Mammalian 293T Cells Research Articles

Identification and pathogenicity analysis of a novel highly virulent strain of spring viraemia of carp virus and production of its structural proteins by the mammalian expression system

Spring viraemia of carp virus (SVCV), a highly pathogenic fish rhabdovirus, caused an acute hemorrhagic and highly contagious disease in cyprinids. In this study, a new strain of SVCV was isolated from diseased zebrafish in Yangling, Shaanxi Province, China, and named SVCV-CG01. Tissue filtrates of diseased fish were detected by Nest PCR using the specific 714 bp and 606 bp fragments from the glycoprotein gene of SVCV. The genes encoding for the N, P, M and G protein were amplified, and the fragments were inserted into pMD-19T vectors to determine the genome of SVCV. Based on phylogenetic analysis of G protein gene, the isolated virus was classified into the Ia genogroup. The titer of the new strain at 48 h and 72 h was approximately 108 and 109 times higher than that of the 0504 strain, respectively. The virus could produce typical cytopathic effects (CPE) and induce apoptosis in EPC cells at 48 and 72 h post infection (hpi). Healthy zebrafish were injected with different doses of SVCV and mass mortality of fish was detected after 4 d infection, with a cumulative death rate of 100% from 107 TCID50 group at 7 d post infection (dpi). Using cultured mammalian 293T cells, an average transfection efficiency of the four structural proteins was 49.1% and 69.6% at 24 and 48 h post transfection (hpt), respectively. This study could be served as a reference for the research on the molecular epidemiology, evolution and pathogenicity of fish viruses.

Filovirus glycoprotein glycosylation analysis and effects on immunogenicity

Abstract Filoviruses are hemorrhagic fever viruses that are some of the deadliest pathogens in the world with a fatality rate up to 90%. Symptoms include fever, vomiting, diarrhea, headache, weakness and can lead to hemorrhaging, multiple organ failure and death. Experimental vaccines use filovirus Surface glycoproteins (GPs) as immunogens to induce protective immune responses. Our laboratory studies the glycosylation patterns of different filovirus GPs to develop improved vaccines. Mammalian 293T cells have more complex N-linked glycosylation machinery compared to Sf9 insect cells but both are capable of O-linked glycosylation. Differences in N- and O-linked glycans on filovirus GPs may induce different immune responses upon vaccination. However, the glycosylation patterns on the different filovirus GPs have not been thoroughly elucidated. Here we report purification and glycan analysis of GPs from filoviruses produced in 293T mammalian cells and Sf9 insect cells. We are currently assessing the effects of different glycosylation of the understudied Lloviu virus GP on immunogenicity in mice.

PL-002 AGTR1 polymorphism is associated with elite endurance athletes: A functional study

Objective To explore the association between the polymorphism of angiotensin II receptor type 1 gene(AGTR1) and elite endurance athlete performance and the mechanism of how the polymorphism works.
 Methods (1) Polymorphism of AGTR1 rs5182 between 122 elite Chinese endurance athletes and 222 controls were analyzed by MALDI-TOF-MS. (2) Aerobic capacity of 79 elite Chinese endurance athletes such as VC/FEV1/MVV/ VO2AT/ HRAT/ VAT/ VO2max/ HRmax/ VVO2max were measured and association between rs5182 polymorphism and the performance was analyzed. (3) PcDNA3.1-AGTR1 -T and pcDNA3.1-AGTR1-C plasmid were build and the plasmids was transfected into mammalian 293T cells. mRNA levels were detected after 48 hours. Statistical analysis was performed using SPSS software version 15.0. Values of P < 0.05 were considered statistically significant. Continuous data were expressed as mean ± SD, while categorical data were expressed as frequencies. Genotype distribution and allele frequencies between athletes and control subjects were compared using χ2 tests. Aerobic performance data was analyzed with One-Way ANOVA if it conformed to normal distribution and homogeneity of variance otherwise Non-parametric test of independent sample was used.
 Results (1) Genotype frequencies of AGTR1 rs5182 are significant differences between the athletes and control subjects (p = 0.040), the Word-Class athletes and control subjects (p = 0.018), 5km athletes and control subjects (p =0.015), 10km athletes and control subjects (p = 0.026), male athletes and male controls(p=0.045). (2) Association is found between Genotype distribution and MV(L/min) though others not (Genotype: MV; CC: 122.514±6.767; CT:117.187±17.961; TT:119.688±20.226, p=0.047). (3) Transiently transfectedpcDNA3.1-AGTR1-T and pcDNA3.1-AGTR1-C plasmids into 293T cells successfully. The differences of mRNA levels between the groups were not significant (p = 0.991).
 Conclusions AGTR1 gene rs5182 could be a candidate genetic mark of selection elite endurance athletes in Han Population from Northern China, but this polymorphism does not affect AT1R protein function through changing its mRNA level.

Production and crystallization of the C-propeptide trimer from human procollagen III.

The C-propeptide domains of the fibrillar procollagens, which are present throughout the Metazoa in the form of ∼90 kDa trimers, play crucial roles in both intracellular molecular assembly and extracellular formation of collagen fibrils. The first crystallization of a C-propeptide domain, that from human procollagen III, is described. Following transient expression in mammalian 293T cells of both the native protein and a selenomethionine derivative, two crystal forms of the homotrimer were obtained: an orthorhombic form (P2(1)2(1)2(1)) that diffracted to 1.7 Å resolution and a trigonal form (P321) that diffracted to 3.5 Å resolution. Characterization by MALDI-TOF mass spectrometry allowed the efficiency of selenomethionine incorporation to be determined.

PA residues in the 2009 H1N1 pandemic influenza virus enhance avian influenza virus polymerase activity in mammalian cells.

The 2009 pandemic influenza virus (pH1N1) is a swine-origin reassortant containing human, avian, and swine influenza genes. We have previously shown that the polymerase complex of the pH1N1 strain A/California/04/2009 (Cal) is highly active in mammalian 293T cells, despite the avian origin of both its PA and PB2. In this study, we analyzed the polymerase residues that are responsible for high pH1N1 polymerase activity in the mammalian host. Characterization of polymerase complexes containing various combinations of Cal and avian influenza virus A/chicken/Nanchang/3-120/01 (H3N2) (Nan) by reporter gene assay indicates that Cal PA, but not PB2, is a major contributing factor to high Cal polymerase activity in 293T cells. In particular, Cal PA significantly activates the otherwise inactive Nan polymerase at 37 and 39°C but not at the lower temperature of 34°C. Further analysis using site-directed mutagenesis showed that the Cal PA residues 85I, 186S, and 336M contribute to enhanced activity of the Cal polymerase. Recombinant A/WSN/33 (H1N1) (WSN) viruses containing Nan NP and polymerase (PA, PB1, PB2) genes with individual mutations in PA at residues 85, 186, and 336 produced higher levels of viral protein than the virus containing wild-type (WT) Nan PA. Interestingly, compared to the WT, the virus containing the 85I mutation grew faster in human A549 cells and the 336M mutation most significantly enhanced pathogenicity in a mouse model, among the three PA mutations tested. Our results suggest that multiple mutations in PA, which were rarely present in previous influenza isolates, are involved in mammalian adaptation and pathogenicity of the 2009 pH1N1.

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6. Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15. The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity. Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method with some modifications. Compared with the lipid transfection method, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory. The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

Construction and eukaryotic expression of recombinant plasmid pEGFP-N1-SrV+encoding the variable region of the surface protein of Streptococcus mutans

Objective To construct the recombinant plasmid pEGFP-NI-SrV ＋ and evaluate the expression of SrV ＋ in mammalian 293T cells. Methods sty ＋ , a gene encoding the variable region of the surface protein of the Streptococcus mutans OMZ175, was cloned chemically based on its reported nucleotide sequence. The eukaryotic expression plasmid, pEGFP-N1-SrV ＋ , was constructed by introducing the srv ＋ gene into the Kpn 1/Xho I site of pEGFP-Nt. The recombinant plasmid pEGFP-N1-SrV ＋ was transfected into 293T cells with lipofectamine and the expression level of SrV ＋ was evaluated. Results The eukaryotic expression plasmid pEGFP-N1-SrV ＋ was constructed successfully. GFP was observed by green fluorescent microscope, and a 72 x 103 protein was detected by Western blot. Real-time RT-PCR analysis revealed that the expression of the pEGFP-N1-SrV ＋ in 293T was excellent and significant compared the control group. Conclusion The recombinant plasmid pEGFP-N1-SrV ＋ was successfully constructed, which could encode the expression of SrV ＋ after transfeeted into the mammalian 293T Cells. Key words: Streptococcus mutans ; Variable region of surface proteins ; Eukaryotic expression vec- tor plasmid pEGFP-N1 ; 293T cells

Differential expression of the Ebola virus GP 1, 2 protein and its fragments in E. coli

Bacterial expression platforms are frequently used for the expression and production of different recombinant proteins. The full length Ebola virus (EBOV) GP 1, 2 gene and subfragments of the GP 1 gene were cloned in a bacterial expression vector as a C-terminal His 6 fusion protein. Surprisingly, the full length EBOV GP 1, 2 gene could not be expressed in Escherichia coli. The subfragments of GP 1 were only expressed in small amounts with the exception of one small fragment (subfragment D) which was expressed at very high levels as inclusion bodies. This was seen even in the in vitro translation system with no expression of full length GP 1, 2 , GP 1 subfragments A and C and low level expression of subfragment B. Only the subfragment D showed high level of expression. In E. coli (Top10), the recombinant GP 1 subfragment D protein was expressed exclusively as an insoluble ∼25 kDa His 6 fusion protein, which is the expected size for a non-glycosylated recombinant protein. The IMAC purified and refolded non-glycosylated protein was used to immunize mice for the development of monoclonal anti-EBOV antibodies which successfully yielded several monoclonal antibodies with different specificities. The monoclonal and polyclonal antiserum derived from the animals immunized with this recombinant GP 1 subfragment D protein was found to specifically recognize the full length glycosylated EBOV GP 1, 2 protein expressed in mammalian 293T cells, thus, demonstrating the immunogenicity of the recombinant subfragment.

Investigation of the Dimer Interface and Substrate Specificity of Prolyl Dipeptidase DPP8

DPP8 belongs to the family of prolyl dipeptidases, which are capable of cleaving the peptide bond after a penultimate proline residue. Unlike DPP-IV, a drug target for type II diabetes, no information is available on the crystal structure of DPP8, the regulation of its enzymatic activity, or its substrate specificity. In this study, using analytical ultracentrifugation and native gel electrophoresis, we show that the DPP8 protein is predominantly dimeric when purified or in the cell extracts. Four conserved residues in the C-terminal loop of DPP8 (Phe(822), Val(833), Tyr(844), and His(859)), corresponding to those located at the dimer interface of DPP-IV, were individually mutated to Ala. Surprisingly, unlike DPP-IV, these single-site mutations abolished the enzymatic activity of DPP8 without disrupting its quaternary structure, indicating that dimerization itself is not sufficient for the optimal enzymatic activity of DPP8. Moreover, these mutations not only decreased k(cat), as did the corresponding DPP-IV mutations, but also dramatically increased K(m). We further show that the K(m) effect is independent of the substrate assayed. Finally, we identified the distinctive and strict substrate selectivity of DPP8 for hydrophobic or basic residues at the P2 site, which is in sharp contrast to the much less discriminative substrate specificity of DPP-IV. Our study has identified the residues absolutely required for the optimal activity of DPP8 and its unique substrate specificity. This study extends the functional importance of the C-terminal loop to the whole family of prolyl dipeptidases.

Dimerization of CtIP, a BRCA1- and CtBP-interacting Protein, Is Mediated by an N-terminal Coiled-coil Motif

CtIP is a transcriptional co-regulator that binds a number of proteins involved in cell cycle control and cell development, such as CtBP (C terminus-binding protein), BRCA1 (breast cancer-associated protein-1), and LMO4 (LIM-only protein-4). The only recognizable structural motifs within CtIP are two putative coiled-coil domains located near the N and C termini of the protein. We now show that the N-terminal coiled coil (residues 45-160), but not the C-terminal coiled coil, mediates homodimerization of CtIP in mammalian 293T cells. The N-terminal coiled coil did not facilitate binding to LMO4 and BRCA1 proteins in these cells. A protease-resistant domain (residues 27-168) that minimally encompasses the putative N-terminal coiled coil was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This region is predicted to contain two smaller coiled-coil regions. The CtIP-(45-160) dimerization domain is helical and dimeric, indicating that the domain does form a coiled coil. The two smaller domains, CtIP-(45-92) and CtIP-(93-160), also formed dimers of lower binding affinity, but with less helical content than the longer peptide. The hydrodynamic radius of CtIP-(45-160) is the same as those of CtIP-(45-92) and CtIP-(93-160), implying that CtIP-(45-160) does not form a single long coiled coil, but a more compact structure involving homodimerization of the two smaller coiled coils, which fold back as a four-helix bundle or other compact structure. These results suggest a specific model for CtIP homodimerization via its N terminus and contribute to an improved understanding of how this protein might assemble other factors required for its role as a transcriptional corepressor.