基因作用低于2F2R,两个22R之间连接碱基数对活化GFP基因作用没有明显的影响。22R(5′-GTGAAAAAAATGCTTTATTTGT-3′)含有不完整的回文序列,可以形成不完整的茎环结构,包括一个3碱基loop、3 bp第一茎、2碱基泡和3 bp第二茎。改变22R茎环结构的碱基突变明显影响其活化GFP基因的作用,过多互补和过少互补的茎环结构均不利于活化基因,提示适当的不完整茎环结构与活化基因有关。     

Generation of an influenza A virus vector expressing biologically active human interleukin-2 from the NS gene segment

Engineering of the influenza A virus NS1 protein became an attractive approach to the development of influenza vaccine vectors since it can tolerate large inserts of foreign proteins. However, influenza virus vectors expressing long foreign sequences from the NS1 open reading frame (ORF) are usually replication deficient in animals due to the abrogation of their NS1 protein function. In this study, we describe a bicistronic expression strategy based on the insertion of an overlapping UAAUG stop-start codon cassette into the NS gene, allowing the reinitiation of translation of a foreign sequence. Although the expression level of green fluorescent protein (GFP) from the newly created reading frame was significantly lower than that obtained previously from an influenza virus vector expressing GFP from the NS1 ORF, the bicistronic vector appeared to be replication competent in mice and showed outstanding genetic stability. All viral isolates derived from mouse lungs at 10 days postinfection were still capable of expressing GFP in infected cells. Utilizing this bicistronic approach, we constructed another recombinant influenza virus, allowing the secretion of biologically active human interleukin-2 (IL-2). Although this virus also replicated to high titers in mouse lungs, it did not display any mortality rate in infected animals, in contrast to control viruses. Moreover, the IL-2-expressing virus showed an enhanced CD8+ response to viral antigens in mice after a single intranasal immunization. These results indicate that influenza viruses could be engineered for the expression of biologically active molecules such as cytokines for immune modulation purposes.     

An Attenuated Coxsackievirus B3 Vector: A Potential Tool for Viral Tracking Study and Gene Delivery

Cardiomyocytes are quite resistant to gene transfer using standard techniques. We developed an expression vector carrying an attenuated but infectious and replicative coxsackievirus B3 (CVB3) genome, and unique ClaI-StuI cloning sites for an exogenous gene, whose product can be released from the nascent viral polyprotein by 2A^pro cleavage. This vector was tested as an expression vehicle for green fluorescent protein (GFP). The vector transiently expressed GFP in cell cultures for at least ten passages and delivered functional GFP to the infected cardiomyocytes for at least 6 days. Moreover, the recombinant viruses showed virulence attenuation in vitro and in vivo. The findings suggest that the recombinant CVB3 vector could be a useful tool for viral tracking study and delivering exogenous proteins to cardiomyocytes.     

GFP工程酵母菌的构建及其对Cu2+的荧光响应

从酿酒酵母基因组DNA中克隆到金属硫蛋白启动子(PCUP1)片段,将绿色荧光蛋白(GFP)基因置于PCUP1的调控下,构建重组质粒pCUP9K-GFP,并通过氯化锂法转化毕赤酵母,获得工程菌株。工程菌细胞及其发酵液中可检出GFP荧光,表明PCUP1能启动外源基因GFP转录,使工程菌表达并分泌GFP。研究发现,工程菌培养液中分别加入10μmol/L的铜、铬、镉和砷离子后,铜处理组GFP荧光强度明显增加,其余三种离子对工程菌荧光强度影响不大;用铜离子诱导后,工程菌发酵上清液的荧光强度明显增强,并与铜离子浓度(0～1mmol/L)呈正相关。研究表明,该工程菌中启动子PCUP1受铜离子诱导,GFP的表达对铜离子具有剂量依赖性,在一定浓度范围内,GFP荧光强度与铜离子浓度呈正相关。     

羊BLG基因5′和3′调控区克隆及其调控GFP基因在乳腺细胞的表达

乳球蛋白 (ＢＬＧ)是反刍家畜的一种主要乳清蛋白质。将外源基因置于ＢＬＧ 5′调控区下游 ,能够控制外源基因在动物乳腺的组织特异性表达。现已清楚在ＢＬＧ 5′端有多个乳核因子 1(ＮＦ1)和转录因子Ｓｔａｔ5的识别位点 ,此外还存在多个激素作用位点[1 ] 。具有这些调控成分的ＢＬＧ 5′翼端 (5′ｆｌａｎｋｉｎｇ)能够控制外源基因在乳腺的表达 ,但受整合位点、外源基因结构以及转基因拷贝数及其排列方式的影响[2 ] 。研究结果表明 ,仅含有 5′调控区的乳腺表达构件 ,还不能使外源基因的表达达到内源性ＢＬＧ的表达水平 ,因为在 5′远端(…     

Improved primate foamy virus vectors and packaging constructs

Foamy virus (FV) vectors that have minimal cis-acting sequences and are devoid of residual viral gene expression were constructed and analyzed by using a packaging system based on transient cotransfection of vector and different packaging plasmids. Previous studies indicated (i) that FV gag gene expression requires the presence of the R region of the long terminal repeat and (ii) that RNA from packaging constructs is efficiently incorporated into vector particles. Mutants with changes in major 5' splice donor (SD) site located in the R region identified this sequence element as responsible for regulating gag gene expression by an unidentified mechanism. Replacement of the FV 5' SD with heterologous splice sites enabled expression of the gag and pol genes. The incorporation of nonvector RNA into vector particles could be reduced to barely detectable levels with constructs in which the human immunodeficiency virus 5' SD or an unrelated intron sequence was substituted for the FV 5' untranslated region and in which gag expression and pol expression were separated on two different plasmids. By this strategy, efficient vector transfer was achieved with constructs that have minimal genetic overlap.     

Transmissible Gastroenteritis Coronavirus Genome Packaging Signal Is Located at the 5′ End of the Genome and Promotes Viral RNA Incorporation into Virions in a Replication-Independent Process

Preferential RNA packaging in coronaviruses involves the recognition of viral genomic RNA, a crucial process for viral particle morphogenesis mediated by RNA-specific sequences, known as packaging signals. An essential packaging signal component of transmissible gastroenteritis coronavirus (TGEV) has been further delimited to the first 598 nucleotides (nt) from the 5′ end of its RNA genome, by using recombinant viruses transcribing subgenomic mRNA that included potential packaging signals. The integrity of the entire sequence domain was necessary because deletion of any of the five structural motifs defined within this region abrogated specific packaging of this viral RNA. One of these RNA motifs was the stem-loop SL5, a highly conserved motif in coronaviruses located at nucleotide positions 106 to 136. Partial deletion or point mutations within this motif also abrogated packaging. Using TGEV-derived defective minigenomes replicated in trans by a helper virus, we have shown that TGEV RNA packaging is a replication-independent process. Furthermore, the last 494 nt of the genomic 3′ end were not essential for packaging, although this region increased packaging efficiency. TGEV RNA sequences identified as necessary for viral genome packaging were not sufficient to direct packaging of a heterologous sequence derived from the green fluorescent protein gene. These results indicated that TGEV genome packaging is a complex process involving many factors in addition to the identified RNA packaging signal. The identification of well-defined RNA motifs within the TGEV RNA genome that are essential for packaging will be useful for designing packaging-deficient biosafe coronavirus-derived vectors and providing new targets for antiviral therapies.     

Evaluation of baculovirus expression vectors with enhanced stability in continuous cascaded insect-cell bioreactors

Continuous protein production with baculovirus expression vectors in insect-cell bioreactors is characterized by a dramatic drop in heterologous protein production within a few weeks. This is mainly due to the spontaneous deletion of the heterologous gene(s) from the baculovirus genome and/or to the rapid accumulation of defective interfering baculoviruses (DIs). Cell culture experiments with bacmid-derived baculoviruses showed that spontaneous deletions in the foreign bacterial artificial chromosome (BAC) sequences readily occurred. These deletions correlated with a low density of baculovirus homologous (repeat) regions (hrs), which are located dispersed throughout the baculovirus genome and are believed to act as origins of viral DNA replication (oris). To test the hypothesis that deletions are more likely to occur in regions with a low ori density, the properties of bacmid-derived baculoviruses with an additional hr in the unstable BAC sequences were compared to the standard bacmid-derived baculovirus in a continuous cascaded insect-cell bioreactor configuration. All viruses were equipped with a green fluorescent protein (GFP) gene and a gene encoding the classical swine fever virus E2 glycoprotein (CSFV-E2). The insertion of an extra hr in the BAC vector led to improved genetic stability of adjacent sequences, resulting in prolonged protein expression. The maintenance of the BAC sequences appeared to be dependent on the orientation of the inserted hr. The advantages of the utilization of hrs to improve the stability of baculovirus expression vectors for the large-scale protein production in insect-cell bioreactors are discussed.     

高通量筛选雌激素类化合物重组绿色荧光蛋白酵母细胞测评体系

以载体双表达的方式构建重组酵母环境雌激素的评价体系, 用于快速筛选雌激素类化合物。在表达载体中, 用3-磷酸甘油醛脱氢酶(GPD)启动子驱动a人雌激素受体基因(hERa)的表达; 在报告载体中, 用雌激素效应元件(ERE)调控的绿色荧光蛋白(yEGFP)作为报告基因。将两者转化于酵母细胞(W303-1A)中, 构建成重组绿色荧光蛋白酵母细胞。该酵母细胞经不同浓度的雌激素类化合物作用后, 发现GFP的表达量与此类受试物具有明显的剂量效应关系。与其他环境雌激素酵母评价体系相比, 该重组酵母评价细胞, 在应用时不需要破坏细胞壁, 也不需要底物和相关试剂, 可直接在96孔板中操作完成, 具有快速、高通量、敏感性高、重现性好及廉价等特点。