Magnetic microsphere and its use in biochemical separation and analysis

Magnetic microsphere comprises a magnetically responsive metal or metal oxide core surrounded by a polymer shell with active groups. Nowadays, methods of directly coating polymer, monomer polymerazation, impregnation, extrusion and biological synthesis are generally used to prepare magnetic particles. This kind of superparamagnetic microspheres can be attached to chemical, biochemical and biological substances by their active groups, then applying a magnetic field to separate from the media. Preparation and utilization of magnetic microspheres in immunoassay, nucleic acid hybrization assay, gene sequencing, cell isolation, enzyme immoblization, receptor isolation and other Gelds are reviewed with 44 references in this paper. Also, the further development is outlooked.

Progress in electrochemiluminescent analysis

The recent progress in electrochemiluminescent (ECL) assay is reviewed. This review begins with the fundamental researches in ECL, including the discovery of new ECL-active species, such as biochemical, organic and metallorganic materials, digital modeling of ECL process, the flow cells used in ECL assay, and electrochemiluminescent sensor. The application of ECL in environmental analysis, immunoassay, nucleotide acid hybridization sensor. The applications of ECL in environmental analysis, immunooassay, nucleic acid hybridization assay, and other aspects are reviewed with the latest references in detail. Finally, the main problems in the further investigation are outlooked, so are its prospects.

Molecular Cloning and Expression Analysis of a Cytosolic Hsp70 gene from Laminaria japonica (Laminariaceae, Phaeophyta)

In this study, a full-length cytosolic heat shock protein 70 complementary DNA (cDNA) of Laminaria japonica (designated as LJHsp70) was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) coupled with rapid amplification of cDNA ends. The full length of LJHsp70 cDNA was 2,918 bp, with a 5' untranslated region of 248 bp, a 3' untranslated region of 696 bp, and an open reading frame of 1,974 bp encoding a polypeptide of 657 amino acids with an estimated molecular mass of 72.03 kDa and an estimated isoelectric point of 4.97. There was highly repeated sequence of CAA in 5' untranslated region of LJHsp70. The result of phylogenetic tree of Hsp70s, the BLAST program, analysis and cytosolic Hsp70-specific motif of LJHsp70 verified that the cloned LJHsp70 belonged to cytosolic Hsp70 family. Three typical Hsp70 signature motifs were detected in LJHsp70 by InterPro analysis. Under different stress conditions, messenger RNA (mRNA) expression levels of LJHsp70 were quantified by quantitative RT-PCR. To L. japonica sporophytes kept in different temperatures for 1 h, the expression level of LJHsp70 at 30A degrees C was highest and twofold higher than that at 10A degrees C. To L. japonica sporophytes kept at 25A degrees C for different times, the mRNA expression level of LJHsp70 reached a maximum level after 7 h and then dropped progressively. The expression level of LJHsp70 at 0 or 5aEuro degrees salt concentration for 2 h was twofold higher than that at 30aEuro degrees salt concentration for 2 h. The results showed that LJHsp70 may be a kind of potential biomarker used to monitor environment conditions.

An improved PCR method for direct identification of Porphyra (Bangiales, Rhodophyta) using conchocelis based on a RUBISCO intergenic spacer

An improved method of PCR in which the small segment of conchocelis is amplified directly without DNA extraction was used to amplify a RUBISCO intergenic spacer DNA fragment from nine species of red algal genus Porphyra (Bangiales, Rhodophyta), including Porphyra yezoensis (Jiangsu, China), P. haitanensis (Fujian, China), P. oligospermatangia (Qingdao, China), P. katadai (Qingdao, China), P. tenera (Qingdao, China), P. suborboculata (Fujian, China), P. pseudolinearis (Kogendo, Korea), P. linearis (Devon, England), and P. fallax (Seattle, USA). Standard PCR and the method developed here were both conducted using primers specific for the RUBISCO spacer region, after which the two PCR products were sequenced. The sequencing data of the amplicons obtained using both methods were identical, suggesting that the improved PCR method was functional. These findings indicate that the method developed here may be useful for the rapid identification of species of Porphyra in a germplasm bank. In addition, a phylogenetic tree was constructed using the RUBISCO spacer and partial rbcS sequence, and the results were in concordant with possible alternative phylogenies based on traditional morphological taxonomic characteristics, indicating that the RUBISCO spacer is a useful region for phylogenetic studies.

Expression profiles of penaeidin from Fenneropenaeus chinensis in response to WSSV and vibrio infection by real-time PCR

Penaeidin from Chinese shrimp (Fenneropenaeus chinensis) has proved to be one of the most important antimicrobial peptides in the bodies of animals. The relative quantitative real-time PCR method is developed to study through time, the mRNA expression profile of penaeidin in the muscle and haemocyte tissue of Chinese shrimp infected with vibrio (Vibrio anguillarum) and WSSV (white spot syndrome virus). Research results showed that the same pathogens infection experiments produced similar gene expression profile in different tissues while different expression profiles appeared in the same tissues infected by different exterior pathogens. In vibrio infection experiments, a "U" Re expression profile resulted. Expression levels of penaeidin increased and surpassed the non-stimulated level, indicating that penaeidin from Chinese shrimp has noticeable antimicrobial activities. In WSSV infection experiments, the expression profile appeared as an inverse "U" with the expression of penaeidin gradually decreasing to below baseline level after 24 h. The expression of antimicrobial peptides gene in mRNA level in response to virus infection in shrimp showed that international mechanisms of virus to haemocytes and microbial to haemocytes are completely different. Decline of penaeidins expression levels may be due to haemocytes being destroyed by WSSV or that the virus can inhibit the expression of penaeidins by yet undiscovered modes. The expression profiles of penaeidin in response to exterior pathogen and the difference of expression profiles between vibrio and WSSV infection provided some clues to further understanding the complex innate immune mechanism in shrimp.

Rapid detection of virulent protease secreted by Vibrio anguillarum by dot enzyme-linked immunosorbent assay

Dot enzyme-linked immunosorbent assay (dot-ELISA), indirect ELISA and Western blot were performed to detect the virulent protease secreted by Vibrio anguillarum which was isolated from the diseased left-eyed flounder, Paralichthys olivaceous. Sensitivity results showed that dot-ELISA is a more sensitive, rapid and simple technique for the protease detection. The minimal detectable amount of protease is about 7 pg in the dot-ELISA test, while 7.8 ng in the indirect ELISA and 6.25 ng in the Western blot respectively. Protease could be detected 2 h after incubation of V. anguillarum in the 2216E liquid medium but enzyme activity was very low at that period. From 6 to 12 h, the amount and enzyme activity of protease increased markedly and reached maximum at stationary phase. Analysis of serum samples periodically collected from the infected flounders showed that after 2 h of infection by V. anguillarum, the pathogenic bacteria could be detected in the blood of the infected flounders but no protease was found. It was 5 similar to 6 h after infection that the protease was detected in blood and then the amount increased as infection advanced. Quantitative detection of protease either incubation in the medium or from the blood of infected flounders could be accomplished in virtue of positive controls of quantificational protease standards ("marker") so that the alterations of protease secretion both in vitro and in vivo could be understood generally. In addition, the indirect ELISA and dot-ELISA were also performed to detect V. anguillarum cells. Results indicated that the sensitivity of indirect ELISA to bacteria cells is higher than that of the dot-ELISA, and that the minimal detectable amount is approximately 10(4) cell/mL in the indirect ELISA, while 10(5) cell/mL in the dot-ELISA.

White spot syndrome virus (WSSV) infects specific hemocytes of the shrimp Penaeus merguiensis

White spot syndrome virus (WSSV) was specifically detected by PCR in Penaeus merguiensis hemocytes, hemolymph and plasma. This suggested a close association between the shrimp hemolymph and the virus. Three types of hemocyte from shrimp were isolated using flow cytometry. Dynamic changes of the hemocyte subpopulations in P. merguiensis at different times after infection were observed, indicating that the WSSV infection selectively affected specific subpopulations. Immunofluorescence assay (IFA) and a Wright-Giemsa double staining study of hemocyte types further confirmed the cellular localization of the virus in the infected hemocytes. Electron microscopy revealed virus particles in both vacuoles and the nucleus of the semigranular cells (SGC), as well as in the vacuoles of the granular cells (GC). However, no virus could be detected in the hyaline cells (HC). Our results suggest that the virus infects 2 types of shrimp hemocytes-GCs and SGCs. The SGC type contains higher virus loads and exhibits faster infection rates, and is apparently more susceptible to WSSV infection.

Analysis of three marine fish cell lines by RAPD assay

We tested the applicability of the random amplified polymorphic deoxyribonucleic acid (RAPD) analysis for identification of three marine fish cell lines FG. SPH, and RSBK and as a possible tool to detect cross-contamination. Sixth commercial 10-mer RAPD primers were tested on the cell lines and on samples collected from individual fish. The results obtained showed that the cell lines could be identified to the correspondent species on the basis of identical Patterns produced by 35-48% of the primers tested. the total mean similarity indices for cell lines versus correspondent species of individual fish ranged from 0.825 to 0.851. indicating the existence of genetic variation in these cell lines in relation to the species of their origin. Also, four primers, which gave a monomorphic hand pattern within species/line, but different among the species/line, were obtained. These primers can be useful for identification of these cell lines and for characterization of the genetic variation of these cell lines in relation to the species of their origin. This supported the use of RAPD analysis as an effective tool in species identification and cross-contamination test among different cell lines.

A prophenoloxidase from the Chinese mitten crab Eriocheir sinensis: Gene cloning, expression and activity analysis

Prophenoloxidase (proPO) is a conserved copper-containing enzyme that plays important roles in immune response of crustaceans and insects. In the present study, the full-length cDNA of a prophenoloxidase (designated EsproPO) was cloned from haemocytes of Chinese mitten crab Eriocheir sinensis by expressed sequence tag (EST) and PCR techniques. The isolated 3549 bp full-length cDNA of EsproPO contained a 2040 bp open reading frame (ORF) encoding a putative proPO protein of 679 amino acids, a 5'-untranslated region (UTR) of 68 bp, and a long 3'-UTR of 1441 bp. Two putative copper-binding sites, a proteolytic activation site, and a complement-like motif (GCGWPQHM) were identified in the deduced amino acid sequence of EsproPO. Homology analysis revealed that EsproPO was highly similar to other proPOs from crustaceans with identities from 52% to 68%. The conserved domains and motifs, and higher similarity with other proPOs suggested that EsproPO was a member of the proPO family. The mRNA expression of EsproPO and PO specific activities in the tissues of hepatopancreas, gill, gonad, muscle, heart, eye and haemocytes were measured by quantitative real-time PCR and colorimetric assay, respectively. The mRNA transcripts of EsproPO and PO specific activities could be detected in all the examined tissues with the highest level both in hepatopancreas. Three peaks of EsproPO mRNA expression were recorded at 2 h, 12 h and 48 h in haemocytes of Chinese mitten crab post Vibrio anguillarum challenge, which was consistent with the temporal profile of PO specific activity. The mRNA expression pattern and the activity fluctuation of EsproPO post V. anguillarum stimulation indicated that it was potentially involved in the acute response against invading bacteria in Chinese mitten crab. (c) 2007 Elsevier Ltd. All rights reserved.

Species identification in salted products of red snappers by semi-nested PCR-RFLP based on the mitochondria 12S rRNA gene sequence

A molecular approach was developed to distinguish species of red snappers among commercial salted fish products. The specific fragments of the mitocliondrial 12S rRNA gene, which were about 450 bp, were obtained using the semi-nested polymerase chain reaction (semi-nested PCR). Subsequently, PCR amplicons were sequenced, aiming to select restriction endonucleases that generated species-specific restriction fragment length polymorphism (RFLP) profiles. Discrimination of red snappers Lutjanus sanguineus, L. erythopterus from L. argentintaculatus, L. malabarius and other morphologically similar fishes such as Lethrinus leutjanus and Pinjalo pinjalo was feasible by one restriction digestion reaction with three endonucleases Hae III, Sca I and SnaB I, however, for differentiation of L. sattguineus and L. erythopterus, another restriction digestion reaction with single restriction endonuclease Mae II was needed. The seminested PCR-RFLP was demonstrated to be reliable in species identification of salted fish products in this study. (c) 2005 Published by Elsevier Ltd.

The mitochondrial manganese superoxide dismutase gene in Chinese shrimp Fenneropenaeus chinensis: Cloning, distribution and expression

Manganese superoxide dismutase (MnSOD) plays an important role in crustacean immune defense reaction by eliminating oxidative stress. Knowledge on MnSOD at molecular level allows us to understand its regulatory mechanism in crustacean immune system. A novel mitochondrial manganese superoxide dismutase (mMnSOD) was cloned from hepatopancreas of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 1185 bp with a 660 bp open reading frame, encoding 220 amino acids. The deduced amino acid sequence contains a putative signal peptide of 20 amino acids. Sequence comparison showed that the mMnSOD of F. chinensis shares 88% and 82% identity with that of giant freshwater prawn Macrobrachium rosenbergii and blue crab Callinectes sapidus, respectively. mMnSOD transcripts were detected in hepatopancreas, hemocytes, lymphoid organ, intestine, ovary, muscle and gill by Northern blotting. RT-PCR analysis indicated that mMnSOD showed different expression profiles in shrimp hemocytes and hepatopancreas after artificial infection with while spot syndrome virus (WSSV). In addition, a fusion protein containing mMnSOD was produced in vitro. LC-ESI-MS analysis showed that two peptide fragments (-GDVNTVISLAPALK- and -NVRPDYVNAIWK-) of the recombinant protein were identical to the corresponding sequence of M. rosenbergii mMnSOD, and the enzyme activity of the refolded recombinant protein was also measured. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular cloning, expression of a big defensin gene from bay scallop Argopecten irradians and the antimicrobial activity of its recombinant protein

Antimicrobial peptides are important components of the host innate immune responses by exerting broad-spectrum microbicidal activity against pathogenic microbes. The first mollusk big defensin (designated AiBD) cDNA was cloned from bay scallop Argopecten irradians by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The scallop AiBD consisted of 531 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, encoding a polypeptide of 122 amino acids. The high similarity of AiBD deduced amino acid sequence with big defensin from Tachypleus tridentatus and Branchiostoma belcheri tsingtaunese indicated that AiBD should be a member of big defensin family. The expression of AiBD in various tissues was measured by using Northern blotting analysis. mRNA transcripts of AiBD could be detected in haemocytes of unchallenged scallops. The temporal expression of AiBD in haemolymph after Vibrio anguilarum challenge was recorded by quantitative real time PCR. The relative expression level of AiBD in haemolymph was up-regulated evenly in the first 8 h, followed by a drastic increase, and increased 131.1-fold at 32 h post-injection. These results indicated that AiBD could be induced by bacterial challenge, and it should participate in the immune responses of A. irradians. Biological activity assay revealed that recombinant AiBD could inhibit the growth of both Gram-positive and Gram-negative bacteria, and also showed strong fungicidal activity towards the expression host. Recombinant expression of AiBD made it possible to further characterize its functions involved in immune responses, and also provided a potential therapeutic agent for disease control in aquaculture. (c) 2006 Elsevier Ltd. All rights reserved.

CfLGBP, a pattern recognition receptor in Chlamys farreri involved in the immune response against various bacteria

Lipopolysaccharide and beta-1, 3-glucan binding protein (LGBP) is a kind of pattern recognition receptor, which can recognize and bind LPS and beta-1, 3-glucan, and plays curial roles in the innate immune defense against Gram-negative bacteria and fungi. In this study, the functions of LGBP from Zhikong scallop Chlamys farreri performed in innate immunity were analyzed. Firstly, the mRNA expression of CfLGBP in hemocytes toward three typical PAMPS stimulation was examined by realtime PCR. It was up-regulated extremely (P < 0.01) post stimulation of LPS and beta-glucan, and also exhibited a moderate up-regulation (P < 0.01) after PGN injection. Further PAMPs binding assay with the polyclonal antibody specific for CfLGBP proved that the recombinant CfLGBP (designated as rCfLGBP) could bind not only LPS and beta-glucan, but also PGN in vitro. More importantly, rCfLGBP exhibited obvious agglutination activity towards Gram-negative bacteria Escherichia coil, Gram-positive bacteria Bacillus subtilis and fungi Pichia pastoris. Taking the results of immunofluorescence assay into account, which displayed CfLGBP was expressed specifically in the immune cells (hemocytes) and vulnerable organ (gill and mantle), we believed that LGBP in C farreri, serving as a multi-functional PRR, not only involved in the immune response against Gram-negative and fungi as LGBP in other invertebrates, but also played significant role in the event of anti-Gram-positive bacteria infection. As the first functional research of LGBP in mollusks, our study provided new implication into the innate immune defense mechanisms of C. farreri and mollusks. (C) 2010 Elsevier Ltd. All rights reserved.

Discovery of the genes in response to white spot syndrome virus (WSSV) infection in Fenneropenaeus chinensis through cDNA microarray

We used microarray technology to study differentially expressed genes in white spot syndrome virus (WSSV)-infected shrimp. A total of 3136 cDNA targets, including 1578 unique genes from a cephalothorax cDNA library and 1536 cDNA clones from reverse and forward suppression subtractive hybridization (SSH) libraries of Fenneropenaeus chinensis, plus 14 negative and 8 blank control clones, were spotted onto a 18 x 18 mm area of NH2-modified glass slides. Gene expression patterns in the cephalothorax of shrimp at 6 h after WSSV injection and moribund shrimp naturally infected by WSSV were analyzed. A total of 105 elements on the arrays showed a similar regulation pattern in artificially infected shrimp and naturally infected moribund shrimp; parts of the results were confirmed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The up-regulated expression of immune-related genes, including heat shock proteins (HSP70 and HSP90), trehalose-phosphate synthase (TPS), ubiquitin C, and so forth, were observed when shrimp were challenged with WSSV. Genes including myosin LC2, ATP synthase A chain, and arginine kinase were found to be down-regulated after WSSV infection. The expression of housekeeping genes such as actin, elongation factor, and tubulin is not stable, and so these genes are not suitable as internal standards for semiquantitative RT-PCR when shrimp are challenged by WSSV. As a substitute, we found that triosephosphate isomerase (TPI) was an ideal candidate of interstandards in this situation.