[Characterization and immunofluorescence localization analysis of carboxypeptidase A in molt fluid of silkworm].

Molting is an important physiological phenomenon of many metamorphosis insects, during which the old and new epidermis are separated by enzymes present in the molting fluid. Various proteomic studies have discovered the presence of Bombyx mori carboxypeptidase A (Bm-CPA) in the molting fluid of silkworm, but its function remains unclear. In order to better understand the role of Bm-CPA in the molting process of silkworm, Bm-CPA was analyzed by bioinformatics analysis, real-time fluorescence quantitative PCR, antibody preparation, immunofluorescence staining, and expression in Pichia pastoris. The results showed that Bm-CPA had a conserved M14 zinc carboxypeptidase domain and glycosylation site. Its expression was regulated by ecdysone 20E, and large expression was observed in the epidermis of the upper cluster stage. Immunofluorescence staining showed that Bm-CPA was enriched in the epidermis during the molting stage, and the inhibitor of Bm-CPA led to the larval death due to the inability to molt. We also successfully obtained a large number of recombinant Bm-CPA proteins by Pichia pastoris expression in vitro. These results may facilitate further understanding the molting development process of silkworm.

Characterization of the novel glucose-methanol-choline (GMC) oxidoreductase EnOXIO1 in Eimeria necatrix.

Eimeria species are intracellular obligate parasites, among the most common pathogens affecting the intensive poultry industry. Oxidoreductases are members of a class of proteins with redox activity and are widely found in apicomplexan protozoans. However, there have been few reports related to Eimeria species. In this study, total RNA was extracted from the gametocytes of E. necatrix Yangzhou strain to amplify the EnOXIO1 gene using reverse-transcription polymerase chain reaction. After cloning and sequence analysis, the prokaryotic expression vector pET-28a(+)-EnOXIO1 was constructed and transformed into Escherichia coli BL21(DE3), and the recombinant protein rEnOXIO1 was expressed by induction with isopropyl ß-D-1-thiogalactopyranoside. The full length EnOXIO1 gene was 2535 bp encoding 844 amino acids, and the EnOXIO1 protein had a molecular weight of about 100 kDa and was mainly expressed in inclusion bodies. Western blot analysis indicated that the rEnOXIO1 protein had good antigenicity and cross-reactivity and was specifically recognized by a 6 ×HIS labeled monoclonal antibody, mouse anti-recombinant protein polyclonal antibody, and recovery serum from chickens infected with E. necatrix, E. acervulina, and E. tenella sporulated oocysts. The results of laser confocal immunofluorescence localization showed that the EnOXIO1 protein was mainly located on the wall-forming bodies in gametocytes and played an important role in the formation of the oocyst wall. Quantitative PCR analysis revealed that transcript levels of EnOXIO1 were highest in the gametocyte stage. Protein expression levels of EnOXIO1 were higher in the gametocyte stage than in other developmental stages according to western blot analysis. Vaccination of chickens against E. necatrix was achieved with recombinant protein rEnOXIO1, which triggered humoral immunity and antibody production, increased average body weight gain, reduced oocyst output and alleviated lesions after E. necatrix infection. The highest ACI value (172.36) was observed in chickens that received 200 µg rEnOXIO1 compared with other immunization groups.

Expression, localization, and function of P4HB in the spermatogenesis of Chinese mitten crab (Eriocheir sinensis).

Background: The sperm of Chinese mitten crab (Eriocheir sinensis) have special noncondensed nuclei. The formation and stability of the special nuclei are closely related to the correct folding of proteins during spermatogenesis. P4HB plays a key role in protein folding, but its expression and role in the spermatogenesis of E. sinensis are unclear. Objective: To investigate the expression and distribution characteristics of P4HB in the spermatogenesis of E. sinensis as well as its possible role. Methods: The testis tissues of adult and juvenile E. sinensis were used as materials. We utilized a variety of techniques, including homology modeling, phylogenetic analysis, RT-qPCR, western blotting, and immunofluorescence staining to predict the protein structure and sequence homology of P4HB, analyze its expression in the testis tissues, and localize and semi-quantitatively assess its expression in different male germ cells. Results: The sequence of P4HB protein in E. sinensis shared a high similarity of 58.09% with the human protein disulfide isomerase, and the phylogenetic tree analysis indicated that the protein sequence was highly conserved among crustaceans, arthropods, and other animals species. P4HB was found to be expressed in both juvenile and adult E. sinensis testis tissues, with different localization patterns observed all over the developmental stages of male germ cells. It was higher expressed in the spermatogonia, spermatocytes, and stage I spermatids, followed by the mature sperm than in the stage II and III spermatids. The subcellular localization analysis revealed that P4HB was predominantly expressed in the cytoplasm, cell membrane, and extracellular matrix in the spermatogonia, spermatocytes, stage I and stage II spermatids, with some present in specific regions of the nuclei in the spermatogonia. In contrast, P4HB was mainly localized in the nuclei of stage III spermatids and sperm, with little expression observed in the cytoplasm. Conclusion: P4HB was expressed in the testis tissues of both adult and juvenile E. sinensis, but the expression and localization were different in male germ cells at various developmental stages. The observed differences in the expression and localization of P4HB may be an essential factor in maintaining the cell morphology and structure of diverse male germ cells in E. sinensis. Additionally, P4HB expressed in the nuclei of spermatogonia, late spermatids, and sperm may play an indispensable role in maintaining the stability of the noncondensed spermatozoal nuclei in E. sinensis.

[Gene expression and immunolocalization of chitin deacetylase BmCDA2 in silkworm].

Deacetylation of chitin is closely related to insect development and metamorphosis. Chitin deacetylase (CDA) is a key enzyme in the process. However, to date, the CDAs of Bombyx mori (BmCDAs), which is a model Lepidopteran insect, were not well studied. In order to better understand the role of BmCDAs in the metamorphosis and development of silkworm, the BmCDA2 which is highly expressed in epidermis was selected to study by bioinformatics methods, protein expression purification and immunofluorescence localization. The results showed that the two mRNA splicing forms of BmCDA2, namely BmCDA2a and BmCDA2b, were highly expressed in the larval and pupal epidermis, respectively. Both genes had chitin deacetylase catalytic domain, chitin binding domain and low density lipoprotein receptor domain. Western blot showed that the BmCDA2 protein was mainly expressed in the epidermis. Moreover, fluorescence immunolocalization showed that BmCDA2 protein gradually increased and accumulated with the formation of larval new epidermis, suggesting that BmCDA2 may be involved in the formation or assembly of larval new epidermis. The results increased our understandings to the biological functions of BmCDAs, and may facilitate the CDA study of other insects.

Molecular characterization and immunological properties of Echinococcus granulosus sensu stricto (G1) ADK1 and ADK8.

Adenylate kinases (ADKs) are one of the important enzymes regulating adenosine triphosphate (ATP) metabolism in Echinococcus granulosus sensu lato. The objective of the present study was to explore the molecular characteristics and immunological properties of E. granulosus sensu stricto (G1) adenylate kinase 1 (EgADK1) and adenylate kinase 8 (EgADK8). EgADK1 and EgADK8 were cloned and expressed, and the molecular characteristics of EgADK1 and EgADK8 were analyzed through different bioinformatics tools. Western blotting was used to examine the reactogenicity of recombinant adenylate kinase 1 (rEgADK1) and recombinant adenylate kinase 8 (rEgADK8) and to evaluate their diagnostic value. The expression profiles of EgADK1 and EgADK8 in 18-day-old strobilated worms and protoscoleces were analyzed by quantitative real-time PCR, and their distribution in 18-day-old strobilated worms, the germinal layer, and protoscoleces was determined by immunofluorescence localization. EgADK1 and EgADK8 were successfully cloned and expressed. Bioinformatics analysis predicted that EgADK1 and EgADK8 have multiple phosphorylation sites and B-cell epitopes. Compared with EgADK8, EgADK1 and other parasite ADKs have higher sequence similarity. In addition, both cystic echinococcosis (CE)-positive sheep sera and Cysticercus tenuicollis-infected goat sera could recognize rEgADK1 and rEgADK8. EgADK1 and EgADK8 were localized in protoscoleces, the germinal layer, and 18-day-old strobilated worms. EgADK1 and EgADK8 showed no significant difference in their transcription level in 18-day-old strobilated worms and protoscoleces, suggesting that EgADK1 and EgADK8 may play an important role in the growth and development of E. granulosus sensu lato. Since EgADK1 and EgADK8 can be recognized by other parasite-positive sera, they are not suitable as candidate antigens for the diagnosis of CE.

Gene expression and immunolocalization of chitin deacetylase BmCDA2 in silkworm / 生物工程学报

Deacetylation of chitin is closely related to insect development and metamorphosis. Chitin deacetylase (CDA) is a key enzyme in the process. However, to date, the CDAs of Bombyx mori (BmCDAs), which is a model Lepidopteran insect, were not well studied. In order to better understand the role of BmCDAs in the metamorphosis and development of silkworm, the BmCDA2 which is highly expressed in epidermis was selected to study by bioinformatics methods, protein expression purification and immunofluorescence localization. The results showed that the two mRNA splicing forms of BmCDA2, namely BmCDA2a and BmCDA2b, were highly expressed in the larval and pupal epidermis, respectively. Both genes had chitin deacetylase catalytic domain, chitin binding domain and low density lipoprotein receptor domain. Western blot showed that the BmCDA2 protein was mainly expressed in the epidermis. Moreover, fluorescence immunolocalization showed that BmCDA2 protein gradually increased and accumulated with the formation of larval new epidermis, suggesting that BmCDA2 may be involved in the formation or assembly of larval new epidermis. The results increased our understandings to the biological functions of BmCDAs, and may facilitate the CDA study of other insects.

Characterization and immunofluorescence localization analysis of carboxypeptidase A in molt fluid of silkworm / 生物工程学报

Molting is an important physiological phenomenon of many metamorphosis insects, during which the old and new epidermis are separated by enzymes present in the molting fluid. Various proteomic studies have discovered the presence of Bombyx mori carboxypeptidase A (Bm-CPA) in the molting fluid of silkworm, but its function remains unclear. In order to better understand the role of Bm-CPA in the molting process of silkworm, Bm-CPA was analyzed by bioinformatics analysis, real-time fluorescence quantitative PCR, antibody preparation, immunofluorescence staining, and expression in Pichia pastoris. The results showed that Bm-CPA had a conserved M14 zinc carboxypeptidase domain and glycosylation site. Its expression was regulated by ecdysone 20E, and large expression was observed in the epidermis of the upper cluster stage. Immunofluorescence staining showed that Bm-CPA was enriched in the epidermis during the molting stage, and the inhibitor of Bm-CPA led to the larval death due to the inability to molt. We also successfully obtained a large number of recombinant Bm-CPA proteins by Pichia pastoris expression in vitro. These results may facilitate further understanding the molting development process of silkworm.

Confocal Fluorescence Microscopy Investigation for the Existence of Subdomains within Protein Storage Vacuoles in Soybean Cotyledons.

In legumes, the seed storage proteins accumulate within specialized organelles called protein storage vacuoles (PSVs). In several plant species, PSVs are differentiated into subdomains that accumulate different kinds of proteins. Even though the existence of subdomains is common in cereals and legumes, it has not been reported in soybean PSVs. The two most abundant seed proteins of soybean, 7S and 11S globulins, have different temporal accumulation patterns and exhibit considerable solubility differences that could result in differential accretion of these proteins within the PSVs. Here, we employed confocal fluorescent microscopy to examine the presence or absence of subdomains within the soybean PSVs. Eosin-stained sections of FAA-fixed paraffin embedded soybean seeds, when viewed by confocal fluorescence microscopy, revealed the presence of intricate subdomains within the PSVs. However, fluorescence immunolabeling studies demonstrated that the 7S and 11S globulins were evenly distributed within the PSVs and failed to corroborate the existence of subdomains within the PSVs. Similarly, confocal scanning microscopy examination of free-hand, vibratome and cryostat sections also failed to demonstrate the existence of subdomains within PSVs. The subdomains, which were prominently seen in PSVs of FAA-fixed soybean seeds, were not observed when the seeds were fixed either in glutaraldehyde/paraformaldehyde or glutaraldehyde. Our studies demonstrate that the apparent subdomains observed in FAA-fixed seeds may be a fixation artifact.

The molecular identification, odor binding characterization, and immunolocalization of odorant-binding proteins in Liriomyza trifolii.

The Liriomyza trifolii is a highly invasive polyphagia pest. Understanding the physiological functions of odorant binding proteins (OBPs) in the chemical communication of L. trifolii can lead to effective pest management strategies. Seven full-length OBPs were identified by transcriptome screening of L. trifolii adults. Bioinformatics analyses classified the seven OBPs into two subfamilies (six classic OBPs, one minus-C OBP). The analysis of their expression in different development stages revealed that LtriOBP5 was highly expressed in the larval stage, LtriOBP4 in the pupa stage, and LtriOBP1, 2, 3, 6, 7 in the adult stage; the expression levels were higher in male adults than in females. The analysis of different tissues showed high expression of LtriOBP1, 3, 6, 7 in the antennae, which were selected for in vitro purification. To explore the ligand compounds of OBPs, fluorescence competitive binding experiments were performed. Immunofluorescence localization revealed that LtriOBP1, 3, 6, 7 showed strong binding abilities to plant volatiles and were located in the antennae, implying that LtriOBP1, 3, 6, 7 may play key roles in olfaction, such as host location. LtriOBP6 and LtriOBP7 had strong binding abilities to specific herbivore-induced plant volatiles, suggesting LtriOBP6 and LtriOBP7 may also play critical roles in chemoreception. This study provides preliminary exploration of the olfactory perception mechanism of L. trifolii, which can be used as a basis to design insect behavior regulators and develop highly effective insecticides using mixture of ligands and known pesticides.

Preparation of ribosomal protein S14 polyclonal antibody in broiler pulmonary artery: Its application in broiler ascites syndrome.

RPS14 (ribosomal protein S14) gene maintains the normal physiological activities of the body by regulating the biosynthesis of ribosomes and the translation of important proteins. This study aims to explore the potential role of RPS14 in broiler ascites syndrome (BAS). We successfully prepared polyclonal antibody against RPS14 and studied the localization and expression of RPS14 protein in a variety of animal key tissues. In this experiment, the recombinant expression plasmid PET28a-RPS14 was constructed using the prokaryotic expression technology of foreign genes. Under the conditions of IPTG induction, a His-RPS14 protein with a molecular weight of about 22 kDa was expressed, and the purified recombinant protein was used as an antigen to prepare rabbit anti-chicken serum. Western blot results showed that the serum could specifically identify RPS14 protein in important tissues of broilers. Immunofluorescence combined with homology analysis showed that the antiserum had significant species specificity. Compared with other species, the expression of this protein in key tissues of broilers and ducks was more significant. More importantly, western blotting and immunofluorescence showed that BAS significantly reduced the expression level of RPS14. This further indicated that RPS14 protein can be used as one of the important entry points for BAS research.

Molecular characterization and expression analysis of annexin B3 and B38 as secretory proteins in Echinococcus granulosus.

BACKGROUND: Cystic echinococcosis is a parasitic zoonotic disease, which poses a threat to public health and animal husbandry, and causes significant economic losses. Annexins are a family of phospholipid-binding proteins with calcium ion-binding activity, which have many functions. METHODS: Two annexin protein family genes [Echinococcus granulosus annexin B3 (EgAnxB3) and EgAnxB38] were cloned and molecularly characterized using bioinformatic analysis. The immunoreactivity of recombinant EgAnxB3 (rEgAnxB3) and rEgAnxB38 was investigated using western blotting. The distribution of EgAnxB3 and EgAnxB38 in protoscoleces (PSCs), the germinal layer, 18-day strobilated worms and 45-day adult worms was analyzed by immunofluorescence localization, and their secretory characteristics were analyzed preliminarily; in addition, quantitative real-time reverse transcription polymerase chain reaction was used to analyze their transcript levels in PSCs and 28-day strobilated worms stages. The phospholipid-binding activities of rEgAnxB3 and rEgAnxB38 were also analyzed. RESULTS: EgAnxB3 and EgAnxB38 are conserved and contain calcium-binding sites. Both rEgAnxB3 and rEgAnxB38 could be specifically recognized by the serum samples from E. granulosus-infected sheep, indicating that they had strong immunoreactivity. EgAnxB3 and EgAnxB38 were distributed in all stages of E. granulosus, and their transcript levels were high in the 28-day strobilated worms. They were found in liver tissues near the cysts. In addition, rEgAnxB3 has Ca2+-dependent phospholipid-binding properties. CONCLUSIONS: EgAnxB3 and EgAnxB38 contain calcium-binding sites, and rEgAnxB3 has Ca2+-dependent phospholipid-binding properties. EgAnxB3 and EgAnxB38 were transcribed in PSCs and 28-day strobilated worms. They were expressed in all stages of E. granulosus, and distributed in the liver tissues near the hydatid cyst, indicating that they are secreted proteins that play a crucial role in the development of E. granulosus.

Chemical structure of semiochemicals and key binding sites together determine the olfactory functional modes of odorant-binding protein 2 in Eastern honey bee, Apis cerana.

Insects can exhibit flexible olfaction that is sensitive to complex natural chemical environments. Odorant-binding proteins (OBPs) in insects' antennal chemosensilla can act as transporters of plant volatiles and pheromones across the sensillar lymph. Although the physiological functions of OBPs have been widely reported, it is still unclear how OBP binds to ligands with various structures in detail. Here, we further investigated the ligand-binding modes and characteristics of AcerOBP2 from the Eastern honey bee (Apis cerana). The results showed that, as a specific protein distributed below the base of chemosensilla on the antennal surface, AcerOBP2 was strongly bound with the candidate floral volatiles and bee pheromones. By docking analysis and site-directed mutagenesis, four different binding modes were found in the five AcerOBP2 mutants between six ligands. Two key amino acids, Ser123 and Lys51, play a key role in AcerOBP2 binding to odors, depending on the presence or absence of hydrogen bonds. In addition, the binding modes depend on their chemical structures and the binding poses of the diverse ligands. These results not only further prompted the functional basis of the relationship between the chemical structures of odorants and bee OBPs, but also revealed the complexity of the flexible behavioral modes of odor binding in insect olfactory systems.

Expression and functional characterization of missense mutations in ATP8A2 linked to severe neurological disorders.

ATP8A2 is a P4-ATPase (adenosine triphosphate) that actively flips phosphatidylserine and phosphatidylethanolamine from the exoplasmic to the cytoplasmic leaflet of cell membranes to generate and maintain phospholipid asymmetry. Mutations in the ATP8A2 gene have been reported to cause severe autosomal recessive neurological diseases in humans characterized by intellectual disability, hypotonia, chorea, and hyperkinetic movement disorders with or without optic and cerebellar atrophy. To determine the effect of disease-associated missense mutations on ATP8A2, we expressed six variants with the accessory subunit CDC50A in HEK293T cells. The level of expression, cellular localization, and functional activity were analyzed by western blot analysis, immunofluorescence microscopy, and ATPase activity assays. Two variants (p.Ile376Met and p.Lys429Met) expressed at normal ATP8A2 levels and preferentially localized to the Golgi-recycling endosomes, but were devoid of ATPase activity. Four variants (p.Lys429Asn, pAla544Pro, p.Arg625Trp, and p.Trp702Arg) expressed poorly, localized to the endoplasmic reticulum, and lacked ATPase activity. The expression of these variants was increased twofold by the addition of the proteasome inhibitor MG132. We conclude that the p.Ile376Met and p.Lys429Met variants fold in a native-like conformation, but lack key amino acid residues required for ATP-dependent lipid transport. In contrast, the p.Lys429Asn, pAla544Pro, p.Arg625Trp, and p.Trp702Arg variants are highly misfolded and undergo rapid proteosomal degradation.

Molecular Characterization of Annexin B2, B3 and B12 in Taenia multiceps.

Coenurus cerebralis, the metacestode of Taenia multiceps, causes coenurosis, a disease severely affecting goat, sheep, cattle and yak farming and resulting in huge economic losses annually. Annexins bind calcium ions and play an important role in flatworm parasite development. To explore potential functions of annexins in T. multiceps, three homologous genes, namely, TmAnxB2, TmAnxB3 and TmAnxB12, were screened from the transcriptome dataset, amplified from C. cerebralis cDNA and subjected to bioinformatics analysis. Then, polyclonal antibodies recognizing the recombinant TmAnxB2 (rTmAnxB2) and rTmAnxB3 were prepared for localization of TmAnxB2 and TmAnxB3 in different tissues and developmental stages by immunofluorescence. The transcription of all three genes was also measured by relative fluorescent quantitative PCR. The sizes of rTmAnxB2, rTmAnxB3 and rTmAnxB12 were 58.00, 53.06 and 53.51 kDa, respectively, and rTmAnxB12 was unstable. Both rTmAnxB2 and rTmAnxB3 were recognized by goat-positive T. multiceps sera in Western blots. Immunofluorescence revealed that TmAnxB2 and TmAnxB3 were localized in the protoscolex and cyst wall and TmAnxB3 was also detected in adult cortex. TmAnxB2 and TmAnxB12 mRNA levels were determined to be highest in oncospheres and protoscolex, whereas transcription of TmAnxB3 was highest in scolex and immature segments. Taken together, these findings indicate that TmAnxB2 and TmAnxB12 may play critical roles in T. multiceps larvae, while TmAnxB3 may have important functions in adults. These results will lay the foundation for functional research of annexins in T. multiceps.

[Preparation and purification of polyclonal antibody against Toxoplasma gondii microneme protein 16 and its application in subcellular localization].

OBJECTIVE: To prepare and purify the rabbit anti-TgMIC16 polyclonal antibody, so as to apply it in subcellular localization. METHODS: New Zealand white rabbits were immunized with purified recombinant TgMIC16 mixing with the same volume of Freund's adjuvant for three times, respectively. The rabbit serum was collected on the 14th day after the last immunization. The polyclonal antibody in rabbit serum was purified with Protein A affinity purification column. ELISA and Western blotting were used to detect the antibody titer and specificity of polyclonal antibody. The polyclonal antibody was used to the localization of TgMIC16 by the immunofluorescence method. RESULTS: Indirect ELISA showed that the antibody titer was 1∶512 000. Western blotting showed that the recombinant TgMIC16 protein was recognized by the specific polyclonal antibody. IFA showed that TgMIC16 was located in the microneme of Toxoplasma gondii. CONCLUSIONS: The rabbit anti-TgMIC16 is prepared and purified, and successfully applied to immunofluorescence localization of TgMIC16 in T. gondii.

Various Bee Pheromones Binding Affinity, Exclusive Chemosensillar Localization, and Key Amino Acid Sites Reveal the Distinctive Characteristics of Odorant-Binding Protein 11 in the Eastern Honey Bee, Apis cerana.

Odorant-binding proteins (OBPs) are the critical elements responsible for binding and transporting odors and pheromones in the sensitive olfactory system in insects. Honey bees are representative social insects that have complex odorants and pheromone communication systems relative to solitary insects. Here, we first cloned and characterized OBP11 (AcerOBP11), from the worker bees antennae of Eastern honey bee, Apis cerana. Based on sequence and phylogenetic analysis, most sequences homologous to AcerOBP11 belong to the typical OBPs family. The transcriptional expression profiles showed that AcerOBP11 was expressed throughout the developmental stages and highly specifically expressed in adult antennae. Using immunofluorescence localization, AcerOBP11 in worker bee's antennae was only localized in the sensilla basiconica (SB) near the fringe of each segment. Fluorescence ligand-binding assay showed that AcerOBP11 protein had strong binding affinity with the tested various bee pheromones components, including the main queen mandibular pheromones (QMPs), methyl p-hydroxybenzoate (HOB), and (E)-9-oxo-2-decanoic acid (9-ODA), alarm pheromone (n-hexanol), and worker pheromone components. AcerOBP11 also had strong binding affinity to plant volatiles, such as 4-Allylveratrole. Based on the docking and site-directed mutagenesis, two key amino acid residues (Ile97 and Ile140) were involved in the binding of AcerOBP11 to various bee pheromones. Taken together, we identified that AcerOBP11 was localized in a single type of antennal chemosensilla and had complex ligand-binding properties, which confer the dual-role with the primary characteristics of sensing various bee pheromones and secondary characteristics of sensing general odorants. This study not only prompts the theoretical basis of OBPs-mediated bee pheromones recognition of honey bee, but also extends the understanding of differences in pheromone communication between social and solitary insects.

Exodermis and endodermis are the sites of xanthone biosynthesis in Hypericum perforatum roots.

Xanthones are specialized metabolites with antimicrobial properties, which accumulate in roots of Hypericum perforatum. This medicinal plant provides widely taken remedies for depressive episodes and skin disorders. Owing to the array of pharmacological activities, xanthone derivatives attract attention for drug design. Little is known about the sites of biosynthesis and accumulation of xanthones in roots. Xanthone biosynthesis is localized at the transcript, protein, and product levels using in situ mRNA hybridization, indirect immunofluorescence detection, and high lateral and mass resolution mass spectrometry imaging (AP-SMALDI-FT-Orbitrap MSI), respectively. The carbon skeleton of xanthones is formed by benzophenone synthase (BPS), for which a cDNA was cloned from root cultures of H. perforatum var. angustifolium. Both the BPS protein and the BPS transcripts are localized to the exodermis and the endodermis of roots. The xanthone compounds as the BPS products are detected in the same tissues. The exodermis and the endodermis, which are the outermost and innermost cell layers of the root cortex, respectively, are not only highly specialized barriers for controlling the passage of water and solutes but also preformed lines of defence against soilborne pathogens and predators.

Preparation and purification of polyclonal antibody against Toxoplasma gondii microneme protein 16 and its application in subcellular localization / 中国血吸虫病防治杂志

To prepare and purify the rabbit anti-TgMIC16 polyclonal antibody, so as to apply it in subcellular localization.New Zealand white rabbits were immunized with purified recombinant TgMIC16 mixing with the same volume of Freund’s adjuvant for three times, respectively. The rabbit serum was collected on the 14th day after the last immunization. The polyclonal antibody in rabbit serum was purified with Protein A affinity purification column. ELISA and Western blotting were used to detect the antibody titer and specificity of polyclonal antibody. The polyclonal antibody was used to the localization of TgMIC16 by the immunofluorescence method.Indirect ELISA showed that the antibody titer was 1∶512 000. Western blotting showed that the recombinant TgMIC16 protein was recognized by the specific polyclonal antibody. IFA showed that TgMIC16 was located in the microneme of Toxoplasma gondii.The rabbit anti-TgMIC16 is prepared and purified, and successfully applied to immunofluorescence localization of TgMIC16 in T. gondii.

Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages.

The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity in H. perforatum leaf protein extract. Immunofluorescence localization revealed that both CHS and BPS are located in the mesophyll. The maximum fluorescence levels were observed in approx. 0.5 and 1 cm long leaves, respectively. The fluorescence intensity observed for CHS significantly exceeded that for BPS. Using histochemical staining, flavonoids were detected in the mesophyll, indicating that the sites of biosynthesis and accumulation coincide. Our results help understand the biosynthesis and underlying regulation of active H. perforatum constituents.

[Gene cloning and expression characteristics of vacuolar-type ATPase subunit B in Bombyx mori].

Vacuolar-type ATPase (V-ATPase), located in the membrane and organelle membrane, is one of important H⁺-transporting proteins. It keeps the proton balance by transporting H⁺ into vacuole, vesicle, or extracellular using the energy from ATP hydrolysis. The subunit B of the vacuolar-type ATPase (BmV-ATPase B) contains the ATP catalytic site, and plays an important role in this process. To study the function of V-ATPase B in Bombyx mori (BmV-ATPase B), we cloned its coding gene from the midgut of the 5th instar silkworm larvae. Then we constructed prokaryotic expression vector and produced the recombinant protein in E. coli. The recombinant protein was identified as BmV-ATPase B by mass spectrometry and purified using Ni-NTA affinity chromatography. This purified protein was used to immunize rabbit to generate polyclonal antibodies of BmV-ATPase B. Finally, the expression patterns of BmV-ATPase B in the silk gland were analyzed by western blotting and immunofluorescence. The full length CDS sequence of BmV-ATPase B was 1 473 bp. BmV-ATPase B was 55 kDa with a PI of 5.3. We analyzed the expression patterns of BmV-ATPase B in different sections of silk gland from the silkworm on the 3rd day of 5th instar and 1st day of wander stage by western blotting. BmV-ATPase B was expressed in all sections of the silk gland and it was abundant in the anterior silk gland (ASG) both in these two developmental stages. Furthermore, immunofluorescence indicated that BmV-ATPase B was located in the silk gland cells. Laser confocal scanning microscopy analysis revealed that BmV-ATPase B was mainly expressed in the cytomembrane of silk gland cells. These data elucidated the expression patterns of BmV-ATPase B in the silk gland of silkworm, which provides a good basis for further studies on the function of V-ATPase B in silk fiber formation.