Modelling baculovirus infection of insect cells in culture

Conclusions Infection of insect cells with baculovirus is a potentially attractive means for producing both viral insecticides and recombinant proteins. The continuation of mathematical modelling studies such as those reviewed in this paper are essential in order to realise the full potential of the system. Through mathematical models it is possible to predict complex behaviours such as those observed when infecting cells at low MOI or when propagating virus in a continuous culture system. A purely empirical analysis of the same phenomena is very difficult if not impossible.The present three models are — despite their complexity and the effort that has gone into developing them — all first generation models. They summarise, to a large extent, our present quantitative understanding of the interaction between baculovirus and insect cells, when looked upon as a black box system. The binding and initial infection processes are still quantitatively poorly understood and further work in this area is much needed. On the longer term, a second generation of models is likely to consider interior processes such as viral DNA and RNA accumulation in much more detail using a structured model of the infection cycle.     

利用多基因缺失型杆状病毒在昆虫细胞中生产腺相关病毒

腺相关病毒(adeno-associated virus, AAV)是基因治疗领域最常使用的病毒载体之一,产量低、成本高是该产业面临的关键瓶颈问题。本研究旨在基于多基因缺失型杆状病毒,建立双病毒感染昆虫细胞以生产AAV的技术体系。首先,进行AAV生产用多基因缺失型重组杆状病毒的构建和扩增,并检测杆状病毒滴度及其感染细胞的效果;然后,使用双杆状病毒共感染昆虫细胞,并优化感染条件;最后,基于优化条件进行AAV生产,并检测评估产量、质量等相关指标。结果表明,AAV生产用多基因缺失型杆状病毒滴度较野生型无差异,感染后细胞存活率下降明显减缓。使用双病毒路线进行AAV优化生产,Bac4.0-1的基因组滴度为1.63×10¹¹ VG/mL,Bac5.0-2的基因组滴度为1.02×10¹¹ VG/mL,较野生型产量分别提升了240%和110%。电镜下,3组均具有正常的AAV病毒形态,且转导活性相近。本研究建立了基于多基因缺失型杆状病毒感染昆虫细胞的AAV生产体系,显著提高了AAV产量,具有一定的应用价值。     

昆虫细胞制备AAV-ITR基因表达微载体

AAV-ITR基因表达微载体是只含有腺相关病毒(Adeno-associated virus,AAV)倒置末端重复序列(Inverted terminal repeats,ITR)、基因表达顺式元件和目的基因,而不含有其他外源DNA序列的双链或单链DNA。本研究利用杆状病毒表达系统,制备得到两种重组杆状病毒Bac-ITR-EGFP和Bac-inrep,并将二者的P3代病毒共同感染昆虫细胞Spodoptera frugiperda(Sf9),抽提小分子量DNA,获得AAV-ITR-EGFP基因表达微载体,2×107的Sf9细胞抽提可以得到100μg AAV-ITR-EGFP基因表达微载体,核酸电泳显示AAV-ITR-EGFP基因表达微载体主要以单体和二聚体的形式存在。将AAV-ITR-EGFP基因表达微载体通过polyethylenimine(PEI)转染HEK 293T细胞,24 h后荧光显微镜观察有EGFP表达,48 h后达到高峰,转化效率达到65%。     

Production of adeno-associated viral vectors in insect cells using triple infection: optimization of baculovirus concentration ratios

The production of viral vectors or virus-like particles for gene therapy or vaccinations using the baculovirus expression system is gaining in popularity. Recently, reports of a viral vector based on adeno-associated virus (AAV) produced in insect cells using the baculovirus expression vector system have been published. This system requires the triple infection of cells with baculovirus vectors containing the AAV gene for replication proteins (BacRep), the AAV gene for structural proteins (BacCap), and the AAV vector genome (BacITR). A statistical approach was used to investigate the multiplicities of infection of the three baculoviruses and the results were extended to the production of AAVs containing various transgenes. Highest AAV yields were obtained when BacRep and BacCap, the baculovirus vectors containing genes that code for proteins necessary for the formation of the AAV vector, were added in equal amounts at high multiplicities of infection. These combinations also resulted in the closest ratios of infectious to total AAV particles produced. Overexpression of the AAV structural proteins led to the production of empty AAV capsids, which is believed to overload the cellular machinery, preventing proper encapsidation of the AAV vector transgene, and decreased the viability of the insect cells. Delaying the input of BacCap, to reduce the amount of capsids produced, resulted in lower infectious AAV titers then when all three baculoviruses were put into the system at the same time. The amount of BacITR added to the system can be less than the other two without loss of AAV yield.     

Enzyme kinetics of a highly purified mitochondrial creatine kinase in comparison with cytosolic forms

Summary Mitochondrial creatine kinase (CK) purified from canine myocardium showed a single protein band on SDS-PAGE and was free of MMCK. Its amino acid composition was different than MMCK or BBCK and did not react to antiserum to MMCK or BBCK. Using purified mitochondrial, MM and BBCK, the velocity of reaction (V) was estimated for creatine phosphate (CP), creatine (C), adenosine triphosphate (ATP) and adenosine diphosphate (ADP) over a wide range of concentrations including those at V_max. The values for K_m (mM/L) derived from Lineweaver-Burke plots are shown: The affinity of mitochondrial CK for C is much greater than MMCK which is compatible with the energy shuttle hypothesis, namely ATP is converted by mitochondrial CK to CP, and then diffuses to the myofibril for conversion to ATP for utilization.     

Strong buffering capacity of insect cells. Implications for the baculovirus expression system

Insect cells are widely used for expression of a variety of different proteins by using the baculovirus expression system. The applicability of this system depends on production of proteins which have biological properties similar to their native counterparts. One application has been the expression of viral capsid proteins and their assembly into empty capsid structures to provide new viral immunogens which retain complex antigenic sites. An important parameter for efficient folding and assembly of proteins into viral procapsids may be the intracellular pH, particularly for acid-labile particles such as foot-and-mouth disease virus (FMDV). Benzoic acid was used as an effective indicator of intracellular pH in insect cells and 3-O-methyl glucose to measure cell volumes. We have determined the intracellular volume of theSpodoptera frugiperda IPLB-Sf21 insect cells 0.50±0.08 pL per cell. Using the distribution of [¹⁴C]-benzoic acid, we show that the intracellular pH remains constant at pH 7.0 when the cells are grown in media with pH values ranging from 6.2 to 6.8 and, moreover, is not affected by baculovirus infection. These results suggest that insect cells are suitable to express and produce acid-labile structures via the baculovirus expression system and that assembly of proteins and viral procapsids could occur.     

Production of human c-myc protein in insect cells infected with a baculovirus expression vector.

A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells. By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera. The insect-derived proteins were compared with recombinant human c-myc-encoded proteins synthesized in Escherichia coli and Saccharomyces cerevisiae cells. The c-myc gene product was found predominantly in the nucleus by subcellular fractionation of infected insect cells.     

Synthesis of the herpes simplex virus type 1 trans-activator Vmw65 in insect cells using a baculovirus vector

Vmw65, the Herpes Simplex Virus trans-activator of immediate-early genes, was expressed in insect cells using a recombinant baculovirus expression vector and partially purified. Insect cell-derived Vmw65 was shown to be indistinguishable from authentic Vmw65 present in purified HSV-1 virions based on electrophoretic mobility, immunoreactivity with a monoclonal antibody, and ability to interact with cellular factors to form a protein/DNA complex with oligonucleotides containing a TAATGARAT element.Abbreviations AcNPV Autographica californica nuclear polyhedrosis virus - HSV Herpes Simplex Virus - IE Immediate Early - moi multiplicity of infection - Sf9 Spodoptera frugiperda cells     

Genetic modification of a baculovirus vector for increased expression in insect cells

Generating large amounts of recombinant protein in transgenic animals is often challenging and has a number of drawbacks compared to cell culture systems. The baculovirus expression vector system (BEVS) uses virus-infected insect cells to produce recombinant proteins to high levels, and these are usually processed in a similar way to the native protein. Interestingly, since the development of the BEVS, the virus most often used (Autographa californica multi-nucleopolyhedovirus; AcMNPV) has been little altered genetically from its wild-type parental virus. In this study, we modified the AcMNPV genome in an attempt to improve recombinant protein yield, by deleting genes that are non-essential in cell culture. We deleted the p26, p10 and p74 genes from the virus genome, replacing them with an antibiotic selection cassette, allowing us to isolate recombinants. We screened and identified recombinant viruses by restriction enzyme analysis, PCR and Western blot. Cell viability analysis showed that the deletions did not improve the viability of infected cells, compared to non-deletion viruses. However, expression studies showed that recombinant protein levels for the deletion viruses were significantly higher than the expression levels of non-deletion viruses. These results confirm that there is still great potential for improving the BEVS, further increasing recombinant protein expression yields and stability in insect cells.     

以杆状病毒为载体在鸡原代骨骼肌细胞中表达IBDV VP2基因

摘要：【目的】本研究旨在构建在鸡原代骨骼肌细胞中表达IBDV病毒VP2基因的重组杆状病毒。【方法】从IBDV适应细胞毒中提取RNA,用RT-PCR技术扩增VP2基因,将其克隆到自主构建的杆状病毒转移载体的CMV启动子之下,通过Bac-to-Bac系统获得VP2重组Bacmid,并将其转染Sf9昆虫 细胞,获得了VP2重组杆状病毒。重组病毒经扩增后以50个MOI感染鸡原代骨骼肌细胞,接种72h后裂解细胞收获蛋白。【结果】蛋白样品经SDS-PAGE和Western blot证实VP2蛋白获得表达,分子量约48kDa,与预测蛋白大小一致,且能被IBDV阳性血清所识别。【结论】重组杆状病毒可以有效地将VP2基因导入鸡原代细胞,并在CMV的启动下表达具有抗原性的VP2蛋白,本研究为研制IBDV及其他重要禽类传染病的杆状病毒载体疫苗奠定了基础。     

Assessment of virus production and chloramphenicol acetyl transferase expression by insect cells in serum-free and serum-supplemented media using a temperature-sensitive baculovirus

Spodoptera frugiperda insect cells were grown in Sf-900 serum-free medium and two kinds of serum-supplemented media (IPL -41 and Grace's). The specific growth rates of uninfected cells were found to be 0.024, 0.35, and 0.034 h(-1) respectively, at 33 degrees C. The IPL -41 medium supported to highest maximum cell density (10.6 x 10(6) cells/mL) compared to 3.5 x 10(6) and 8.7 x 10(6) cells/mL with the Grace's and serum-free media, respectively. In temperature shifdown experiments with a temperature-sensitive baculo-virus (acts10YM1CAT), virus titer and chloramphenicol acetyl transferase (CAT) expression were highest in the IPL -41 (5.1 x 10(7) PFU/mL and 20000 U/mL). Use of Grace's medium gave higher virus titers than the serum-free medium (4.4 x 10(6) vs 4.1 x 10(5) PFU/mL) as well as higher CAT titers (7050 vs 1980 U/mL). Interestingly, in the three media used, the highest virus and CAT titers were obtained at MOI (multiplicity of infection) of 0.02 At MOI of 2.0 virtually no increase in virus of CAT titer was observed. This result is contrary to those obtained at constant-temperature (27 degrees C) infection and cell culture, in which higher virus titers and recombinant protein expression and obtained at higher MOI.     

人类细小病毒B19壳蛋白VP2在昆虫杆状病毒表达系统中的表达

利用BAC-TO-BAC系统获得了人类细小病毒B19壳蛋白VP2的重组昆虫杆状病毒,并在sf9细胞中表达出VP2。用蚀斑法纯化病毒,终末稀释法测定病毒滴度为3.6×108。Western印迹检测证实了表达蛋白的特异性,间接免疫荧光法可观察到细胞胞浆中的表达蛋白颗粒。     

Thyroid hormones and the creatine kinase system in cardiac cells

The paper reviews the current evidence on the role of thyroid hormones in regulating the creatine kinase energy transfer system at multiple structures in cardiac cells. 1) Thyroid hormones modulate the overall synthesis of phosphocreatine (PCr) by increasing the rate of mitochondrial oxidative phosphorylation. 2) Thyroid hormones regulate the total activity of creatine kinase and its isoenzyme distribution. In comparison with normal thyroid state (euthyroidism), hypothyroidism is characterized by decreased total creatine kinase activity owing to diminished fraction of creatine kinase. On the other hand, hyperthyroidism, while causing no change in total creatine kinase activity, leads to increased fractions of neonatal isoforms of creatine kinase, and, in case of prolonged hyperthyroidism, to decreased fraction of mitochondrial creatine kinase. The latter change is associated with partial uncoupling between mitochondrial creatine kinase and adenine nucleotide translocase reflected by decreased PCr/O ratio. 3) Hyperthyroidism leads to increased passive sarcolemmal permeability due to which the leakage of creatine along its concentration gradient occurs. As a result of (i) increased sarcolemmal permeability for creatine, (ii) uncoupling of mitochondrial PCr synthesis, and (iii) increased energy utilization rate the steady state intracellular PCr content decreases under hyperthyroidism which, in turn, increases the myocardial susceptibility to hypoxic damage. Thyroid state also modulates the protective effects of exogenous PCr on energetically depleted myocardium.     

Production of selenomethionyl-derivatized proteins in baculovirus-infected insect cells

A protocol is described for the production of both intracellularly expressed and secreted selenomethionyl-derivatized recombinant proteins in baculovirus-infected insect cells. The method results in the production of recombinant soluble proteins with an SeMet occupancy of approximately 75% and with a recovery of approximately 20% that of native protein expression. The method is independent of the percentage methionine content of the protein and is reliable and consistent. Similar results are obtained using either Spodoptera frugiperda Sf9 or Trichoplusia ni High Five insect cells as the expression host, and when cultures are grown in either shake flasks or in Wave BioReactors.     

Modeling and optimization of the baculovirus expression vector system in batch suspension culture

A mathematical model has been developed that predicts the cell population dynamics and production of recombinant protein and infective extracellular virus progeny by insect cells after infection with baculovirus in batch suspension culture. Infection in the model is based on the rate of virus attachment to suspended insect cells under culture conditions. The model links the events following infection with the sequence of gene expression in the baculovirus replicative cycle. Substrate depletion is used to account for the decrease in product yield observed when infecting at high cell densities. Model parameters were determined in shaker flasks for two media: serum-supplemented IPL-41 medium and serum free Sf900II medium. There was good agreement between model predictions and the results from an independent series of experiments performed to validate the mode. The model predicted: (1) the optimal time of infection at high multiplicity of infection: (2) the timing and magnitude of recombinant protein production in a 2-L bioreactor; and (3) the timing and magnitude of recombinant protein production at multiplicities of infection from 0.01 to 100 plaque-forming units per cell. Through its ability to predict optimal infection strategies in batch suspension culture, the model has use in the design and optimization of large-scale systems for the production of recombinant products using the baculovirus expression vector system. (c) 1994 John Wiley & Sons, Inc.     

Expression of the human EGF receptor with ligand-stimulatable kinase activity in insect cells using a baculovirus vector.

The mechanism by which the binding of epidermal growth factor (EGF) to specific cell surface receptors induces a range of biological responses remains poorly understood. An important part of the study of signal transduction in this system involves the production of sufficient native and mutant EGF receptor species for X-ray crystallographic and spectroscopic analysis. Baculovirus vectors containing the cDNA encoding the human EGF receptor protein have here been utilized to infect insect cells. This results in expression of a 155-kb transmembrane protein which is recognized by four antibodies against different regions of the human EGF receptor. Studies with tunicamycin, monensen and endoglycosidase H show the difference in size between the recombinant and the native receptor is due to alterations in glycocsylation. Studies of [125I] EGF binding shows a Kd of 2 X 10(-9) M in intact infected insect cells which falls to 2 X 10(-7) M upon detergent solubilization. The recombinant protein exhibits an EGF-stimulated tyrosine protein kinase activity and an analysis of tryptic peptides shows that the phosphate acceptor sites are similar to those of the EGF receptor isolated from A431 cells. These observations indicate that functional EGF receptor can be expressed in insect cells, and furthermore, this system can be used for large-scale production.