The establishment of new cell lines from Pseudaletia unipuncta with differential responses to baculovirus infection

Summary Six insect cell lines from Pseudaletia unipuncta embryos were established and characterized, and their susceptibility to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infection was investigated. These embryonic P. unipuncta cell lines had characteristics distinct from each other in morphology and growth, and showed differential responses to AcMNPV infection. Among the six cell lines, two were highly susceptible to virus infection. One of these two cell lines, BTI-Pu-A7S, produced over 100 AcMNPV occlusion bodies per cell, on average. Three cell lines showed an apoptotic response following AcMNPV infection. One cell line did not support complete virus replication through the late phase of virus growth and did not exhibit apoptosis. The P. unipuncta cell lines could be distinguished from SF21 and BTI-Tn-5B1-4 cells by their isozyme markers.     

Virus-like particle production at low multiplicities of infection with the baculovirus insect cell system

The baculovirus insect cell expression system (BEVS) was used for the production of self-forming Porcine parvovirus-like particles (VLPs) in serum-free medium. A low multiplicity of infection (MOI) strategy was used to overcome an extra virus amplification step, undesirable in industrial production, and to minimize the virus passage effect. It was confirmed that the time of infection (TOI) and MOI are dependent variables. Higher cell densities were obtained at low MOIs, keeping a constant TOI; however, both volumetric and specific productivities were lower. In synchronous infection, at high MOI, the specific productivity decreased when the cells were infected in the late phase of growth. Product degradation due to cell lysis strongly influenced the optimal time of harvest (TOH). Time of harvest was found to be highly dependent on the MOI, and a direct relationship with the cell yield was obtained.Analysis of the culture medium reveals that glutamine depletion occurs in the late phase of the growth. Supplementation of glutamine to uninfected cell cultures resulted in an increased cell yield. Its addition to cultures infected in the middle phase of the growth curve was also able to restore the productivity levels, but addition to cells in their stationary phase caused no observable effect on product expression. The study clearly shows that for a specific TOI it is not obvious what the correct MOI should be to obtain the best volumetric productivity.     

Manipulation of baculovirus vectors

Baculovirus expression vectors provide an excellent system for the synthesis of recombinant proteins in insect cells. This article presents sufficient background information to allow the nonspecialist to understand the basic principles of the technology and the development of baculovirus expression vectors. A summary of the most commonly used plasmids and viruses is presented. Detailed techniques are described to enable recombinant baculoviruses to be constructed. These methods include the protocols required for propagating insect cells in culture and their subsequent infection with viruses.     

杆状病毒AcMNPV vp39基因的克隆、表达与抗体制备

目的:构建苜蓿丫纹夜蛾核多角体病毒(Autographa californica nucleopolyhedro virus,AcMNPV)VP39的原核表达载体,表达、纯化蛋白并制备多克隆抗体。方法:用PCR方法扩增vp39基因,并将其克隆至pET-21a( )上,转化到大肠杆菌BL21(DE3)中进行诱导表达,采用割胶回收的方法纯化融合蛋白,纯化的融合蛋白作为抗原,免疫新西兰大白兔,Western blot检测抗体活性。结果:构建了pET-VP39原核表达质粒,含有该质粒的大肠杆菌经IPTG诱导超量表达了一个与预期理论值相符的约为40kDa的融合蛋白。对制备的抗体进行免疫印迹分析表明该抗血清能与感染苜蓿丫纹夜蛾核多角体病毒的细胞蛋白样品发生特异性反应。结论:获得了兔抗AcMNPV-VP39多克隆抗体,为进一步深入研究VP39在病毒侵染过程中与宿主因子的相互作用提供了检测工具。     

Mutational analysis of a baculovirus major late promoter

We have used a linker-scan mutation strategy to analyze P_cap99, the proximal promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) gene encoding the major capsid protein. A series of recombinant viruses expressing the cat reporter gene under the control of selected mutants of this promoter was constructed. Only mutations that altered bases within a region extending from 8 bp upstream to 6 bp downstream from a TAAG sequence had a significant effect on expression from the late gene promoter. A synthetic promoter consisting of only these 18 bp (P_capmin) was sufficient to direct late expression. Aside from this small region surrounding the TAAG, no evidence for distinct late activating or repressing sequence elements was obtained. Experiments comparing and combining late and very late gene promoter sequences suggest that late expression is intrinsically determined by the presence and immediate context of a TAAG sequence and that very late expression [as previously shown in Ooi et al. J. Mol. Biol. 210 (1989) 721–736] results from additional modulation of TAAG-dependent expression by downstream promoter elements placed in an appropriate context. A compact combination promoter (95 bp), constructed by fusing P_capmin to linker-modified very late polyhedrin promoter, directs strong expression at late and very late times post-infection.     

Evaluation of viral contamination in a baculovirus expression system

Insect expression systems based on baculovirus are widely used for generating recombinant proteins. Here, the infectivity of baculoviruses under the physiological stresses of ‘freeze–thaw’ and sonication and the baculoviral contamination of recombinant proteins after protein purification were evaluated. Our findings suggest that Nonidet P‐40 (NP‐40) treatment of baculoviruses completely abolishes their infectivity and that recombinant proteins purified with affinity beads do not include infectious baculoviruses. We therefore suggest that baculovirus is completely inactivated by NP‐40 treatment and that recombinant proteins are unlikely to be contaminated with infectious baculoviruses after their affinity purification.

     

A suspended cell line from Trichoplusia ni (Lepidoptera): Characterization and expression of recombinant proteins

A suspended cell line from Trichoplusia ni embryos was established, and its susceptibility to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infection was investigated. This cell line had characteristics distinct from the BTI‐Tn5Bl‐4 cell line (Tn5Bl‐4) from T. ni in growth, and showed approximately the same responses to AcMNPV infection, production of occlusion bodies, and levels of recombinant protein expression. No clumps were observed at maximum cell density at late‐log phase in shake‐flask or T‐flask cultures, and thus the cells represent a useful new contribution for baculovirus research. The cells consist of two major morphological types: approximately 70% spindle‐shaped cells and 30% round cells. The cell line was highly susceptible to virus infection and produced around 107 AcMNPV occlusion bodies per cell, on average. Production of β‐galactosidase and secreted alkaline phosphatase was high with 3.97 ± 0.13 × 10⁴IU/mL and 3.48 ± 0.40 IU/mL, respectively. This cell line may be applicable for studies of scale‐up production of viruses or baculovirus‐insect cell expression. We also believe the new line can be a source for cell clones with higher production of virus and recombinant proteins compared to the parent or other existing cell lines such as Tn5Bl‐4.     

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.     

Human monocyte chemoattractant protein-1 expressed in a baculovirus system

Human monocyte chemoattractant protein-1 (hMCP-1) was produced using a baculovirus system. The hMCP-1 cDNA was inserted into the genomic DNA of Autographa californica nuclear polyhedrosis virus (AcNPV) using a transfer vector, pJVP10Z. Spodoptera frugiperda insect cells, which were infected with this recombinant virus, secreted recombinant hMCP-1 (re-hMCP-1) at the level of 10–20 μg/ml of culture medium. This product was shown to chemoattract monocytes. Three distinct bands of 11,11.5 and 12 kDa were revealed by immunoblotting analysis, and this heterogeneity was assigned to differences in carbohydrate processing. N-terminal amino-acid sequence analysis of the purified product revealed identity with hMCP-1. Thus, in this system, re-hMCP-1 was produced in large quantities and modified in a manner similar to native hMCP-1.     

Expression and purification of the matrix protein of Nipah virus in baculovirus insect cell system

Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N‐terminally His‐tagged NiV M protein in insect cells. A time‐course study demonstrated that the highest yield of recombinant M protein (400–500 μg) was expressed from infected cells 3 days after infection. A single‐step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme‐linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:171–177, 2016     

Cell lines used for the selection of recombinant baculovirus

Summary Four insect cell lines were used to isolate two recombinant baculoviruses which had theβ-galactosidase (β-gal) gene for colorimetric assay purposes. Plaque assays were performed using twoTrichoplusia ni cell lines: BTI-TN-5B1-4 and TN-368, and twoSpodoptera frugiperda cell lines: IPLB-SF-21AE and SF9. The number of plaques (occlusion positive and blueβ-gal⁺ recombinants) formed in theTrichoplusia cells was higher than in theSpodoptera cells. The appearance ofAutographa californica NPV polyhedra was also faster in theT. ni cell lines. The effect of cell passage on the plaque formation proved to be critical when two different passages of the SF9 cells were tested. The higher passage produced a lower viral titration. The size and time of appearance of the plaques was also different.     

昆虫杆状病毒表达载体系统在疫苗研究中的应用进展

昆虫杆状病毒表达载体系统(Baculovirus expression vector system,BEVS)已成功应用于多种蛋白的表达,并为疫苗开发提供了充足的原材料。相比其他表达系统,BEVS具有许多优势:杆状病毒专一寄生于无脊椎动物,安全性高;重组蛋白表达水平高;可对重组蛋白进行正确折叠和翻译后修饰,获得具有生物活性的蛋白;适应于多基因表达如病毒样颗粒(Virus-like particle)的复杂设计;适用于大规模无血清培养等。为了更好地理解BEVS在疫苗研究中的应用前景,文中将从BEVS的发展及其在疫苗研究中的应用等方面进行综述。     

Protein production using the baculovirus‐insect cell expression system

The baculovirus‐insect cell expression system is widely used in producing recombinant proteins. This review is focused on the use of this expression system in developing bioprocesses for producing proteins of interest. The issues addressed include: the baculovirus biology and genetic manipulation to improve protein expression and quality; the suppression of proteolysis associated with the viral enzymes; the engineering of the insect cell lines for improved capability in glycosylation and folding of the expressed proteins; the impact of baculovirus on the host cell and its implications for protein production; the effects of the growth medium on metabolism of the host cell; the bioreactors and the associated operational aspects; and downstream processing of the product. All these factors strongly affect the production of recombinant proteins. The current state of knowledge is reviewed. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:1–18, 2014     

杆状病毒表达系统的发展

杆状病毒是近年来被广泛用于高效表达外源蛋白的载体系统,本文就杆状病毒表达系统的生物学特性、转染载体、重组病毒的筛选、基因表达调控及其发展应用等方面作一概述。     

Generation of baculovirus vectors for the high-throughput production of proteins in insect cells

The baculovirus expression system is one of the most popular methods used for the production of recombinant proteins but has several complex steps which have proved inherently difficult to adapt to a multi-parallel process. We have developed a bacmid vector that does not require any form of selection pressure to separate recombinant virus from non-recombinant parental virus. The method relies on homologous recombination in insect cells between a transfer vector containing a gene to be expressed and a replication-deficient bacmid. The target gene replaces a bacterial replicon at the polyhedrin loci, simultaneously restoring a virus gene essential for replication. Therefore, only recombinant virus can replicate facilitating the rapid production of multiple recombinant viruses on automated platforms in a one-step procedure. Using this vector allowed us to automate the generation of multiple recombinant viruses with a robotic liquid handler and then rapidly screen infected insect cell supernatant for the presence of secreted proteins.     

杆状病毒表达系统研究进展

介绍了杆状病毒表达系统的构建策略,载体发展情况及其表达外源基因的影响因素.杆状病毒表达系统在基因工程、药物开发、疫苗生产等方面发挥了越来越重要的作用,其表达效率高,表达产物与天然产物有相似的结构和活性,且对人畜无害,为当今基因工程研究中最有发展前途的表达系统.