共查询到20条相似文献,搜索用时 31 毫秒
1.
Yung-Shyeng Tsao Russell Condon Eugene Schaefer Peggy Lio Zhong Liu 《Cytotechnology》2001,37(3):189-198
Human Embryonic Kidney 293 (HEK293) cells were adapted into a serum-free suspension medium through steps of gradual serum
weaning for the production of adenoviral (AdV) gene therapy vectors. The presence of sodium heparin in the medium formulation
reduced cell clumping dramatically in suspension culture. The adapted cells were ready to grow either in serum-containing
medium as an attached culture or in serum-free medium in suspension culture. A scalable production process was developed in
shake flasks and was then evaluated in stirred tank bioreactors. This process includes a growth phase in batch-mode followed
by a production phase involving medium perfusion and supplementation. Fortification with calcium chloride post viral inoculation
resulted in an increase in virus production by at least one fold. Addition of stimulating agents such as sodium butyrate,
N-acetyl-L-cysteine (NAC), dimethyl sulfoxide(DMSO), or ethyl alcohol post infection was shown to further improve virus production
in a dose-dependent manner. The serum-free suspension process described here should be suitable for the manufacturing of other
E1-deleted AdV vectors and could potentially be used for the production of recombinant proteins by HEK293 cells.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
2.
Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells 总被引:2,自引:0,他引:2
This article describes the step-wise approach undertaken to select a serum-free medium (SFM) for the efficient production of a recombinant adenoviral vectors expressing beta-galactosidase (Ad5 CMV-LacZ), in the complementing human embryonic kidney 293S cells. In the first step, a 293S-derived transfectoma, secreting a soluble epidermal growth factor receptor sEGFr (D2-22), was used to estimate the potential of selected serum-free formulations to support the production of a recombinant protein as compared to serum-containing medium. Assays showed that only one among six commercial serum-free formulations could support both sEGFr production and cell growth in static or suspension culture. In commercially available calcium-containing serum-free formulations, the cell aggregates reached up to 3 mm in diameter. In the second step, 293S cells were gradually adapted to a low-calcium version of the selected medium (LC-SFM). Cells were cloned, then screened according to their ability to grow at a rate and an extent comparable to parental cells in serum-containing medium (standard) as single cells or small aggregates. The 293SF-3F6 clone, first adapted to and then cloned in the selected serum-free medium, was selected for further experiments. Bioreactor run performed with the 293SF-3F6 clone showed similar growth curve as in the shake-flask controls. In the final step, the recombinant viral vector productivity of the 293S cells and the 293SF-3F6 clone was tested. The 293SF-3F6 cells infected by Ad5 CMV-LacZ in 3 L-scale bioreactor maintained the specific productivities of both beta-galactosidase and adenoviral vector equivalent to the shake-flask controls in suspension culture. Results from this study clearly demonstrate that the 293SF-3F6 cell line thus selected may be used either for establishing stable transfected cell line or for the production of adenoviral vectors required for gene therapy studies. 相似文献
3.
悬浮培养HEK-293 N3S细胞生产重组腺病毒Ad-GFP的实验研究 总被引:1,自引:0,他引:1
利用5L生物反应器悬浮培养HEK-293N3S细胞生产携带绿色荧光蛋白基因的重组腺病毒(recombinant adenovirus-greenfluorescent protein,Ad-GFP),为规模化生产腺病毒基因药物建立一种稳定可行的生产工艺。复苏的种子细胞进行逐级放大最后接入5L搅拌式生物反应器中,采用含5%胎牛血清(FBS)的DMEM/F12培基灌流培养293N3S细胞,当细胞密度达到(2~4)×106个/mL时感染Ad-GFP,48h后收获细胞,经两步氯化铯超速离心获得纯化的Ad-GFP。采用紫外分光光度计比色法和高压液相色谱法(HPLC)测定病毒颗粒数和纯度,采用组织培养半数感染剂量(TCID50)法检测腺病毒的感染滴度。连续培养10~12d,细胞密度可达到(2~4)×106个/mL左右,纯化的Ad-GFP感染滴度和颗粒数分别为1.0×1011IU/mL和1.68×1012VP/mL,比活性为6.0%,A260/A280比值为1.33,产品纯度达到99.2%。建立了5L生物反应器悬浮培养293N3S细胞生产重组腺病毒Ad-GFP的生产工艺,对携带其他基因的重组腺病毒药物生产具有一定的指导意义。 相似文献
4.
Comparison of manufacturing techniques for adenovirus production 总被引:2,自引:0,他引:2
We have compared three different production methods, which may be suitable for the large scale production of adenovirus vectors
for human clinical trials. The procedures compared 293 cells adapted to suspension growth in serum-free medium in a stirred
tank bioreactor, 293 cells on microcarriers in serum-containing medium in a stirred tank bioreactor, and 293 cells grown in
standard tissue culture plasticware. With a given virus, yields varied between 2000 and 10,000 infectious units/cell. The
stirred tank bioreactor routinely produced between 4000 and 7000 infectious units/cell when 293 cells were grown on microcarriers.
The 293 cells adapted to suspension growth in serum-free medium in the same stirred tank bioreactor yielded between 2000 and
7000 infectious units/cell. Yields obtained from standard tissue culture plasticware were up to 10,000 infectious units/cell.
Cell culture conditions were monitored for glucose consumption, lactate production, and ammonia accumulation. Glucose consumption
and lactate accumulation correlated well with the cell growth parameters. Ammonia production does not appear to be significant.
Based on virus yields, ease of operation and linear scalability, large-scale adenovirus production seems feasible using 293
cells (adapted to suspension/serum free medium or on microcarriers in serum containing medium) in a stirred tank bioreactor.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
5.
Transient gene expression in suspension HEK-293 cells: application to large-scale protein production
Baldi L Muller N Picasso S Jacquet R Girard P Thanh HP Derow E Wurm FM 《Biotechnology progress》2005,21(1):148-153
Recent advances in genomics, proteomics, and structural biology raised the general need for significant amounts of pure recombinant protein (r-protein). Because of the difficulty in obtaining in some cases proper protein folding in bacteria, several methods have been established to obtain large amounts of r-proteins by transgene expression in mammalian cells. We have developed three nonviral DNA transfer protocols for suspension-adapted HEK-293 and CHO cells: (1) a calcium phosphate based method (Ca-Pi), (2) a calcium-mediated method called Calfection, and (3) a polyethylenimine-based method (PEI). The first two methods have already been scaled up to 14 L and 100 L for HEK-293 cells in bioreactors. The third method, entirely serum-free, has been successfully applied to both suspension-adapted CHO and HEK-293 cells. We describe here the application of this technology to the transient expression in suspension cultivated HEK-293 EBNA cells of some out of more than 20 secreted r-proteins, including antibodies, dimeric proteins, and tagged proteins of various complexity. Most of the proteins were expressed from different plasmid vectors within 5-10 days after the availability of the DNA. Transfections were successfully performed from the small scale (1 mL in 12-well microtiter plates) to the 2 L scale. The results reported made it possible to establish an optimized cell culture and transfection protocol that minimizes batch-to-batch variations in protein expression. The work presented here proves the applicability and robustness of transient transfection technology for the expression of a variety of recombinant proteins. 相似文献
6.
Ghani K Garnier A Coelho H Transfiguracion J Trudel P Kamen A 《Biotechnology and bioengineering》2006,95(4):653-660
Recombinant retroviruses are now an established tool for gene delivery. Presently they are mainly produced using adherent cells. However, due to the restrictive nature of adherent cell culture, this mode of production is hampered by low cell-specific productivity and small production units. The large-scale production of retroviral vectors could benefit from the adaptation of retrovirus packaging cell lines to suspension culture. Here, we describe the ability of a 293 packaging cell line to produce retroviral vectors in suspension culture at high titer. Adherent 293GPG cells, producing a Moloney Murine Leukemia Virus (MoMLV) retrovirus vector pseudotyped with the vesicular stomatitis virus G (VSVG) envelope protein and expressing a TK-GFP fusion protein, were adapted to suspension culture in calcium-free DMEM. At a cell density similar to adherent cell culture, the suspension culture produced retroviral vector consistently in the range of 1 x 10(7) infectious viral particles/mL (IVP/mL), with a specific productivity threefold higher than adherent culture. Furthermore, at the same medium replacement frequency, the suspension producer cells could be cultured at higher density than their adherent counterparts, which resulted in virus titer of 3-4 x 10(7) IVP/mL at 11.0 x 10(6) cells/mL. This corresponds to a 10-fold increase in viral concentration compared to adherent cells. The capacity to up scale the retroviral vector production was also demonstrated by performing a 2 VVD perfusion culture for 9 days in a 3L Chemap bioreactor. The combination of suspension and perfusion led to a 20-fold increase in maximum virus productivity compared to the adherent culture. 相似文献
7.
The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells. 相似文献
8.
The HEK293 cell line has been used for the production of adenovirus vectors to be used in the potential treatment of alcoholism using a gene therapy strategy. Culture optimization and scale-up has been achieved by first adapting the cells to serum-free media and secondly by growing them in suspension. Adenovirus production after infection was increased, resulting in higher specific glucose consumption and lactate accumulation rates compared to the growth phase. We applied media design tools and Metabolic Flux Analysis (MFA) to compare the metabolic states of cells during growth and adenovirus production and to optimize culture media according to the metabolic demand of the cells in terms of glucose and glutamine concentrations. This allowed obtaining a higher maximum cell concentration and increased adenovirus production by minimizing the production of metabolites that can have an inhibitory effect on cell growth. We have proposed a stoichiometric equation for adenovirus synthesis. MFA results allowed determination of how these changes in composition affected the way cells distribute their nutrient resources during cell growth and virus production. Virus purification was successfully achieved using chromatography and Aqueous Two-Phase Systems (ATPS). 相似文献
9.
猪口蹄疫病毒多抗原表位重组腺病毒的构建与鉴定 总被引:1,自引:0,他引:1
本研究设计构建了含有猪O型口蹄疫病毒VP1(21—60)-(141-160)-(200—213)位氨基酸的基因的重组腺病毒质粒pAd-VP,经PacI酶切后转染HEK-293A细胞,3次噬斑纯化获得了重组腺病毒rAd—VP。该重组腺病毒于HEK-293A细胞连续传代至20代效价稳定,TCID50为10^-10/mL。RT—PCR检测证明目的基因在mRNA水平上可有效表达;应用O型口蹄疫病毒标准阳性血清进行间接荧光抗体试验,在rAdVP感染的HEK-293A细胞的胞质可见清晰荧光。证明该重组腺病毒对VP1(21-60)-(141—160)-(200—213)位氨基酸的基因进行了成功的表达,从而为FMDV多抗原表位腺病毒活载体疫苗的研究奠定了基础。 相似文献
10.
A novel method for the production of adenoviral vectors on a scale sufficient to support most research applications and early phase clinical trials is presented. This method utilizes serum-free cell culture medium and a hollow fiber cell culture apparatus. Significantly less time and space are required than in conventional methods, and the resulting adenovirus is collected in a much smaller volume, simplifying the purification steps. The protocol described is a reproducible, convenient, biologically safe, and environmentally sound method for the production of adenoviral vectors for laboratory use and has the potential to scale-up the adenovirus production for clinical use. 相似文献
11.
In gene therapy, retrovirus and adenovirus vectors are extensively used as gene-delivery vehicles and further large-scale processing of these viral vectors will be increasingly important. This study examined stationary and microcarrier cell culture systems with respect to the production of a retrovirus vector (encoding a monounit hammerhead ribozyme gene with an intron) and an adenovirus vector (encoding a reporter lacZ gene). Cytodex 1 and Cytodex 3 solid microcarriers were found to be able to provide good cell growth and high-titer vector production in suspension cultures. Porous microcarriers such as Cytopore 2 gave slightly lower but still efficient growth but produced significantly lower titers of retrovirus and adenovirus vector from the producer cells. The specific retrovirus production was not proportionally related to the specific growth rate of the producer cells. High MOI infection was essential for high-titer production of adenovirus vector in 293 cells. Hydrodynamic shear forces on microcarrier-grown cells increased the production yield for retrovirus vector but decreased for adenovirus vector. The cellular productivity was much more efficient for adenovirus vector produced in 293 cells as compared to the retrovirus vector produced in PA317-RCM1 cells. These findings can provide further insight into the feasibility of applying microcarrier cell culture technology to produce gene-therapy virus vectors. 相似文献
12.
New adenovirus vectors for protein production and gene transfer 总被引:9,自引:0,他引:9
Bernard Massie Dick D. Mosser Maria Koutroumanis Isabelle Vitté-mony Linda Lamoureux France Couture Luc Paquet Claire Guilbault Julie Dionne Dounia Chahla Pierre Jolicoeur Yves Langelier 《Cytotechnology》1998,28(1-3):53-64
Based on two new adenovirus expression cassettes, we have constructed a series of Ad transfer vectors for the overexpression
of one or two genes either in a dicistronic configuration or with separate expression cassettes. Inclusion of the green or
blue fluorescent protein in the vectors accelerates the generation of adenovirus recombinants and facilitates the functional
characterization of genes both in vitro and in vivo by allowing easy quantification of gene transfer and expression. With
our optimized tetracycline-regulated promoter (TR5) we have generated recombinant adenoviruses expressing proteins, that are
either cytotoxic or which interfere with adenovirus replication, at levels of 10–15% of total cell protein. Proteins that
are not cytotoxic can be produced at levels greater than 20% of total cell protein. As well, these levels of protein production
can be achieved with or without adenovirus replication. This yield is similar to what can be obtained with our optimized human
cytomegalovirus-immediate early promoter-enhancer (CMV5) for constitutive protein expression in non-complementing cell lines.
Using the green fluorescent protein as a reporter, we have shown that a pAdCMV5-derived adenovirus vector expresses about
6-fold more protein in complementing 293 cells and about 12-fold more in non- complementing HeLa cells than an adenovirus
vector containing the standard cytomegalovirus promoter. Moreover, a red-shifted variant of green fluorescent protein incorporated
in one series of vectors was 12-fold more fluorescent than the S65T mutant, making the detection of the reporter protein possible
at much lower levels of expression.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
13.
Human 293S cells culture for recombinant adenovirus production is traditionally carried out in batch at a maximum of 6 x 10(5) cells/mL. A previous report demonstrated that fed-batch, applied to the adenovirus/293S cells system, improves the volumetric production of viral proteins by increasing the cell density at which cells can be infected, up to 2 x 10(6) cells/mL, without reducing the per-cell yield of product. To increase this cell density limit, the adenovirus production was performed in a perfusion system where the cells were separated by means of a tangential flow filtration device. 293S cell growth to 14 x 10(6) cells/mL was achieved in 10 days, at a medium renewal rate of 1 volume of medium per reactor volume and day (VVD). For adenovirus production, three 293S cell cultures were perfused at 1 VVD in parallel and infected at an average density of 8 x 10(6) cells/mL. One of the cultures was set at 37 degrees C and the two others at 35 degrees C. After a rapid initial cell loss, the average cell density stabilized at 5.75 x 10(6) cells/mL, 12 h postinfection, which was 8 times higher than the cell density in the batch control. This allowed the production of 3.2 x 10(9) infectious viral particles/mL (IVP/mL) at 37 degrees C and 7.8 x 10(9) IVP/mL at 35 degrees C, this last result being 5.5 times higher than the control. To our knowledge, this nonconcentrated titer is the highest value that has ever been published for adenovirus vector production. These observations lead to the conclusion that perfusion is an efficient tool to maintain, at high cell density, a specific production rate level sufficient to increase significantly the adenovirus volumetric production. Furthermore, it shows that perfusion at 35 degrees C can improve viral titer by 2.4-fold compared to 37 degrees C, in accordance with a previous study on adenovirus batch production. 相似文献
14.
A high-yielding serum-free, suspension cell culture process to manufacture recombinant adenoviral vectors for gene therapy 总被引:3,自引:0,他引:3
G. Schoofs T. J. Monica J. Ayala J. Horwitz T. Montgomery G. Roth F. J. Castillo 《Cytotechnology》1998,28(1-3):81-89
We have developed an efficient, reproducible, and scaleable cell culture process for a recombinant adenoviral vector expressing
therapeutic transgenes for clinical trials. HEK 293 cells – which support the propagation of E1 deficient adenovirus – were
first adapted to serum free media and suspension growth. Subsequent studies focused on the infection, virus production and
harvest from suspension culture bioreactors. Future studies are planned to address the kinetics of adenovirus production in
HEK 293 as well as in other cell lines.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
15.
Adeno-associated virus (AAV)-based vectors belong to the most promising gene transfer vectors in clinical studies. To provide
vector for late-stage clinical trials as well as for a potential commercial phase, a scalable, cGMP-compliant process is required.
Nearly all vector production protocols currently approved in Phase I clinical trials rely on AAV production in adherent HEK
293 cells in the presence of serum. In this study, we present a helper- and serum-free production method of AAV vectors in
suspension-adapted HEK 293 cells. The method is based on plasmid transfection with 25 kDa linear polyethyleneimine. Compared
to existing methods, our system is highly scalable as cells grow in suspension, does not require animal-derived products or
the use of an exogenous virus (adenovirus or baculovirus) and yields genomic titers equal to those obtained in adherent HEK
293 cells in the presence of serum. Most importantly, work load and cost could be dramatically reduced in comparison to earlier
methods, when comparing the production of equivalent volumes of cell culture media. Thus, our protocol should appeal to both
basic research laboratories and cGMP manufacturing units. 相似文献
16.
Emily S. Seo Thomas Vargues David J. Clarke Duan Uhrín Dominic J. Campopiano 《Protein expression and purification》2009,65(2):179-184
Apoptosis is a major problem in animal cell cultures during production of biopharmaceuticals, such as recombinant proteins or viral vectors. A 293 cell line constitutively expressing vMIA (viral mitochondria-localized inhibitor of apoptosis) was constructed and examined on production of a model recombinant protein, green fluorescent protein (GFP) in the adenovirus-293 expression system, and on production of a model infectious adenoviral vector. vMIA-293 cells were more resistant than the parental 293 cells to apoptosis induced by either oxidative stress, or by adenovirus infection. The yield of GFP produced in vMIA-293 cell cultures was consistently higher (140%) compared to that in the parental cells. vMIA reduced production of adenovirus infectious particles, which was not due to a decline of adenovirus replication, since adenoviral DNA replication rate in vMIA-293 cells was higher than that in the parental cells.In conclusion, introduction of the vMIA gene into the 293 cell line is a promising strategy to improve recombinant protein production in the adenovirus-293 expression system. 相似文献
17.
Dormond E Meneses-Acosta A Jacob D Durocher Y Gilbert R Perrier M Kamen A 《Biotechnology and bioengineering》2009,102(3):800-810
Safety requirements for adenoviral gene therapy protocols have led to the development of the third generation of vectors commonly called helper-dependent adenoviral vectors (HDVs). HDVs have demonstrated a high therapeutic potential; however, the poor efficiency and reliability of the actual production process hampers further large-scale clinical evaluation of this new vector. The current HDV production methods involve a preliminary rescue step through transfection of adherent cell cultures by an HDV plasmid followed by a helper adenovirus (HV) infection. Amplification by serial co-infection of complementary cells allows an increase in the HDV titer. Using a HEK293 FLP/frt cell system in suspension culture, an alternative protocol to the current transfection/infection procedure was evaluated. In this work, the adenofection uses the HDV plasmid linked to the HV with the help of polyethylenimine (PEI) and has shown to outperform standard protocols by producing higher HDV yield. The influence of complex composition on the HDV production was examined by a statistical design. The optimized adenofection and amplification conditions were successively performed to generate HDV at the 3 L bioreactor scale. Following only two serial co-infection passages, up to 1.44 x 10(8) HDV infectious units/mL of culture were generated, which corresponded to 26% of the total particles produced. This production strategy, realized in cell suspension culture, reduced process duration and therefore the probability of vector recombination by introducing a cost-effective transfection protocol, ensuring production of high-quality vector stock. 相似文献
18.
Bandaranayake AD Correnti C Ryu BY Brault M Strong RK Rawlings DJ 《Nucleic acids research》2011,39(21):e143
A key challenge for the academic and biopharmaceutical communities is the rapid and scalable production of recombinant proteins for supporting downstream applications ranging from therapeutic trials to structural genomics efforts. Here, we describe a novel system for the production of recombinant mammalian proteins, including immune receptors, cytokines and antibodies, in a human cell line culture system, often requiring <3 weeks to achieve stable, high-level expression: Daedalus. The inclusion of minimized ubiquitous chromatin opening elements in the transduction vectors is key for preventing genomic silencing and maintaining the stability of decigram levels of expression. This system can bypass the tedious and time-consuming steps of conventional protein production methods by employing the secretion pathway of serum-free adapted human suspension cell lines, such as 293 Freestyle. Using optimized lentiviral vectors, yields of 20-100 mg/l of correctly folded and post-translationally modified, endotoxin-free protein of up to ~70 kDa in size, can be achieved in conventional, small-scale (100 ml) culture. At these yields, most proteins can be purified using a single size-exclusion chromatography step, immediately appropriate for use in structural, biophysical or therapeutic applications. 相似文献
19.
Two Different Serum-free Media and Osmolality Effect Upon Human 293 Cell Growth and Adenovirus Production 总被引:1,自引:0,他引:1
Adenoviruses are promising vectors for gene therapy and vaccination protocols. Consequently, the market demands for adenovirus
are increasing, driving the search for new methodologies for large-scale production of concentrated vectors with warranted
purity and efficacy, in a cost-effective way. Nevertheless, the production of adenovirus is currently limited by the so-called
‘cell density effect’, i.e. a drop in cell specific productivity concomitant with increased cell concentration at infection.
Of two different serum-free culture media (CD293 and EX-Cell), evaluated for their effect on human 293 cells growth and adenovirus
production at cell densities higher than 1×106 cells/ml, EX-Cell proved the better medium for cell growth. Although adenovirus production was equivalent in both media when
the infection was performed at 1×106 cells/ml, at 3×106 cells/ml CD293 was the better. This result related to the high ammonia content in EX-Cell medium at the highest cell concentration
at infection. Besides this, the large-scale production of these vectors at high cell densities often requires re-feed strategies,
which increase medium osmolality. While a negative effect on cell growth was observed with increasing osmolalities, adenovirus
productivity was only affected for osmolalities higher than 430 mOsm.
Revisions requested 24 August 2005; Revisions received 9 September 2005 相似文献
20.
Robert Longley Jr. Laurie Radzniak Marc Santoro Yung-Shyeng Tsao Russell G. G. Condon Peggy Lio Marcio Voloch Zhong Liu 《Cytotechnology》2005,49(2-3):161-171
Conditionally replicating adenoviruses (CRAVs) are a group of recombinant human adenoviruses genetically engineered to replicate
in selected tissues, such as tumors. These viruses could potentially offer significant medicinal benefits, since the restrictive
replication of these viral vectors leads to the lysis of target cells without harm to the surrounding tissues. The in vitro
propagation and amplification of the CRAV vectors often requires special host cells with deregulated growth control pathways.
In order to develop an efficient cell culture process for the scaleable production of a CRAV vector, A549 cells, a human lung
carcinoma cell line normally cultured in adherent culture, were adapted to suspension culture. CRAV production was demonstrated
with the suspension-adapted A549 cells and a baseline production process was developed in shake flasks. The ability to scale-up
virus production was confirmed in stirred tank bioreactors. Molecular characterization of the suspension-adapted A549 cells
indicates no significant changes in cellular mechanisms related to adenovirus infection. 相似文献