Variable Surface Glycoprotein RoTat 1.2 PCR as a specific diagnostic tool for the detection of Trypanosoma evansi infections

Background  

Based on the recently sequenced gene coding for the Trypanosoma evansi (T. evansi) RoTat 1.2 Variable Surface Glycoprotein (VSG), a primer pair was designed targeting the DNA region lacking homology to other known VSG genes. A total of 39 different trypanosome stocks were tested using the RoTat 1.2 based Polymerase Chain Reaction (PCR).     

Purification, biochemistry and molecular cloning of an insect glycosylasparaginase from Spodoptera frugiperda

Glycosylasparaginase (EC 3.5.1.26 [EC] ) from Sf9 cells (Spodopterafrugiperda) was purified to homogeneity with a specific activityof 2.1 unit/mg. The enzyme is composed of two non-identical     

Oligosaccharide processing in the expression of human plasminogen cDNA by lepidopteran insect (Spodoptera frugiperda) cells

A comparison has been made between the Asn289-linked oligosaccharide structures of human plasma plasminogen and a recombinant human plasminogen, expressed in lepidopteran insect (Spodoptera frugiperda) cells, after infection of these cells with a recombinant baculovirus containing the entire human plasminogen cDNA. Using anion-exchange liquid chromatography mapping of the oligosaccharide units cleaved from the proteins by glycopeptidase F, compared with elution positions of standard oligosaccharide structures, coupled with monosaccharide compositional analysis, we find that the human plasma protein contained only bisialo-biantennary complex-type carbohydrate and asialo-biantennary complex carbohydrate, confirming earlier work published by this laboratory. The glycosylation pattern of the insect cell expressed recombinant human plasminogen showed considerable microheterogeneity, with identifiable high-mannose carbohydrate (Man9GlcNAc2) and truncated high-mannose oligosaccharide (Man5GlcNAc2, Man4GlcNAc2, and Man3GlcNAc2). Of major importance, approximately 40% of the oligosaccharide population consisted of complex carbohydrate (bisialo-biantennary), identical in structure with that of the human plasma protein. This is the first direct identification of complex carbohydrate in proteins produced in insect cells and demonstrates that trimming and processing of high-mannose carbohydrate into complex-type oligosaccharide can occur. Our data indicate that both normal and alternate pathways exist in these cells for incorporation and trimming of high-mannose oligosaccharides and that mannosidases, as well as galactosyl-, hexosaminidasyl-, and sialyltransferases are present, and/or can be induced, in these cells. From these observations, we conclude that amino acid sequences and/or protein conformational properties can control oligosaccharide processing events.     

Recombinant scorpion insect excitatory toxin BmK IT accelerates the growth of insect Spodoptera frugiperda 9 cells

Several scorpion insect toxins are selectively active on the lepidopterous and dipterous insects. The gene encoding insect excitatory neurotoxin (BmK IT) from the scorpion Buthus martensii Karsch was expressed in Escherichia coli BL21(DE3) at a high level of 3 mg/0.5 L using the prokaryotic expression system pTWIN1. Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), whole-cell patch-clamp technique and immunofluorescence assays were used to evaluate the toxicity of rBmK IT to insect Spodoptera frugiperda 9 (Sf9) cells and to analyze the potential mechanism of this toxicity. rBmK IT accelerated the growth of Sf9 cells in a dose-dependent manner. Voltage-gating sodium channel activity could not be detected in Sf9 cells using a whole-cell patch-clamp technique. However, immunofluorescence analysis clearly showed co-localization of tetrodotoxin (TTX) and rBmK IT on the Sf9 cell membrane, which demonstrated that rBmK IT could bind to and act on the voltage-gated sodium channels on the Sf9 cells by the high affinity action power. The findings presented in this study are essential for further study of this peptide.     

Correlation of LNCR rasiRNAs expression with heterochromatin formation during development of the holocentric insect Spodoptera frugiperda

Repeat-associated small interfering RNAs (rasiRNAs) are derived from various genomic repetitive elements and ensure genomic stability by silencing endogenous transposable elements. Here we describe a novel subset of 46 rasiRNAs named LNCR rasiRNAs due to their homology with one long non-coding RNA (LNCR) of Spodoptera frugiperda. LNCR operates as the intermediate of an unclassified transposable element (TE-LNCR). TE-LNCR is a very invasive transposable element, present in high copy numbers in the S. frugiperda genome. LNCR rasiRNAs are single-stranded RNAs without a prominent nucleotide motif, which are organized in two distinct, strand-specific clusters. The expression of LNCR and LNCR rasiRNAs is developmentally regulated. Formation of heterochromatin in the genomic region where three copies of the TE-LNCR are embedded was followed by chromatin immunoprecipitation (ChIP) and we observed this chromatin undergo dynamic changes during development. In summary, increased LNCR expression in certain developmental stages is followed by the appearance of a variety of LNCR rasiRNAs which appears to correlate with subsequent accumulation of a heterochromatic histone mark and silencing of the genomic region with TE-LNCR. These results support the notion that a repeat-associated small interfering RNA pathway is linked to heterochromatin formation and/or maintenance during development to establish repression of the TE-LNCR transposable element. This study provides insights into the rasiRNA silencing pathway and its role in the formation of fluctuating heterochromatin during the development of one holocentric organism.     

草地贪夜蛾PP2A的克隆、序列分析及在大肠杆菌中的表达

为建立斑蝥素等PP2A抑制剂的体外活性测定方法,用于以杀虫剂为目的的药物快速活性评价,本研究首次同源克隆了鳞翅目昆虫草地贪夜蛾Spodoptera frugiperda(J.E.Smith)的PP2A催化亚基cDNA全长序列,探索了该基因在大肠杆菌中的表达。结果显示,S.frugiperda的PP2A编码一条309个氨基酸的肽链,预测其蛋白质分子量为35.46 ku,等电点5.37。分析蛋白质氨基酸序列,没有发现信号肽和跨膜结构,推测该PP2A主要存在于胞质中。多重比对分析S.frugiperda及其它昆虫的PP2A,表明PP2A的保守性较高,可以用S.frugiperda PP2A作为研究斑蝥素等抑制剂的药物筛选靶标酶,用于杀虫剂的筛选。利用该基因构建pET30a-PP2A原核表达载体转化至E.coli BL21(DE3)中,在16³0℃,0.230℃,0.2⁰.8 mmol/L的IPTG下均能成功诱导PP2A-His表达,经Ni-琼脂糖柱纯化后可以得到SDS-PAGE呈现单一条带的纯化蛋白,电泳纯度大于90%,为进一步活性测定奠定了基础。     

Potential of adultOrius insidiosus [Hemiptera: Anthocoridae] as a predator of the fall armyworm,Spodoptera frugiperda [Lepidoptera: Noctuidae]

Orius insidiosus (Say) was observed to successfully prey on eggs and earlier instar larvae of the fall armyworm (FAW),Spodoptera frugiperda (J. E. Smith). Significantly more neonates of FAW were killed per 24 h than either 1, 2 or 3 d old larvae. Female and male predators exhibited a typical type-II functional response when preying on varying densities of eggs or neonates of FAW.      

草地贪夜蛾半胱氨酸蛋白酶Sf-caspase-3基因克隆与表达分析

半胱氨酸蛋白酶caspases是细胞凋亡过程中重要的酶类,参与调控昆虫变态发育及响应外界压力等多种生理过程。本研究通过转录组数据及逆转录PCR鉴定并验证得到Sf-caspase-3基因编码区全长。该基因编码区长840 bp,编码279个氨基酸,预测蛋白分子量为31.43 kDa。氨基酸序列分析表明Sf-caspase-3具有保守的QACRG五肽序列,且与Lep-caspase-3高度保守。进化树分析表明Sf-caspase-3与甜菜夜蛾Spodoptera exigua Se-caspase-3亲缘关系最近。RT-qPCR检测结果表明Sf-caspase-3在草地贪夜蛾Spodoptera frugiperda各个龄期均有表达,其中3~6龄幼虫期表达量最高,蛹期和卵期表达量最低。此外Sf-caspase-3在6龄幼虫中肠组织中表达量最高,而在表皮、脂肪体及马氏管中表达量极低。本研究成功构建Sf-caspase-3原核表达重组质粒,在28℃下0.4 mmol/L异丙基硫代半乳糖苷（IPTG）诱导重组菌株4 h得到约50 kDa大小条带,与预期条带大小相符。蛋白可溶性分析表明Sf-caspase-3主要以不可溶的形式存在包涵体中。本研究为草地贪夜蛾Sf-caspase-3功能及鳞翅目昆虫细胞凋亡机制研究提供一定的理论依据。     

A novel medium for expression of proteins selectively labeled with 15N-amino acids in Spodoptera frugiperda (Sf9) insect cells

Whereas bacterial expression systems are widely used for production of uniformly or selectively ¹⁵N-labeled proteins the usage of the baculovirus expression system for labeling is limited to very few examples in the literature. Here we present the complete formulations of the two insect media, IML406 and 455, for the high-yield production of selectively ¹⁵N-labeled proteins in insect cells. The quantities of ¹⁵N-amino acids utilized in the production of labeled GST were similar in the case of bacterial and viral expression. For the most studied amino acids essential for insect cells the ¹⁵N-HSQC spectra, recorded with GST labeled in insect cells, showed no cross labeling and provided therefore spectra of better quality compared to NMR spectra of GST expressed in E. coli. Also in the case of amino acids not essential for Sf9 cells we were able to label a defined number of amino acid species. Therefore the selective labeling using the baculovirus expression vector system represents a complement or even an alternative to the bacterial expression system. Based on these findings we can provide a first simple overview of the network of the amino acid metabolism in E. coli and insect cells focused on nitrogen. For some amino acids the expression of labeled proteins in insect cells can replace the cell-free protein expression.     

Overexpression of full-length human glucocorticoid receptor in Spodoptera frugiperda cells using the baculovirus expression vector system

We have expressed a full-length human glucocorticoid receptor (hGR) in Spodoptera frugiperda (Sf9) cells using the baculovirus expression vector system (BEVS). The level of expression is approximately 100-fold greater than in CEM-C7 cells. Between 0.5-1.0 mg hGR can be generated per liter of Sf9 cell culture. The expressed hGR is capable of binding glucocorticoids with specificity and high affinity. Covalent labeling with 3H-dexamethasone mesylate and Western blot analysis using a polyclonal antibody indicate that the molecular weight of the expressed protein is approximately 94 k. The nonactivated receptor sediments as a 8-9S complex in sucrose gradients and can be heat activated to a 4S form. The activated receptor is capable of retarding the migration of a 23 base-pair DNA fragment containing the glucocorticoid response element from the tyrosine aminotransferase gene. These data indicate that the expressed GR displays characteristics identical to those of GR from mammalian cells. By scaling up this culture we can, for the first time, obtain enough purified full-length receptor for crystallographic and functional studies which could provide new insight into exactly how hGR works.     

Trypanosoma evansi: A comparison of PCR and parasitological diagnostic tests in experimentally infected mice

Trypanosoma evansi is the causative agent of equine trypanosomosis, disease that affects horse’s productivity and health. Parasitological and molecular methods are mostly used to detect the infection. The aim of this work was evaluate PCR sensitivity to detect T. evansi using the primers 21/22-mer, ITS1, ESAG 6/7 and TBR 1/2 designed from repetitive (multicopies) genomic sequences. The results were compare with two parasitological tests in mice, micro-haematocrite centrifugation technique and direct microscopic examination. The results shows (a) that the minimum amount of DNA from blood of highly parasitaemic mice that was detectable by PCR was 0.001 ng, using the ESAG6/7 and TBR1/2 primer, (b) using TBR1/2 primer for parasites purified could detect 0.000001 ng and (c) in the prepatent period PCR detect the presence of parasites earlier than parasitological techniques. Nevertheless, the percentage of detection for PCR varies depending on primer employed with 60% and 66% for ITS1 and 21/22-mer, and 80% for ESAG6/7 and TBR1/2. Consequently, TBR1/2 and ESAG6/7 were the best primers to monitor T. evansi infections in mice. For epidemiological application, such comparative evaluation should be made for detection of T. evansi in livestock such as horses.     

Antimicrobial activity of conditioned medium fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni Hi5 insect cells

Concentrated conditioned medium (CM) fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni cells, eluting from a gel filtration column at around 10 kDa, were found to exhibit strong antibacterial activity against Bacillus megaterium and Escherichia coli. The B. megaterium cells incubated in the CM fraction from Sf9 cells rapidly lost viability: after 8 min the viability had decreased to 0.7%, as compared with the control. Addition of the CM fraction to E. coli cells resulted in a less drastic drop in viability: 65% viability was lost after 60 min of incubation. Further, exposure to the CM fraction caused a substantial leakage of intracellular proteins, as demonstrated by SDS-PAGE analysis. Cell lysis was confirmed by optical density measurements, microscopic investigations and flow cytometry. B. megaterium exposed to a CM fraction from T. ni cells lost 97% of their viability in about 40 min. Ubiquitin, thioredoxin and cyclophilin were identified in the antibacterial fraction from Sf9 cells by mass spectrometry and N-terminal amino acid sequencing. Other proteins in the fraction gave no matches in a database search. Since ubiquitin was shown not to cause the antimicrobial effect and thioredoxin and cyclophilin were likely not involved, the responsible agent may be an unknown protein, not yet registered in databases. The antimicrobial effect of the CM fraction from T. ni cells most probably comes from a lysozyme precursor protein.     

Predictive inference on cytoplasmic and mitochondrial thioredoxin peroxidases in the highly radioresistant Lepidopteran insect Spodoptera frugiperda

Lepidopteran insects show remarkable resistance to radiation and chemical stress than insects of other orders. Despite this, the antioxidant machinery of insects of this order is poorly understood. Recently we demonstrated the significance of cytoplasmic NOS and a stronger mitochondrial antioxidant enzyme system in the stress-resistance of Lepidopteran insects. In the present study, we hypothesize two thioredoxin peroxidase orthologues (Sf-TPx1 and Sf-TPx2) in Lepidopteran insect Spodoptera frugiperda and demonstrate their structural/functional features important for cellular antioxidant activity and stress resistance. Results show a higher mitochondrial localization score (WoLFPSORT) of Sf-TPx2 (mitochondria-18.0, cytoplasm-7.0, nucleus-4.0) than its Drosophila orthologue Jafrac2 (secretory-30.0; mitochondria/nucleus/cytoplasm-no signal), which is important for antioxidant activity, and a higher cytoplasmic localization score of Sf-TPx1 (mitochondria-no signal; cytoplasm-22.0; nucleus-3.5) than the Drosophila Jafrac1 (mitochondria-17; nucleus- 11; cytoplasm-no signal). Structural modeling data show certain motifs present in Jafrac1 and Jafrac2 that affect active site conformation and separate cysteine residues at distances not suitable for disulphide bridge formation (5.21Å; 5.73Å). These motifs are absent in Sf-TPx1 and Sf-TPx2, yielding shorter distance (2.01Å; 2.05Å) between the cysteine residues suitable for disulphide bridge formation. Taken together, the disulphide bridge as well as mitochondrial and cytoplasmic localization are crucial for peroxidatic activity of TPx''s. Therefore,we hypothesize that the Spodoptera TPx''s offer potentially stronger anti-oxidant activity than that of Drosophila orthologues, and may contribute in the high radioresistance of Lepidopteran insects.     

Dengue and Zika virus multi-epitope antigen expression in insect cells

Dengue virus and Zika virus are arthropod-borne flaviviruses that cause millions of infections worldwide. The co-circulation of both viruses makes serological diagnosis difficult as they share high amino acid similarities in viral proteins. Antigens are one of the key reagents in the differential diagnosis of these viruses through the detection of IgG antibodies in serological assays during the convalescent-phase of infections. Here, we report the expression of Dengue virus (DENV) and Zika virus (ZIKV) antigens containing non-conserved and immunodominant amino acid sequences using the baculovirus expression vector system in insect cells. We designed DENV and ZIKV antigens based on the domain III of the E protein (EDIII) after analyzing previously reported epitopes and by multiple alignment of the most important flaviviruses. The ZIKV and DENV multi-epitope genes were designed as tandem repeats or impaired repeats separated by tetra- or hexa-glycine linkers. The biochemical analyses revealed adequate expression of the antigens. Then, the obtained multi-epitope antigens were semi-purified in a sucrose gradient and tested using patients’ sera collected during the convalescent-phase that were previously diagnosed positive for anti-DENV and -ZIKV IgG antibodies. The optimal serum dilution was 1:200, and the mean absorbance values in the preliminary tests show that multi-epitope antigens have been recognized by human sera. The production of both antigens using the multi-epitope strategy in the eukaryotic system and based on the EDIII regions provide a proof of concept for the use of antigens in the differentiation between DENV and ZIKV.

     

Induction and inhibition of an apparent neuronal phenotype in Spodoptera frugiperda insect cells (Sf21) by chemical agents

The goal of this research was to induce neuron-like properties in Sf21 cells, an insect ovarian cell line, which could lead to a new high-throughput insecticide screening method and a way to mass produce insect neuronal material for basic research. This study applied differentiation agents to produce viable neuron-like cells. In the presence of the molting hormone 20-hydroxyecdysone (20-HE), or insulin, in the growth medium, a maximum of ca. 30?% of Sf21 cells expressed an apparent neuronal morphology of unipolar, bipolar, or multipolar axon-like processes within 2?C3?days. Maximal differentiation occurred after 2?days in the presence of 50???M 20-HE or 3?days in 10???M insulin. Both 20-HE and insulin displayed time- and concentration-dependent differentiation with biphasic curves, suggesting that two binding sites or processes were contributing to the observed effects. In addition, combinations of 20-HE and insulin produced apparent synergistic effects on differentiation. Caffeine, a central nervous system stimulant, inhibited induction of elongated processes by 20-HE and/or insulin, with an IC₅₀ of 9 nM for 20-HE, and the inhibition was incomplete, resulting in about one-quarter of the differentiated cells remaining, even at high concentrations (up to 1?mM). The ability to induce a neural phenotype simplifies the studies of insect cells, compared to either the use of primary nervous tissue or genetic engineering techniques. The presence of ion channels or receptors in the differentiated cells remains to be determined.