PLCζ is a new isoenzyme of the PLC family which plays an important role in activating mammalian oocytes. In recent years, large-scale expression and purification of active PLCζ protein in vitro for structural biology research has not been successful. In this study, the recombinant human PLCζ protein was expressed and purified in the baculovirus expression system. First, the full length of human PLCζ gene was cloned into the pFastBac-HTA plasmid to form the recombinant donor plasmid that was further transformed into DH10Bac Escherichia coli cells to construct the recombined bacmid by the site-specific transposition that was screened by resistance and blue-white spots. Then the bacmid was transfected to Sf9 insect cells via cellfectin to package the recombinant baculovirus. After the amplification of the recombinant baculovirous, the recombinant protein was expressed from the cells transduced by the recombinant baculovirus and was purified by Ni-NTA resin. Purified protein was identified by Western blotting and time-of-flight mass spectrometry and the enzyme activity was determined. The results showed that the recombinant PLCζ protein in the Sf9 cells was achieved at 72 hours after baculovirus infection and expressed in secreted form in cell culture medium. The recombinant protein purified by Ni²⁺ affinity column was identified as PLCζ by Western blotting and ionization time-of-flight mass spectrometry and the enzyme activity was up to 326.8 U/mL. The experimental results provide a reference for the large-scale production and biological application of recombinant human PLCζ protein.
Animals ; Baculoviridae ; Genetic Vectors ; Humans ; Recombinant Proteins ; Sf9 Cells ; Spodoptera

The fall armyworm (FAW), Spodoptera frugiperda, is a destructive pest native to America and has recently become an invasive insect pest in China. Because of its rapid spread and great risks in China, understanding of FAW genetic background and pesticide resistance is urgent and essential to develop effective management strategies. Here, we assembled a chromosome-level genome of a male FAW (SFynMstLFR) and compared re-sequencing results of the populations from America, Africa, and China. Strain identification of 163 individuals collected from America, Africa and China showed that both C and R strains were found in the American populations, while only C strain was found in the Chinese and African populations. Moreover, population genomics analysis showed that populations from Africa and China have close relationship with significantly genetic differentiation from American populations. Taken together, FAWs invaded into China were most likely originated from Africa. Comparative genomics analysis displayed that the cytochrome p450 gene family is extremely expanded to 425 members in FAW, of which 283 genes are specific to FAW. Treatments of Chinese populations with twenty-three pesticides showed the variant patterns of transcriptome profiles, and several detoxification genes such as AOX, UGT and GST specially responded to the pesticides. These findings will be useful in developing effective strategies for management of FAW in China and other invaded areas.
Animals ; China ; Genomics ; Humans ; Male ; Pesticides ; Spodoptera/genetics* ; Transcriptome

Persistent baculovirus infection is observed frequently in insect populations. Persistent infection can be transformed to a replicative and infective state caused by stress factors and plays an important role in regulating the size of insect population and in epizoology of baculoviruses. The aim of this study is to establish a persistently baculovirus-infected cell system to explore the molecular mechanisms of baculoviral persistence. Spodoptera exigua nucleopolyhedrovirus (SeMNPV) was serially undiluted passaged in Se301 cells to reduce virulence. Upon infection of Se301 cells with the SeMNPV up to passage 8, a few cells survived even if most of cells died due to virus infection. The surviving cells were passaged and designated as P8-Se301 cell strain. P8-Se301 cells had a population doubling time of 58-65 hours and grew slower than Se301 cells. Light microscopy and electron microscopy observation showed symptom of baculovirus infection, such as virogenic stroma, viral particles and occlusion bodies, in some of P8-Se301 cells. End-point dilution assay and infectious center assay showed that 4.14% +/- 0.99% cells continually released infectious progeny virus which replicated slower than SeMNPV in Se301 cells. The result indicated that P8-Se301 cells show a typical character trait of baculovirus persistent infection.
Animals ; Cells, Cultured ; Nucleopolyhedrovirus ; growth & development ; physiology ; Spodoptera ; virology ; Virus Cultivation ; methods

Ten strains of the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana were evaluated to find the most effective strain for optimization studies. The first criterion tested for strain selection was the mortality (> 50%) of Spodoptera litura larvae after inoculation of the fungus for 4 days. Results on several bioassays revealed that B. bassiana BNBCRC showed the most virulence on mortality S. litura larvae (80% mortality). B. bassiana BNBCRC also showed the highest germination rate (72.22%). However, its conidia yield (7.2 x 10(8) conidia/mL) was lower than those of B. bassiana B 14841 (8.3 x 10(8) conidia/mL) and M. anisopliae M6 (8.2 x 10(8) conidia/mL). The highest accumulative radial growth was obtained from the strain B14841 (37.10 mm/day) while the strain BNBCRC showed moderate radial growth (24.40 mm/day). M. anisopliae M6 possessed the highest protease activity (145.00 mU/mL) while M. anisopliae M8 possessed the highest chitinase activity (20.00 mU/mL) during 96~144 hr cultivation. Amongst these criteria, selection based on virulence and germination rate lead to the selection of B. bassiana BNBCRC. B. bassiana B14841 would be selected if based on growth rate while M. anisopliae M6 and M8 possessed the highest enzyme activities.
Beauveria ; Biological Assay ; Chitinase ; Fungi ; Germination ; Larva ; Metarhizium ; Patient Selection ; Spodoptera ; Spores, Fungal ; Sprains and Strains

Bovine growth hormone (bGH) gene was expressed in an insect spodoptera frugiperda cell line using a Baculovirus, Hyphantria cunea nuclear polyhedrosis virus (HcNPV). The bGH gene in pbGH plasmid was sequenced and amplified by PCR technique with two primers containing NcoI sites. The bGH gene consisted of 654 bp (217 amino acid residues), the 5'-untranslated region of the cloned bGH cDNA contains 56 bp, and the 3'-untranslated region contains 145 bp and two pallindromic regions. The amplified bGH gene DNA fragment (654 bp) was inserted into the NcoI site of the pHcEVII vector, which was named pHcbGH. The pHcbGH transfer vector DNA and the wild type HcNPV DNA were cotransfected into s. frugiperda cells to construct a recombinant virus. Eight recombinant viruses were selected and named HcbGH. One clone, HcbGH-4-1 showed largest plaque size, therefore the recombinant virus was further studied. The multiplication patters of the recombinant HcbGH-4-1 was similar to that of the wild type HcNPV. The bGH gene DNA in the HcbGH-4-1 recombinant was confirmed by Southern lot hybridization. The amount of the bGH (217 amino acid residues, 21 kDa) produced in S. frugiperda cells infected with the HcbGH-4-1 recombinant was approximately 5.5 ng per ml (106 cells) by radioimmunoassay.
Baculoviridae* ; Cell Line ; Clone Cells ; DNA ; DNA, Complementary ; Growth Hormone* ; Insects ; Nucleopolyhedrovirus* ; Plasmids ; Polymerase Chain Reaction ; Radioimmunoassay ; Spodoptera

OBJECTIVETo express the L1 protein of human papillomavirus type 16 (HPV16) in insect cell suspension culture system.

METHODSOptimized the conditions of suspension culture, recombinant virus amplification and protein expression. Determined the virus tilter by plague analysis and detected the target protein by SDS-PAGE and Western blot; The formation of VLPs by HPV16 L1 protein was observed with TEM.

RESULTSThe Sf9 cells could grow better in suspension culture with seeding density of 5 x 10-5 cell/mL and the maximum expression quantity was obtained by infection of cells with rBacV/HPV16L1 (MOI =10) and harvesting after 72-84 h. HPV16L1 protein could assemble into VLPs in Sf9 cells observed with TEM.

CONCLUSIONThe conditions of cell culture, virus amplification and protein expression were optimized. HPV16 L1 protein could assemble into VLPs in Sf9 cells, which would provide a foundation for further study of the vaccine and diagnosis kits.

Animals ; Capsid Proteins ; biosynthesis ; Cell Proliferation ; Oncogene Proteins, Viral ; biosynthesis ; Recombinant Proteins ; biosynthesis ; Spodoptera ; Suspensions ; Virion ; ultrastructure

Based on site-specific transposition of an expression cassette into a baculovirus shuttle vector (Bacmid) which propagated in Escherichia coli, the Bac-to-Bac System provides a rapid and efficient method to generate recombinant baculoviruses and is widely used for high level expression of heterologous proteins. And the efficiency of recombinant baculovirus infecting cells plays an important role on the protein expression. In this study, we introduced an EGFP expression cassette driven by polyhedrin promoter into the p74 locus of Bacmid by homologous recombination. The target Bacmid-egfp was then transformed into E. coli DH10B containing the transposition helper plasmid to gain a new transposition receipt strain E. coli DH10Bac-egfp. Because of the intact attTn7 sites and lacZ', target gene cloned in a pFastBac vector can be transposed into the Bacmid-egfp shutter vector to construct recombinant baculovirus, which would allow the tracing of the target protein expression and the recombinant Bacmid transfection or recombinant baculoviral infection under fluorescence microscopes. Recombinant virus Bac-egfp-DsRed was constructed by transposing DsRed into the Bacmid-egfp in E. coliDHl0Bac-egfp, and the Sf9 cells infected with the recombinant virus expressed DsRed and EGFP efficiently. Another protein IL-6 fused with 6 x his tag was expressed and purified sucessfully from Sf9 cells infected with recombinant virus Bac-egfp-6 x his-IL6 constructed by the improved Bac-to-Bac system.
Animals ; Baculoviridae ; genetics ; Green Fluorescent Proteins ; genetics ; Interleukin-6 ; biosynthesis ; genetics ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; Spodoptera

It has been reported that baculoviruses could serve as a new gene-transfer vehicle for mammalian cells. We have previously constructed recombinant baculovirus BacV-CMV-EGFPA and have proven that mammalian cells could be effectively infected by the recombinant baculovirus. In this report, we studied the efficiency of baculovirus to deliver exogenous gene into twenty mammalian cells, including twelve human cell lines (WI-38, Hela, HepG2, 293, PLC/PRF/5, 143B, MCF-7, BGC-223, DMS 114, CNE, Raji, LCL-cm), seven murine cell lines (BNL 1ME A.7R.1, CHO-K1, L-929, JC, PT67, NIH3T3, P815) and one monkey cell line (CV1). Results showed that most mammalian cell lines could be transduced by the recombinant baculovirus, the transduction efficiencies of the human and monkey cell lines were markedly higher than that of murine cell lines, and the transduction efficiencies in adherent culture cell lines higher than that of suspend culture cell lines, implying that the infection efficiency of the baculovirus may be correlative with the organism used and the growth properties of the cell lines. The plasmid pcDNA3. 1-EGFP, which contains the CMV promoter and EGFP reporter gene, was next transfected by LipofectAMINE into a number of mammalian cells, especially those cells that were low in the baculovirus transfection. Results showed that the CMV promoter could effectively direct the expression of the reporter gene in these mammalian cells. Therefore the gene-expression efficiencies in different mammalian cell lines by the recombinant baculovirus which contains the same CMV promoter were dictated by the ability of the baculovirus to enter the cell lines. This study suggested that the recombinant baculovirus vector is more suitable for gene expression in primate adherent culture cells than in murine cells and suspend culture cells.
Animals ; Baculoviridae ; genetics ; Cytomegalovirus ; genetics ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; Promoter Regions, Genetic ; Spodoptera

The beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae) is difficult to control using chemical insecticides because of the development of insecticide resistance. Several pest control agents are used to control the beet armyworm. Entomopathogenic fungi are one of the candidates for eco-friendly pest control instead of chemical control agents. In this study, among various entomopathogenic fungal strains isolated from soil two isolates were selected as high virulence pathogens against larva of beet armyworm. Control efficacy of fungal conidia was influenced by conidia concentration, temperature, and relative humidity (RH). The isolates Metarhizium anisopliae FT83 showed 100% cumulative mortality against second instar larvae of S. exigua 3 days after treatment at 1 x 10(7) conidia/mL and Paecilomyces fumosoroseus FG340 caused 100% mortality 6 days after treatment at 1 x 10(4) conidia/mL. Both M. anisopliae FT83 and P. fumosoroseus FG340 effectively controlled the moth at 20~30degrees C. M. anisopliae FT83 was significantly affected mortality by RH: mortality was 86.7% at 85% RH and 13.4% at 45% RH. P. fumosoroseus FG340 showed high mortality as 90% at 45% RH and 100% at 75% RH 6 days after conidia treatments. These results suggest that P. fumosoroseus FG340 and M. anisopliae FT83 have high potential to develop as a biocontrol agent against the beet armyworm.
Beta vulgaris ; Fungi* ; Humidity ; Insecticide Resistance ; Insecticides ; Larva ; Metarhizium* ; Mortality ; Moths ; Paecilomyces* ; Pest Control ; Soil ; Spodoptera* ; Spores, Fungal ; Virulence*

A number of recombinant proteins isolated from cell sources are being produced for biopharmaceuticals. Although most biopharmaceuticals are highly purified, there is a safety concern that such recombinant products could be contaminated with impurities including adventitious virus, mycoplasma, endotoxin and oncogenic DNA. Residual DNA in recombinant biopharmaceuticals is a potential risk factor and must be evaluated and removed to meet the regulatory guidelines. Recombinant HPV type 16 L1 VLPs, recombinant protein produced in Spodoptera frugiperda (Sf) 9 insect cells, is a HPV subunit vaccine candidate which has been studied as a preventive vaccine of cervical cancers. In this study, we performed detection and quantification of residual cellular DNA in the production of recombinant HPV type 16 L1 VLPs. HPV-16 L1 VLPs were purified by processes including detergent lysis, sonication treatment, sucrose cushion centrifugation, CsCl equilibrium density centrifugation, and DNase treatment which was added to inactivate residual cellular DNA after CsCl centrifugation step. We have developed a precise assay based on a dot-blot hybridization using digoxigenin random primed labeling DNA probes for the detection and quantification of residual cellular DNA during the purification process and final products. Detection limit of residual cellular DNA was 0.1 ng in this assay and the amount of residual cellular DNA in the final product was 0.5 ng~1 ng per 100 microgram of protein. This study describes safer and more sensitive methods alternative to radioactive techniques employed for residual cellular DNA quantification of biopharmaceuticals produced by recombinant protein technology and presents method validation data demonstrating precision and reproducibility.
Centrifugation ; Deoxyribonucleases ; Detergents ; Digoxigenin ; DNA Probes ; DNA* ; Human papillomavirus 16* ; Insects ; Limit of Detection ; Mycoplasma ; Recombinant Proteins ; Risk Factors ; Sonication ; Spodoptera ; Sucrose