In vitro and in vivo host range of Anticarsia Gemmatalis multiple nuclear polyhedrosis virus

Summary A clone of the wild type (wt) Anticarsia gemmatalis multiple nuclear polyhedrosis virus AgMNPV, derived from a geographical isolate (Hondrina, Brazil) and designated AgMNPV-CL4-3A1, was used to determine the host range of this virus in six established lepidopteran cell lines: Anticarsia gemmatalis (BCIRL-AG-AM1), Helicoverpa zea (BCIRL-HZ-AM1), Heliothis virescens (BCIRL-HV-AM1), Helicoverpa armigera (BCIRL-HA-AM1), Trichoplusia ni (TN-CL1), Bombyx mori (BMN), and a coleopteran cell line Anthonomus grandis (BRL-AG-1). In addition, the in vivo host range of this clone was also assayed in larvae of Helicoverpa zea, Heliothis virescens, Trichoplusia ni, and the homologous species Anticarsia gemmatalis by probit analysis. On the basis of temporal studies of TCID₅₀ values, BCIRL-HV-AM1 cells gave the highest extracellular virus (ECV) titer (9.7×10⁶ TCID₅₀/ml) followed by BCIRL-HA-AM1 cells (8.3×10⁵ TCID₅₀/ml) and BCIRL-AG-AM1 cells (3.2×10⁵ TCID₅₀/ml). In addition, a low ECV titer of 1.37×10³ TCID₅₀/ml was detected from TN-CL1 cells 96 h postinoculation, while BRL-AG-1, BMN, and BCIRL-HZ-AM1 cells were nonpermissive to AgMNPV-CL4-3A1 on the basis of TCID₅₀ results. AgMNPV-CL4-3A1 and the wild type AgMNPV had similar restriction profiles that were different from wild type AcMNPV. The LC₅₀ values were 96.9, 564.6, 733.3, and 1.1×10⁴ occlusion bodies/cm² of diet for A. gemmatalis, Helicoverpa zea, Heliothis virescens, and T. ni, respectively. This article presents the results of research only. Mention of proprietary products in this article does not indicate endorsement or a recommendation for use by USDA-ARS. All programs and services of the USDA are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, marital status or handicap.     

AcMNPV in permissive, semipermissive, and nonpermissive cell lines from arthropoda

Summary Insect cell lines from Arthropoda represented by Lepidoptera, Coleoptera, Diptera, and Homoptera were evaluated for their ability to support replication of AcMNPV. In addition, some of the cell lines that were refractive to AcMNPV were tested with AcMNPV hsp70 Red, a recombinant carrying the red fluorescent protein (RFP) gene, for their ability to express this protein after inoculation. Of the 10 lepidopteran cell lines tested, only three cell lines from Helicoverpa zea (BCIRL-HZ-AM1), Lymantria dispar (IPLB-LD 65), and Cydia pomonella (CP-169) failed to support detectable viral replication as measured by tissue culture infectious dose 50 (TCID₅₀) assay. Heliothis virescens (BCIRL-HV-AM1) produced the highest viral titer of 2.3±0.1×10⁷ TCID₅₀/ml followed by Heliothis subflexa (BCIRL-HS-AM1) at 4.7±0.1×10⁶ TCID₅₀/ml and Spodoptera frugiperda (IPLB-SF21) at 4.1±0.1×10⁶ TCID₅₀/ml. None of the coleopteran, dipteran, or homopteran cell lines supported AcMNPV replication. However, when studies were performed using AcMNPV hsp70 Red, the dipteran cell lines Aedes aegypti (ATC-10) and Drosophila melanogaster (line 2), both expressed the RFP as well as the refractive lepidopteran cell lines from H. zea and L. dispar. No RFP expression was observed in any of the coleopteran or homopteran cell lines. Cell lines refractive to AcMNPV did not appear to be adversely affected by the virus, as judged by their ability to multiply, nor was there any indication of induced apoptosis, as assessed by deoxyribonucleic acid fragmentation profiles or cell blebbing or both. Disclaimer: Mention of trade names or commercial product in the publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U. S. Department of Agriculture. All programs and services of the U. S. Department of Agriculture are offered on a nondiseriminatory basis without regard to race, color, national origin, religion, sex, age marital status, or handicap.     

Production of selected baculoviruses in newly established lepidopteran cell lines

Summary One key to the in vitro mass production of baculoviruses is the development of insect cell lines capable of producing high levels of extracellular virus (ECV) and/or occlusion bodies (OBs). For this study, 34 newly established cell lines from 10 lepidopteran species were screened for their ability to produce ECV and OBs from a variety of baculoviruses. The selected baculoviruses included: the alfalfa looper virus (AcMNPV); the celery looper virus (AfMNPV); the velvetbean caterpillar virus (AgMNPV), the bollworm virus (HzSNPV), the diamondback moth virus (PxMNPV), and the beet armyworm virus (SeMNPV). ECV titers were determined using TCID₅₀ assays (50% tissue culture infectivity dose), with the presence or absence of OBs being noted. For AcMNPV, 28 new cell lines were tested, with eight producing AcMNPV ECV titers of 1.1–47.3×10⁶ TCID₅₀/ml and 11 producing OBs. For AgMNPV, six new cell lines were tested, with all producing AgMNPV ECV titers of 3.5–62.3×10⁶ TCID₅₀/ml and generating OBs. For HzSNPV, four new cell lines were tested with three lines producing HzSNPV ECV titers of 1.4–5.0×10⁶TCID₅₀/ml, but none generating OBs. For PxMNPV, 10 new cell lines were tested with seven generating PxMNPV ECV titers of 4.7–232.6×10⁶TCID₅₀/ml and eight producing OBs. Lastly, using qualitative or semiquantitative methods, homologous cell lines were tested for AfMNPV and SeMNPV production, all of which produced OBs. Overall, many of the cell lines tested were found to produce OBs and generate moderate to high levels of ECVs of one or more baculoviruses. All programs and services of the USDA Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status or handicap.     

In vitro host range of the Hz-1 nonoccluded virus in insect cell lines

A total of 13 insect cell lines spanning 4 orders (Lepidoptera, Coleoptera, Diptera, and Homoptera) were tested for their ability to replicate the nonoccluded virus Hz-1. Only the Lepidopteran cell lines supported replication of the virus with TN-CL1 and BCIRL-HZ-AM1 producing the highest titers of 2.4 × 10⁸ tissue culture infective dose (TCID)₅₀/ml and 2.0 × 10⁸ TCID₅₀/ml, respectively. A codling moth cell line (CP-169) was the only Lepidopteran cell line that did not replicate the virus and transfection of this cell line with Hz-1 DNA failed to replicate the virus. Also, transfection with DNA from a recombinant baculovirus carrying the red fluorescent protein gene (AcMNPVhsp70 Red) was not expressed in CP-169 cells. The replication cycle of Hz-1 in BCIRL-HZ-AM1 cells showed that this virus replicated rapidly starting at 16 h postinoculation (p.i.) and reaching a peak titer of 1.0 × 10⁸ TCID₅₀/ml 56 h postinoculation. Hz-1 when compared with several other baculoviruses has the widest in vitro host spectrum.     

Replication ofAutographa californica baculovirus (AcMNPV) in a coleopteran cell line

Summary A coleopteran cell line (AGE) derived from the cotton boll weevilAnthonomus grandis supported replication ofAutographa californica multiple nuclear polyhedrosis virus (AcMNPV). The titer of extracellular virus (ECV) and the number of occlusion bodies (OB) produced in AGE cells were approximately equal to those produced by aTrichoplusia ni cell line (TN-CL1), and the OB produced by both cell lines were equally infectious forT. ni larvae. The identity of the AGE cell line was established by chromosome and isoenzyme analyses.     

Effects of long- and short-term passage of insect cells in different culture media on baculovirus replication

Two insect cell lines that had been maintained in both serum-free (SFM) and serum-containing (SCM) media for over 5 years were each tested for their ability to replicate baculovirus. The gypsy moth cell line, IPLB-LdEIta (Ld), produced similar (not statistically different) amounts of gypsy moth nucleopolyhedrovirus (LdMNPV) occlusion bodies (OBs) in the two media (serum-free Ex-Cell 400 and TC-100 with 9% (v/v) fetal bovine serum, SCM(1)) but produced more of the Autographa californica nucleopolyhedrovirus (AcMNPV) OBs in SFM than in SCM(1). When Ld cells normally grown in SCM(1) were switched to SFM, production of OBs from both viruses improved and, after three passages, reached higher levels of AcMNPV production than in cells normally maintained in that medium. Alternatively, cells switched from SFM to SCM(1) initially produced as much (in the case of LdMNPV) or higher (in the case of AcMNPV) levels of virus OBs than cells normally maintained in SCM(1) but productivity dropped off over subsequent passages such that after five passages in SCM(1), cells produced substantially fewer OBs of both viruses. A fall armyworm cell line (IPLB-SF21AE; Sf) showed slightly different effects from long- and short-term passage in SFM (Ex-Cell 400) or SCM(2) (TMN-FH). Cells maintained in SFM produced about 20 times more AcMNPV OBs than cells maintained long-term in SCM. Sf cells switched from SFM to SCM maintained the level of production of that seen in SFM at the first passage, but quickly dropped off OB production levels to that normally seen in SCM. Alternatively, SCM-maintained Sf cells produced higher levels at the first passage in SFM and, within five passages in SFM, reached levels found in cells maintained for long term in this medium. Under the conditions in which these two cell lines were infected, the highest levels of AcMNPV OB production in Ld cells were about five times that of Sf cells. In a separate series of experiments, cells normally grown in SFM were passaged over five times in Ex-Cell 400 to which serum was added; both cell lines produced as much virus as that in SFM. These results suggest that it is not the serum per se but rather some other components which differ between the SFM and the SCM formulations that are responsible for the varied virus production obtained in these studies. The results of these studies suggest that a maintenance and virus production protocol can be developed with Ld cells which could improve overall efficiency of virus production. These studies also suggest that long-term maintenance of cells in SFM was not detrimental to their ability to produce baculoviruses.     

Insect cells and their potential as stabilization barriers for DNA of multiple and single nucleopolyhedroviruses against ultraviolet-B-simulated sunlight inactivation

A cell line from Trichoplusia ni (TN-CL1) infected with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV-HPP) and a cell line from Helicoverpa zea (BCIRL-HZ-AM1) infected with the Helicoverpa zea single nucleopolyhedrovirus (HzSNPV/BrCL2) were subjected to ultraviolet-B (UV-B) irradiation at a predetermined level of exposure that would inactivate greater than 95% of the virus suspended in the liquid. The working hypothesis was that the homologous insect cells would utilize their inherent deoxyribonucleic acid (DNA) repair mechanism(s) to prevent, repair, or at least mitigate the damaging effects of UV-B light on viral DNA synthesis. We attempted to determine this by using infected cells that were subjected to UV-B irradiation at different postinoculation periods under two experimental conditions of exposure: (1) shielded, and (2) nonshielded. Of the two cell lines infected with their respective homologous viruses, the virus from TN-CL1 cells was the least sensitive to UV-B light because the extracellular virus (ECV) and occlusion body (OB) levels of virus-infected TN-CL1 cells were higher than those of the virus-infected BCIRL-HZ-AM1 cells. Production of ECV and OB from both cell lines was lower in the exposed, nonshielded treatment than in the exposed, shielded treatment. However, AcMNPV-HPP was produced in enough quantity to indicate that TN-CL1 might impart a level of protection to the virus against UV light.     

In vitro host range studies with a new baculovirus isolate from the diamondback moth Plutella xylostella (L.) (Plutellidae: Lepidoptera)

Summary The in vitro host range of a newly isolated baculovirus from the diamondback moth Plutella xylostella was tested against six lepidopteran cell lines. Two baculoviruses with host ranges from the alfalfa looper Autographa californica (A. californica multiple nucleopolyhedrovirus, AcMNPV) and the celery looper Anagrapha falcifera (AfMNPV) were also included in this study for comparative purposes. PxMNPV replicated in all six cell lines and produced occlusion bodies, with HV-AMI and TN-CLI cells producing the highest viral titers and greatest number of occlusion bodies. There was no significant replication of AcMNPV and AfMNPV in the HZ-FB33 cell line and thus no production of occlusion bodies. The restriction endonuclease profiles of the three baculoviruses showed similarities but could be readily distinguished from each other. Either HV-AM1 or TN-CL1 would be suitable cell lines for the in vitro production of PxMNPV.     

The establishment of heliothine cell lines and their susceptibility to two baculoviruses

Summary A total of eight cell lines were established from Helicoverpa armigera (3) and H. punctigera (5) embryos and ovaries. Cell lines were established and grown in TC100 and/or TC199-MK containing 10% fetal bovine serum. The serum-free medium ExCell™ 400 was also used, with and without 10% supplemental fetal bovine serum, but failed to generate cell lines from fat bodies, embryos, or ovarian tissues. Cell lines consisted of heterogenous cell types ranging from oval to fibroblast-like. This is the first report on the successful establishment of cell lines from H. punctigera. Cell lines from the two species were distinguishable from each other by DAF-PCR, and noticeable differences in minor bands were observed among cell lines from the same species. All of the established cell lines from both species were susceptible to HzSNPV but did not replicate more virus than that of a H. zea cell line (BCIRL-HZ-AM1-A11). However, an H. punctigera cell line (HP1) replicated AcMNPV to the highest titer (1.0×10⁸ 50% tissue culture infective dose/ml), and only one of the H. armigera cell lines (HA1) was susceptible to this virus.     

Maize Ac/Ds transposon system leads to highly efficient germline transmission of transgenes in medaka (Oryzias latipes)

As the medaka is a popular fish model in genetics, developmental biology and toxicology, the development of an efficient transgenic medaka technique is important for a variety of biological experiments. Here we demonstrated that the maize transposon system, Ac/Ds, greatly improved the transgenesis of microinjected DNA. Using the Ac/Ds system, two types of stable transgenic medaka lines, Tg(hsp70:gfp) and Tg(cyp1a1:gfp), were established with germline transmission rates of 83.3% (10/12) and 100.0% (4/4) from GFP-expressing founders, respectively. The percentages of transgenic progeny ranged between 3.1% and 100.0% in F1 from different transgenic founders. Interestingly, multiple insertions were found from transgenic founders and the cloned insertion sites confirmed the transposition mediated by Ac transposase. In addition, we demonstrated the inducible GFP expression in both GFP transgenic medaka lines. In Tg(hsp70:gfp) whose gfp gene was under the control of a heat shock inducible medaka hsp70 promoter, GFP expression was induced ubiquitously after heat shock. In Tg(cyp1a1:gfp), the gfp gene was driven by medaka cyp1a1 promoter that could be activated by various xenobiotic chemicals including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); indeed, GFP expression was found to be induced in the liver, intestine and kidney by TCDD. Our data presented here demonstrated the highly efficient transgenesis with the aid of the maize Ac/Ds transposon system.     

Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing,cell death and T-DNA loss

The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells fromAgrobacterium tumefaciens. Individual GFP-expressing protoplasts were isolated after 4 days' co-cultivation. The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression. Of the proto-calluses initially expressing GFP, 50% had lost detectable GFP activity during the first 4 weeks of culture. Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity. The remaining 7 cell lines contained no gfp sequences. Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced. In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.     

Expression ofβ-galactosidase and luciferase in insect cell lines infected with a recombinant AcMNPV

Summary An AcMNPV recombinant (Ac-gal-luc) carrying theβ-galactosidase and luciferase genes under the control of the p10 and polyhedrin promoters, respectively, was used to study expression in nine insect cell lines. All AcMNPV-permissive cell lines expressed both reporter genes with the coleopteran cell line,Anthonomus grandis (AGE), producing the highest concentrations ofβ-galactosidase (5.0×10⁶ pg/ml) and luciferase (2.67×10³ pg/ml). Both enzymes were detected as early as 12 h postinoculation in lysate samples of the AGE cell line.Helicoverpa armigera (HA), a nonpermissive cell line, expressedβ-galactosidase at 72 h postinoculation at a concentration of 3.5×10³ pg/ml. However, expression of luciferase was not detected. Expression of luciferase andβ-galactosidase was also not detected in the nonpermissiveHelicoverpa zea (HZ) cell line.     

Serum-Free Suspension Culture of MDCK Cells for Production of Influenza H1N1 Vaccines

Development of serum-free suspension cell culture processes is very important for influenza vaccine production. Previously, we developed a MDCK suspension cell line in a serum-free medium. In the present study, the growth kinetics of suspension MDCK cells and influenza virus production in the serum-free medium were investigated, in comparison with those of adherent MDCK cells in both serum-containing and serum-free medium. It was found that the serum-free medium supported the stable subculture and growth of both adherent and suspension cells. In batch culture, for both cell lines, the growth kinetics in the serum-free medium was comparable with those in the serum-containing medium and a commercialized serum-free medium. In the serum-free medium, peak viable cell density (VCD), haemagglutinin (HA) and median tissue culture infective dose (TCID₅₀) titers of the two cell lines reached 4.51×10⁶ cells/mL, 2.94Log₁₀(HAU/50 μL) and 8.49Log₁₀(virions/mL), and 5.97×10⁶ cells/mL, 3.88Log₁₀(HAU/50 μL), and 10.34Log₁₀(virions/mL), respectively. While virus yield of adherent cells in the serum-free medium was similar to that in the serum-containing medium, suspension culture in the serum-free medium showed a higher virus yield than adherent cells in the serum-containing medium and suspension cells in the commercialized serum-free medium. However, the percentage of infectious viruses was lower for suspension culture in the serum-free medium. These results demonstrate the great potential of this suspension MDCK cell line in serum-free medium for influenza vaccine production and further improvements are warranted.     

Comparative recombinant protein production of eight insect cell lines

Summary A recombinantAutographa californica baculovirus expressing secreted alkaline phosphatase (SEAP) gene was used to evaluate the expression of a secreted glycoprotein in eight insect cell lines derived fromSpodoptera frugiperda, Trichoplusia ni, Mamestra brassicae andEstigmene acrea. Because cell density was found to influence protein production, SEAP production was evaluated at optimal cell densities for each cell line on both a per cell and per milliliter basis. On a per cell basis, theT. ni-derived BTI-TN-5B1-4 cells produced a minimum of 20-fold more SEAP than theS. frugiperda-derived Sf9 or Sf21 cell lines and a minimum of 9-fold more than any of the other cell lines growing in serum-containing medium. On a per milliliter basis, BTI-TN-5B1-4 cells produced a minimum of fivefold more SEAP than any of the other cell lines tested. Using cell lines that were adapted to serum-free medium, SEAP yields were the same or better than their counterparts in serum-containing medium. At 3 days postinoculation, extracellular SEAP activity ranged from 59 to 85% of total SEAP activity with cell lines grown in serum-free and serum-containing media.     

电穿孔介导质粒DNA肿瘤内转移抑制恶性肿瘤生长与转移

利用携带绿色荧光蛋白（green fluorescent protein, GFP）编码基因的表达质粒,测试电穿孔方法介导目的基因活体组织内转移的效率并优化电击参数.在此基础上采用电穿孔技术直接将编码白介素12（IL-12）、白介素2（IL-2）、粒单细胞克隆刺激因子（GM-CSF）等免疫调节因子或反义血管内皮细胞生长因子121（VEGF₁₂₁）、可溶性血管内皮细胞膜受体（sFlk-1及ExTek）等血管生成抑制因子表达质粒转移至肿瘤局部.实验结果表明电穿孔介导GFP表达质粒肌肉内转移的效率较高,GFP可在肌细胞内持续高水平表达3周以上,而在肿瘤细胞内只能表达4～6 d,但高电压短脉冲电击组肿瘤内GFP阳性细胞数比低电压长脉冲组高2.68倍.多次电击介导IL-12表达质粒转移至肿瘤组织内,可有效地抑制小鼠膀胱癌BTT-gfp、人乳腺癌MCF-7及肝癌SMMC 7721-gfp的生长.MCF-7对血管生成抑制因子基因转移治疗较敏感,单独应用反义VEGF₁₂₁、sFlk-1或ExTek即显示明确的治疗效果.SMMC 7721-gfp单独应用sFlk-1有效.小鼠膀胱癌对单独应用反义VEGF₁₂₁、sFlk-1或ExTek治疗效果不理想,但联合应用sFlk-1和ExTek仍然可以有效地抑制肿瘤生长与转移,甚至使肿瘤缩小或消失.提示电穿孔技术是一项高效、安全、经济的体内基因转移方法.     

Lepidopteran cell lines after long-term culture in alternative media: Comparison of growth rates and baculovirus replication

Summary Three insect cell lines, IPLB-LdFB and IPLB-LdEIta from gypsy moth fat body and embryos and UFL-AG-286 from velvetbean caterpillar embryos, have been concurrently maintained for 1 to 12 yr on two media formulations, modified TC-100 containing 9% fetal bovine serum and Ex-cell 400, a commercial serum-free medium (SFM). Cells grown in each medium were tested for susceptibility to and productivity of various multiply embedded nucleopolyhedroviruses. The three lines chosen for these experiments fall into three categories of relative growth in SFM versus TC-100: LdFB cells grew similarly in each medium, LdEIta grew better in Ex-Cell than in TC-100, and AG-286 grew better in TC-100 than in Ex-Cell. The susceptibility of cells to infection also varies, although without any apparent correlation to which medium was best for supporting growth. Endpoint assays suggested that LdFB cells grown in serum-containing medium are more susceptible to virus infection than their SFM counterparts, while the opposite is true for LdEIta cells. Production of virus, based on numbers of occlusion bodies, showed fewer differences with only AcMNPV production with AG-286 in TC-100 being statistically higher than production of the same virus in Ex-cell 400. These studies suggest that long-term passage in alternative media may impact the ability of cells to support virus infection and replication, but the effects on each cell line and virus system need to be determined.     

Green fluorescent protein marks skeletal muscle in murine cell lines and zebrafish

The green fluorescent protein (GFP) acts as a vital dye upon the absorption of blue light. When the gfp gene is expressed in bacteria, flies or nematodes, green fluorescence can be directly observed in the living organism. We inserted the cDNA encoding this 238-amino-acid (aa) jellyfish protein into an expression vector containing the rat myosin light-chain enhancer (MLC-GFP) to evaluate its ability to serve as a muscle-specific marker. Transiently, as well as stably, transfected C2C12 cell lines produced high levels of GFP distributed homogeneously throughout the cytoplasm and was not toxic through several cell passages. Expression of MLC-GFP was strictly muscle-specific, since Cos 7 fibroblasts transfected with MLC-GFP did not fluoresce. When GFP and βGal markers were compared, the GFP signal was visible in the cytoplasm of the living cell, whereas visualization of βGal required fixation and resulted in deformation of the cells. When the MLC-GFP construct was injected into zebrafish embryos, muscle-specific gfp expression was apparent within 24 h of development, gfp expression was never observed in non-muscle tissues using the MLC-GFP construct. Transgenic fish continued to express high levels of gfp in skeletal muscle at 1.5 months, demonstrating that GFP is an effective marker of muscle cells in vivo.     

Temperature influence on fluorescence intensity and enzyme activity of the fusion protein of GFP and hyperthermophilic xylanase

By constructing the expression system for fusion protein of GFPmut1 (a green fluorescent protein mutant) with the hyperthermophilic xylanase obtained from Dictyoglomus thermophilum Rt46B.1, the effects of temperature on the fluorescence of GFP and its relationship with the activities of GFP-fused xylanase have been studied. The fluorescence intensities of both GFP and GFP-xylanase have proved to be thermally sensitive, with the thermal sensitivity of the fluorescence intensity of GFP-xylanase being 15% higher than that of GFP. The lost fluorescence intensity of GFP inactivated at high temperature of below 60°C in either single or fusion form can be completely recovered by treatment at 0°C. By the fluorescence recovery of GFP domain at low temperature, the ratios of fluorescence intensity to xylanase activity (R _gfp/A _xyl) at 15°C and 37°C have been compared. Even though the numbers of molecules of GFP and xylanase are equivalent, the R _gfp/A _xyl ratio at 15°C is ten times of that at 37°C. This is mainly due to the fact that lower temperature is more conducive to the correct folding of GFP than the hyperthermophilic xylanase during the expression. This study has indicated that the ratio of GFP fluorescence to the thermophilic enzyme activity for the fusion proteins expressed at different temperatures could be helpful in understanding the folding properties of the two fusion partners and in design of the fusion proteins.     

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.