犬细小病毒VP2蛋白在真核细胞中的分泌表达及特性

摘要:【目的】利用真核细胞分泌表达犬细小病毒VP2蛋白和研究其特性。【方法】为构建犬细小病毒（Canine parvovirus, CPV）VP2基因的真核分泌型表达载体,首先通过酶切从含有人CD5信号肽序列的质粒中将CD5信号肽基因片段切出,将其连接到真核表达载体pcDNA3.1A的多克隆位点上,构建成pcDNA3.1-CD5sp质粒。然后再通过PCR方法从含有犬细小病毒VP2基因的质粒中扩增VP2基因,并将其插入到pcDNA3.1- CD5sp载体中CD5信号肽的下游,构建成VP2基因的真核分泌型表达载体pcDNA-CD5sp-VP2。经磷酸钙介导转染293T细胞,使其在真核细胞中进行分泌表达,并通过ELISA检测表达的VP2蛋白与犬转铁蛋白受体（TfR）结合的活性。【结果】序列分析结果表明,本实验构建的犬细小病毒VP2基因真核分泌型表达载体结构正确,将该表达载体转染的293T细胞,在培养基中通过Western-blot检测到有VP2重组蛋白的存在。经ELISA检测表明表达的重组VP2蛋白具有与犬转铁蛋白受体结合的活性。【结论】 利用人的CD5信号肽实现了犬细小病毒VP2蛋白在真核细胞中的分泌表达,表达的VP2蛋白具有与犬转铁蛋白受体结合的活性。     

Vaccination of shrimp (Penaeus chinensis) against white spot syndrome virus (WSSV)

Two structural protein genes, VP19 and VP466, of white spot syndrome virus (WSSV) were cloned and expressed in Sf21 insect cells using a baculovirus expression system for the development of injection and oral feeding vaccines against WSSV for shrimps. The cumulative mortalities of the shrimps vaccinated by the injection of rVP19 and rVP466 at 15 days after the challenge with WSSV were 50.2% and 51.8%, respectively. For the vaccination by oral feeding of rVP19 and rVP466, the cumulative mortalities were 49.2% and 89.2%, respectively. These results show that protection against WSSV can be generated in the shrimp, using the viral structural protein as a protein vaccine.     

Prokaryotic expression of chicken infectious anemia apoptin protein and characterization of its polyclonal antibodies

In the present study recombinant VP3 (rVP3) was expressed in E. coli BL21 (DE3) (pLysS) and its polyclonal antibodies were characterized. SDS-PAGE analysis revealed that the expression of recombinant protein was maximum when induced with 1.5 mM IPTG for 6 h at 37 degrees C. The 6xHis-tagged fusion protein was purified on Ni-NTA and confirmed by Western blot using CAV specific antiserum. Rabbits were immunized with purified rVP3 to raise anti-VP3 polyclonal antibodies. Polyclonal serum was tested for specificity and used for confirming expression of VP3 in HeLa cells transfected with pcDNA.cav.vp3 by indirect fluorescent antibody test (IFAT), flow cytometry and Western blot. Available purified rVP3 and polyclonal antibodies against VP3 may be useful to understand its functions which may lead to application of VP3 in cancer therapeutics.     

Self-assembly of the infectious bursal disease virus capsid protein, rVP2, expressed in insect cells and purification of immunogenic chimeric rVP2H particles by immobilized metal-ion affinity chromatography

A gene encoding a structural protein (VP2) of a local strain (P3009) of infectious bursal disease virus (IBDV) was cloned and expressed using the baculovirus expression system to develop a subunit vaccine against IBDV infection in Taiwan. The expressed rVP2 proteins formed particles of approximately 20-30 nm in diameter. Those particles were partially purified employing sucrose density gradient ultracentrifugation, and the purified particles were recognized by a monoclonal antibody against the VP2 protein of IBDV P3009. To facilitate the purification of the particles, the VP2 protein was engineered to incorporate a metal ion binding site (His)(6 )at its C-terminus. The chimeric rVP2H proteins also formed particles, which could be affinity-purified in one step with immobilized metal ions (Ni(2+)). Particle formation was confirmed by direct observation under the electron microscope. The production level of rVP2H protein was determined to be 20 mg/L in a batch culture of Hi-5 cells by quantifying the concentration of the purified proteins. The chicken protection assay was performed to evaluate the immunogenicity of the rVP2H protein. When susceptible chickens were inoculated with the recombinant rVP2H proteins (40 microg/bird), virus-neutralizing antibodies were induced, thereby conferring a high level of protection against the challenge of a very virulent strain of IBDV. In conclusion, the most significant finding in this work is that both of the expressed rVP2 and rVP2H proteins can form a particulate structure capable of inducing a strong immunological response in a vaccinated chicken.     

High level expression of monomeric and dimeric human alpha1,3-fucosyltransferase V

alpha3/4-Fucosyltransferases play a crucial role in inflammatory processes and tumor metastasis. While several human fucosyltransferases (FucTs) with different acceptor substrate specificities have been identified, the design of specific inhibitors for therapeutic approaches is hampered by the lack of structural information. In this study, we evaluated the expression of different constructs of human fucosyltransferase V to generate the large amounts required for structural studies. The truncated constructs lacking the transmembrane region and the cytosolic N-terminus, were expressed in baculovirus-infected Trichoplusia ni (Tn) insect cells and in two non-lytic expression systems, stably transfected human HEK 293 and T. ni cells. Since secretion of some glycosyltransferases is controlled by formation of dimeric molecules via disulfide bonds, one of the fucosyltransferase V constructs contained the N-terminal cysteine residue 64 for dimerization, whereas this residue was replaced in the other construct by serine. In both human and insect cells dimerization did not prove to be essential for efficient expression and secretion. On the basis of enzymatic activity, the yield of secreted fucosyltransferase V was approximately 10-fold higher in stably transfected insect cells than in HEK 293 cells. In particular the monomeric form of the enzyme provides a valuable tool for structural analyses to elucidate the fine specifity of fucosyltransferase V-mediated fucosylation of Lewis type glycans.     

Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose

Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy’s disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particles (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy’s disease and vehicles for the delivery of drugs.     

Problems encountered in bicistronic IRES-GFP expression vectors employed in functional analyses of GC-induced genes

Our laboratory has developed a series of Gateway^? compatible lentiviral expression systems for constitutive and conditional gene knock-down and over-expression. For tetracycline-regulated transgenic expression, we constructed a lentiviral “DEST” plasmid (pHR-TetCMV-Dest-IRES-GFP5) containing a tetracycline-responsive minimal CMV promoter, followed by an attP site-flanked DEST cassette (for efficient cloning of cDNAs by “Gateway^?” recombination cloning) and green fluorescent protein (GFP) driven by an internal ribosomal entry site (IRES).This lentiviral bicistronic plasmid allows immediate FACS identification and characterization of successfully transfected cell lines. Although this system worked well with several cDNAs, we experienced serious problems with SLA, Bam and BMF. Particularly, we cloned the cDNA for human SLA (Src–like adapter), a candidate gene in GC-induced apoptosis, into this plasmid. The resulting construct (pHR-TetCMV-SLA-IRES-GFP5) was transfected into HEK 293-T packaging cells to produce viral particles for transduction of CEM-C7H2-2C8 cells. Although the construct produced many green fluorescent colonies at the HEK 293-T and the CEM-C7H2-2C8 level, we could not detect any SLA protein with α-SLA antibody from corresponding cell lysates. In contrast, the antibody readily detected SLA in whole cell lysate of HEK 293-T cells transfected with a GST-flagged SLA construct lacking IRES-GFP. To directly address the potential role of the IRES-GFP sequence, we cloned the SLA coding region into pHR-TetCMV-Dest, a vector that differs from pHR-TetCMV-Dest-IRES-GFP5 just by the absence of the IRES-GFP cassette. The resulting pHR-TetCMV-SLA construct was used for transfection of HEK 293-T cells. Corresponding lysates were assayed with α-SLA antibody and found positive. These data, in concert with previous findings, suggest that the IRES-GFP cassette may interfere with translation of certain smaller size cDNAs (like SLA) or generate fusion proteins and entail defective virus production in an unpredictable manner.     

Enhancement of sapovirus recombinant capsid protein expression in insect cells

Human sapovirus (SaV) is uncultivable, but expression of the recombinant capsid protein (rVP1) in insect cells results in the formation of virus-like particles (VLPs) that are morphologically similar to the native viruses. However, the SaV rVP1 expression levels are considerably low. We have found that inclusions of short foreign nucleotide sequences inserted directly upstream from the predicted rVP1 AUG start codon lead to increased yield of VLPs. This method allowed us to express a SaV rVP1, which could not have been expressed to measurable or practical levels otherwise.     

Expression of the major capsid protein VP6 of group C rotavirus and synthesis of chimeric single-shelled particles by using recombinant baculoviruses.

VP6 of group C (Cowden strain) rotavirus was expressed in the baculovirus system. The recombinant protein, expressed to a high level in insect cells, was purified by ion-exchange chromatography. The purified protein was proven to be trimeric. The effect of pH on the trimer's stability was investigated. Coexpression of VP6 from group A (bovine strain RF) and VP6 from group C in the baculovirus system did not result in the formation of chimeric trimers. Coexpression of VP2 from group A rotavirus (bovine strain RF) and VP6 from group C in the baculovirus system led to the formation of chimeric, empty, single-shelled particles. These results demonstrate conservation in the domains necessary for binding to VP2 in different serogroups of VP6. The locations of the domains involved in trimerization and in the interaction with VP2 are discussed.     

Production and purification of immunogenic virus-like particles formed by the chimeric infectious bursal disease virus structural protein,rVP2H,in insect larvae

This study describes an alternative approach to produce rVP2H protein using insect larvae of the cabbage looper Trichoplusia ni as hosts for the expression of the protein. The chimeric rVP2H protein, having an extra six histidine residues at the C-terminus of the VP2, a structural protein of infectious bursal disease virus (IBDV), is a vaccine candidate for the prevention of infectious bursal disease. The chimeric rVP2H protein was expressed in insect larvae in form of virus-like particles, in which they maintain their native immunogenic properties. The expression level of rVP2H protein in T. ni larvae was estimated to be approximately 0.4 mg/g of larvae or 0.2 mg/larvae. The rVP2H particles have a uniform morphology of dodecahedral structure with a size of 23 nm in diameter, and the particles could be affinity-purified in one step with immobilized metal-ion affinity chromatography (IMAC) from the larvae homogenate. The recovery of rVP2H protein was approximately 55% following IMAC and the protein was obtained with a purity of around 90%. An additional purification step of ammonium sulphate precipitation was added to speed up the process of microfiltration and ultrafiltration of the homogenate prior to IMAC. This step enhanced the final purity of rVP2H protein to 99%, demonstrating that the purification protocol developed herein was a powerful strategy for obtaining highly pure rVP2H protein from insect larvae. The immunogenicity and protective properties of the larvae-derived rVP2H protein were evaluated using a chicken protection assay. When larvae-derived rVP2H protein was intramuscularly injected into specific-pathogen-free chickens (20 μg/bird), high titres of virus-neutralizing antibodies were induced and the chickens were protected from the infection of a very virulent strain of IBDV isolated locally.     

In vitro expression and monoclonal antibody of RNA-dependent RNA polymerase for infectious bursal disease virus

VP1, the RNA-dependent RNA polymerase of infectious bursal disease virus (IBDV), has been suggested to play an essential role in the replication and translation of viral RNAs. In this study, we first expressed the complete VP1 protein gene in Escherichia coli (E. coli), and then the produced polyclonal antibody and four monoclonal antibodies (mAbs) to recombinant VP1 protein (rVP1) were shown to bind the IBDV particles in chicken embryo fibroblast and Vero cells. The epitopic analysis showed that mAbs 1D4 and 3C7 recognized respectively two distinct antigenic epitopes on the rVP1 protein, but two pair of mAbs 1A2/2A12 and 1E1/1H3 potentially recognized another two topologically related epitopes. Immunocytochemical stainings showed that VP1 protein formed irregularly shaped particles in the cytoplasm of the IBDV-infected cells. These results demonstrated that the mAbs to rVP1 protein could bind the epitopes of IBDV particles, indicating that the rVP1 protein expressed in E. coli was suitable for producing the mAb to VP1 protein of IBDV, and that the cytoplasm could be the crucial site for viral genome replication of IBDV.     

Polyclonal antibodies specific for VP1 and VP3 capsid proteins of Taura syndrome virus (TSV) produced via gene cloning and expression

Capsid protein genes VP1 and VP3 of Taura syndrome virus (TSV) were cloned into pGEX-6P-1 expression vector and transformed into Escherichia coli BL21. After induction, recombinant VP1 (rVP1) and recombinant VP3 (rVP3) were produced, purified by SDS-PAGE and used for immunization of Swiss mice for antisera production. Anti-rVP1 and anti-rVP3 antisera showed specific immunoreactivities to rVP1 and rVP3 proteins, respectively, by Western blot assay and also yielded good results for detection of TSV in various shrimp tissues by immunohistochemistry. This is the first step towards our target of preparing monoclonal antibodies specific to rVP1 and rVP3 for use in simple immuno-diagnostic test kits for TSV detection and identification.     

Efficient expression screening of human membrane proteins in transiently transfected Human Embryonic Kidney 293S cells

It is often an immense challenge to overexpress human membrane proteins at levels sufficient for structural studies. The use of Human Embryonic Kidney 293 (HEK 293) cells to express full-length human membrane proteins is becoming increasingly common, since these cells provide a near-native protein folding and lipid environment. Nevertheless, the labor intensiveness and low yields of HEK 293 cells and other mammalian cell expression systems necessitate the screening for suitable expression as early as possible. Here we present our methodology used to generate constructs of human membrane proteins and to rapidly assess their suitability for overexpression using transiently transfected, glycosylation-deficient GnT I-HEK 293 cells (HEK 293S). Constructs, in the presence or absence of a C-terminal enhanced green fluorescence protein (EGFP) molecule, are made in a modular manner, allowing for the rapid generation of several combinations of fusion tags and gene paralogues/orthologues. Solubilization of HEK 293S cells, using a range of detergents, followed by Western blotting is performed to assess relative expression levels and to detect possible degradation products. Fluorescence-detection size exclusion chromatography (FSEC) is employed to assess expression levels and overall homogeneity of the membrane proteins, to rank different constructs for further downstream expression trials. Constructs identified as having high expression are instantly suitable for further downstream large scale transient expression trials and stable cell line generation. The method described is accessible to all laboratory scales and can be completed in approximately 3 weeks.     

Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells

The human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) is one of the key enzymes that initiate synthesis of hinge-region O-linked glycans of human immunoglobulin A1 (IgA1). We designed secreted soluble form of human GalNAc-T2 as a fusion protein containing mouse immunoglobulin light chain kappa secretory signal and expressed it using baculovirus and mammalian expression vectors. The recombinant protein was secreted by insect cells Sf9 and human HEK 293T cells in the culture medium. The protein was purified from the media using affinity Ni-NTA chromatography followed by stabilization of purified protein in 50mM Tris-HCl buffer at pH 7.4. Although the purity of recombinant GalNAc-T2 was comparable in both expression systems, the yield was higher in Sf9 insect expression system (2.5mg of GalNAc-T2 protein per 1L culture medium). The purified soluble recombinant GalNAc-T2 had an estimated molecular mass of 65.8kDa and its amino-acid sequence was confirmed by mass-spectrometric analysis. The enzymatic activity of Sf9-produced recombinant GalNAc-T2 was determined by the quantification of enzyme-mediated attachment of GalNAc to synthetic IgA1 hinge-region peptide as the acceptor and UDP-GalNAc as the donor. In conclusion, murine immunoglobulin kappa secretory signal was used for production of secreted enzymatically active GalNAc-T2 in insect baculovirus expression system.     

VP37 of white spot syndrome virus interact with shrimp cells

Aims:  To investigate VP37 [WSV 254 of White spot syndrome virus (WSSV) genome] interacting with shrimp cells and protecting shrimp against WSSV infection.
Methods and Results:  VP37 was expressed in Escherichia coli and was confirmed by Western blotting. Virus overlay protein binding assay (VOPBA) technique was used to analyse the rVP37 interaction with shrimp and the results showed that rVP37 interacted with shrimp cell membrane. Binding assay of recombinant VP37 with shrimp cell membrane by ELISA confirmed that purified rVP37 had a high-binding activity with shrimp cell membrane. Binding of rVP37 to shrimp cell membrane was a dose-dependent. Competition ELISA result showed that the envelope protein VP37 could compete with WSSV to bind to shrimp cells. In vivo inhibition experiment showed that rVP37 provided 40% protection. Inhibition of virus infection by rVP37 in primary cell culture revealed that rVP37 counterparted virus infection within the experiment period.
Conclusions:  VP37 has been successfully expressed in E . coli . VP37 interacted with shrimp cells.
Significance and Impact of the Study:  The results suggest that rVP37 has a potential application in prevention of virus infection.     

Development of a Chinese hamster ovary cell line for recombinant adenovirus-mediated gene expression

Recombinant human adenovirus (rhAd) has been used extensively for functional protein expression in mammalian cells including those of human and nonhuman origin. High-level protein production by rhAd vectors is expected in their permissive host cells, such as the human embryonic kidney 293 (HEK293) cell line. This is attributed primarily to the permissiveness of HEK293 to rhAd infection and their ability to support viral DNA replication by providing the missing El proteins. However, the HEK293 cells tend to suffer from cytopathic effect (CPE) as a result of virus replication. Under these circumstances, the host cell function is compromised and the culture viability will be reduced. Consequently, newly synthesized polypeptides may not be processed properly at posttranslational levels. Therefore, the usefulness of HEK293 cells for the expression of complex targets such as secreted proteins could be limited. In the search for a more robust cell line as a production host for rhAd expression vectors, a series of screening experiments was performed to isolate clones from Chinese hamster ovary-K1 (CHO-K1) cells. First, multiple rounds of infection of CHO-K1 cells were performed utilizing an rhAd expressing GFP. After each cycle of infection, a small population of CHO cells with high GFP levels was enriched by FACS. Second, individual clones more permissive to human adenovirus infection were isolated from the highly enriched subpopulation by serial dilution. A single clone, designated CHO-K1-C5, was found to be particularly permissive to rhAd infection than the parental pool and has served as a production host in the successful expression of several secreted proteins.     

免疫荧光法检测原核和真核细胞中表达的轮状病毒外壳蛋白VP4

目的：用免疫荧光法快速检测原核和真核细胞中表达的轮状病毒（RV）外壳蛋白VP4。方法：以抗VP4的抗体为一抗、FITC标记的羊抗豚鼠IgG为二抗,用免疫荧光方法检测在大肠杆菌BL21（DE3）中重组表达的同源RVVP4;检测SA11或Wa株RV感染MA104细胞后不同时间段病毒VP4的合成及其在感染细胞中的分布情况。结果：用免疫荧光法可直接检测到原核细胞中表达的外源蛋白,也可检测到病毒蛋白在真核细胞中的分布情况。结论：免疫荧光法可特异、方便、快速地检测RV VP4在原核和真核细胞中的表达;来源于RV TB—Chen株的VP4抗体可特异性识别同源病毒VP4,交叉识别SA11或Wa株的VP4。     

白细胞介素6重组逆转录病毒载体的构建及鉴定

目的 构建特异性过表达大鼠IL-6基因的重组逆转录病毒载体,并在大鼠嗜铬细胞瘤PC12细胞和人胚肾HEK293细胞中检测IL-6的表达。方法以大鼠骨髓间充质干细胞mRNA为模板,经PCR获得目的基因IL-6,将其定向克隆到逆转录病毒载体pSEB-3H中,构建重组逆转录病毒质粒pSEB—IL-6,经脂质体分别转染到PC12细胞和HEK293细胞中,应用Real—timePCR和ELISA的方法在mRNA和蛋白质水平检测IL-6的表达变化。继而用HEK293细胞中包装获得的含有pSEB—IL-6的病毒颗粒进一步感染PC12细胞,Real—timePCR检测,IL-6mRNA的表达水平变化。结果PCR电泳及酶切鉴定证实目的基因正确克隆至逆转录病毒载体中,其基因序列与Genbank报道一致;Real—timePCR和ELISA结果均显示,逆转录病毒质粒pSEB—IL-6转染PC12细胞和HEK293细胞后,IL-6的表达水平较对照组显著上调;经pSEB—IL-6逆转录病毒颗粒感染的PC12细胞中,IL-6mRNA表达水平较对照组提高4倍。结论成功构建了特异性表达大鼠儿-6基因的重组逆转录病毒载体pSEB—IL-6,并获得了具有感染能力的逆转录病毒颗粒,感染真核细胞后可高表达IL-6,为进一步研究IL-6的功能及其在多种疾病中的免疫调节机制提供重要的分子手段。     

基于逆转录病毒载体的外源基因表达系统的评价和应用

逆转录病毒表达系统是基因治疗研究和RNA干扰技术广泛采用的外源基因表达系统。文中以增强型绿色荧光蛋白 (EGFP) 基因的表达水平和稳定性为指标,比较逆转录病毒表达载体pQCXIN和pcDNA3.1(+) 表达质粒介导的外源基因在HEK293细胞和CHO-K1细胞的表达效率。病毒感染HEK293细胞和CHO-K1细胞的相对荧光强度 (Relative fluorescence intensity,RFI) 均约为对应的质粒转染细胞的2倍。多轮反复感染逆转录病毒表达载体能有效提高HEK293细胞表达EGFP的效率。HEK293细胞经4轮病毒感染后的RFI值较1次病毒感染HEK293细胞的RFI值约提高2倍。此外,逆转录病毒表达载体介导的外源基因表达的稳定性优于质粒转染的外源基因表达。采用携带人重组活性蛋白C (Recombinant human activated protein C,rhAPC) 基因的pQCXIN和HEK293细胞进一步验证了逆转录病毒载体介导的外源基因表达效率,构建了rhAPC表达水平为10~15 mg/(106 cells·d) 的HEK293细胞系。研究结果表明,逆转录病毒表达系统是有应用价值的介导外源基因在哺乳动物细胞高效表达的技术途径。     

A versatile ligation-independent cloning method suitable for high-throughput expression screening applications

This article describes the construction of a set of versatile expression vectors based on the In-Fusion™ cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion™ has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragments (3) efficient over a wide (20-fold) insert concentration range and (4) applicable to expression in multiple hosts. The system enables the precise engineering of (His₆-) tagged constructs with no undesirable vector or restriction-site-derived amino acids added to the expressed protein. The use of a multiple host-enabled vector allows rapid screening in both E. coli and eukaryotic hosts (HEK293T cells and insect cell hosts, e.g. Sf9 cells). These high-throughput screening activities have prompted the development and validation of automated protocols for transfection of mammalian cells and Ni-NTA protein purification.