Clonorchis sinensis: molecular cloning and characterization of 28-kDa glutathione S-transferase

A 28-kDa glutathione S-transferase (Cs28GST) was purified from a Clonorchis sinensis cytosolic fraction through anion-exchange and glutathione-affinity column chromatographies. A monoclonal antibody raised against Cs28GST reacted specifically to the C. sinensis antigen among trematode proteins. A putative peptide of 212 amino residues deduced from a cDNA clone appeared homologous with 28-kDa GST of trematodes, and its secondary structural elements predicted a GSH-binding site. Recombinant Cs28GST showed GST enzyme activity with CDNB substrate and was sensitive to the model inhibitors. The recombinant Cs28GST was antigenically indistinguishable from the native form and was recognized specifically by C. sinensis-infected human sera. The Cs28GST was localized in the tegument and underlying mesenchymal tissues. It is suggested that Cs28GST may play significant physiological roles against bioreactive molecules and be a useful reagent for serodiagnosis of clonorchiasis.     

Clonorchis sinensis: glutathione S-transferase as a serodiagnostic antigen for detecting IgG and IgE antibodies

Human Clonorchis sinensis infection is endemic in East Asian countries. Glutathione S-transferases (GSTs) are anti-oxidant enzymes found in all living creatures as well as in trematodes. In this study, we examined the recombinant 26kDa GST protein of C. sinensis (Cs26GST) for its serodiagnostic antigenicity toward IgG and IgE antibodies by ELISA and immuno-enhanced chemiluminescence, respectively. In IgG ELISA, recombinant Cs26GST showed 33.3% sensitivity and 100% specificity for trematode-infected human sera. In the case of the IgE antibody, recombinant Cs26GST showed 50.0% sensitivity and 93.2% specificity for clonorchiasis infection. We propose that the recombinant Cs26GST is a potent serodiagnostic antigen for detecting C. sinensis-specific IgG and IgE antibodies, and that it be best used as an antigenic cocktail in combination with other antigens.     

Ultrastructural localization of 28 kDa glutathione S-transferase in adult Clonorchis sinensis

Glutathione S-transferase (28GST) with molecular mass of 28 kDa is an antioxidant enzyme abundant in Clonorchis sinensis. In adult C. sinensis, 28GST was localized in tegumental syncytium, cytons, parenchyma, and sperm tails examined by immunoelectron microscopy. C. sinensis 28GST was earlier found to neutralize bioreactive compounds and to be rich in eggs. Accordingly, it is suggested that 28GST plays important roles in phase II defense system and physiological roles in worm fecundity of C. sinensis.     

Antigenic profile and localization of Clonorchis sinensis proteins in the course of infection.

In the course of Clonorchis sinensis infection, antigens presented to the hosts may be in a close relation to growth of the fluke. The antigenic proteins stimulating IgG antibody production were chronologically identified by immunoblot and localized by immunohistochemical staining. In the early stage of infection until 12 weeks post-infection (PI), antigens were proteins with molecular mass larger than 34 kDa which were derived from the tegument, testes and intrauterine eggs. After 20 weeks PI, antigens recognized were 29, 27 and 26 kDa proteins from the intestine, excretory bladder and reproductive organs. It is suggested that the tegumental proteins are the most potent antigens and the excretory-secretory proteins with middle molecular mass of 26-45 kDa contribute to the high level production of antibodies after 20 weeks of the C. sinensis infection.     

Immunohistochemical study on the antigenicity of each organ structure of Clonorchis sinensis

An immunohistochemical study was performed to demonstrate comparative antigenicity of each body structure of the liver fluke, Clonorchis sinensis, such as the digestive tract, reproductive organs, excretory system, tegument and suckers. Indirect immunoperoxidase technique was applied, using formalin-fixed and paraffin-embedded sections of C. sinensis as the antigen. Pooled cat sera obtained 10 weeks after an experimental infection with C. sinensis and peroxidase-conjugated goat anti-cat IgG were used as the primary and secondary antibodies, respectively. The intensity of immunohistochemical stain was very sensitive upon the titers of the primary and secondary antibodies, and their optimum dilutions were found to be 1:1,000-1:2,000 and 1:1,000, respectively. The intestinal epithelial cells, intestinal content and excretory bladder showed strong positive coloring reactions even at lower titer (1:2,000) of the primary antibody, whereas the uterine wall and eggs, vitelline glands, and male reproductive organs showed only weak positive reactions despite an increase in the antibody titer (1:1,000). On the other hand, the suckers, tegument, subtegumental cells and other parenchyma portions did not reveal any positive immunoperoxidase reaction at the same antibody titers. From the above results, it is highly suggested that the most potent antigenicity of C. sinensis occur from their excretory-secretory substances originated from the digestive and excretory organs.     

Prokaryotic Expression and Bioinformatics Analysis of Cytosolic Malate Dehydrogenase from Camellia sinensis(Theaceae)

The complete gene of cytosolic malate dehydrogenase (cMDH) from Camellia sinensis, called Cs cMDH, was obtained by RT PCR and rapid amplification of cDNA ends (GenBank accession number GQ845406). This gene was 1 235 bp in length, encoding a protein of 332 amino acids with the putative molecular weight of 355 kD. The Ecoli Rosetta (DE3) harboring pGEX MDH was induced by 05 mmol·L 1 IPTG at 32℃ for 3 hours, and a 615 kD glutathione Stransferase (GST) fused MDH was obtained in soluble form. The results of NCBI BLAST revealed that Cs cMDH shared 88%-93% of amino acid sequence identity with other cMDH from different higher plants. According to the multiple sequence alignment based on the three dimensional structure of protein, Cs cMDH was predicted to be a dimer with thirteen β sheet and thirteen α helix of each subunit. Cs cMDH contains typical fingerprint sequence (G12AAGQIG18) as all MDHs. The amino acid D43 in Cs cMDH is conserved in all NAD MDHs. Cs cMDH also has some conserved sequence units homologous to other NAD MDHs, such as NAD+ binding sites, catalytic motif and substrate binding sites. Moreover, Cs cMDH contains six Cys which are highly conserved in all plant NAD cMDHs. Therefore, Cs cMDH was inferred to be NAD dependent cMDH. The present study may provide the fundament for the further functional characterization of Cs cMDH.     

Identification and characterization of paramyosin from cyst wall of metacercariae implicated protective efficacy against Clonorchis sinensis infection

Human clonorchiasis has been increasingly prevalent in recent years and results in a threat to the public health in epidemic regions, motivating current strategies of vaccines to combat Clonorchis sinensis (C. sinensis). In this study, we identified C. sinensis paramyosin (CsPmy) from the cyst wall proteins of metacercariae by proteomic approaches and characterized the expressed recombinant pET-26b-CsPmy protein (101 kDa). Bioinformatics analysis indicated that full-length sequences of paramyosin are conserved in helminthes and numerous B-cell/T-cell epitopes were predicted in amino acid sequence of CsPmy. Western blot analysis showed that CsPmy was expressed at four life stages of C. sinensis, both cyst wall proteins and soluble tegumental components could be probed by anti-CsPmy serum. Moreover, immunolocalization results revealed that CsPmy was specifically localized at cyst wall and excretory bladder of metacercaria, as well as the tegument, oral sucker and vitellarium of adult worm. Both immunoblot and immunolocalization results demonstrated that CsPmy was highly expressed at the stage of adult worm, metacercariae and cercaria, which could be supported by real-time PCR analysis. Both recombinant protein and nucleic acid of CsPmy showed strong immunogenicity in rats and induced combined Th1/Th2 immune responses, which were reflected by continuous high level of antibody titers and increased level of IgG1/IgG2a subtypes in serum. In vaccine trials, comparing with control groups, both CsPmy protein and DNA vaccine exhibited protective effect with significant worm reduction rate of 54.3% (p<0.05) and 36.1% (p<0.05), respectively. In consistence with immune responses in sera, elevated level of cytokines IFN-γ and IL-4 in splenocytes suggested that CsPmy could induce combined cellular immunity and humoral immunity in host. Taken together, CsPmy could be a promising vaccine candidate in the prevention of C. sinensis regarding its high immunogenicity and surface localization.     

Molecular identification and characterization of leucine aminopeptidase 2, an excretory-secretory product of Clonorchis sinensis

Aminopeptidases serve vital roles in metabolism of hormones, neurotransmission, turnover of proteins and immunological regulations. Leucine aminopeptidases catalyze the hydrolysis of amino-acid residues from the N-terminus of proteins and peptides. In the present study, leucine aminopeptidase 2 (LAP2) gene of Clonorchis sinensis (C. sinensis) was isolated and identified from an adult cDNA library of C. sinensis. Recombinant CsLAP2 was expressed and purified in Escherichia coli BL21. The open reading frame of LAP2 contains 1,560?bp equivalent to 519 amino acids, a similarity analysis showed a relatively low homology with Homo sapiens (19.0?%), Trypanosoma cruzi (18.0?%), Mus musculus (19.3?%), and relatively high homology with Schistosoma mansoni (65.6?%). The optimum condition of rCsLAP2 enzyme activity was investigated using a fluorescent substrate of Leu-MCA at 37?°C and pH 7.5. The K (m) and V (max) values of rCsLAP2 were 18.2?μM and 10.7?μM/min, respectively. CsLAP2 gene expression can be detected at the stages of the adult worm, metacercaria, excysted metacercaria and egg of C. sinensis using real-time PCR, no difference was observed at the stages of the adult worm, metacercaria and egg. However, CsLAP2 showed a higher expression level at the stage of excysted metacercaria than the adult worm (3.90-fold), metacercaria (4.60-fold) and egg (4.59-fold). Histochemistry analysis showed that CsLAP2 was located at the tegument and excretory vesicle of metacercaria, and the tegument and intestine of adult worm. The immune response specific to rCsLAP2 was characterized by a mixed response patterns of Th1 and Th2, indicating a compounded humoral and cellular immune response. The combined results from the present study indicate that CsLAP2 was an important antigen exposed to host immune system, and probably implicated as potential role in interaction with host cells in clonorchiasis.     

冬虫夏草成熟过程中冬虫夏草菌及其突变基因型在子座和僵虫体中的差异表达

文献报道了从冬虫夏草(Cs)中同时检出蝙蝠蛾拟青霉(Ph)、中华被毛孢(Hs)和冬虫夏草菌(Os)的多个基因型;Cs的成熟伴有化学成分的改变和僵虫体中Hs竞争性菌落形成能力的下降.同时检验了Cs成熟过程中Os突变基因型生物量的变化.应用Southern杂交检验发现:Cs的成熟伴有Ph和2组Os基因型生物量的显著增加.o...     

Characterization of glutathione S-transferase of Taenia solium.

A Taenia solium glutathione-S-transferase fraction (SGSTF) was isolated from a metacestode crude extract by affinity chromatography on reduced glutathione (GSH)-sepharose. The purified fraction displayed a specific glutathione S-transferase (GST) activity of 2.8 micromol/min/mg and glutathione peroxidase selenium-independent activity of 0.22 micromol/min/mg. Enzymatic characterization of the fraction suggested that the activity was closer to the mammalian mu-class GSTs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and enzyme activity analysis showed that the fraction was composed of a major band of Mr = 26 kd and that the active enzyme was dimeric. Immunohistochemical studies using specific antibodies against the major 26-kd band of the SGSTF indicated that GST protein was present in the tegument, parenchyma, protonephridial, and tegumentary cytons of the T. solium metacestode. Antibodies generated against the SGSTF tested in western blot showed cross-reactivity against GSTs purified from Taenia saginata, T. taeniaeformis, and T. crassiceps, but did not react with GSTs from Schistosoma mansoni, or mice, rabbit, and pig liver tissue. Furthermore, immunization of mice with SGSTF reduced the metacestode burden up to 74.2%. Our findings argue in favor of GST having an important role in the survival of T. solium in its hosts.     

Mutagenesis of the active-site cysteine in the ubiquitin-specific protease contained in large tegument protein pUL36 of pseudorabies virus impairs viral replication in vitro and neuroinvasion in vivo

Herpesviruses specify a ubiquitin-specific protease activity located within their largest tegument protein. Although its biological role is still largely unclear, mutation within the active site abolished deubiquitinating (DUB) activity and decreased virus replication in vitro and in vivo. To further elucidate the role of DUB activity for herpesvirus replication, the conserved active-site cysteine at amino acid position 26 within pUL36 of Pseudorabies virus (PrV) (Suid herpesvirus 1), a neurotropic alphaherpesvirus, was mutated to serine. Whereas one-step growth kinetics of the resulting mutant virus PrV-UL36(C(26)S) were moderately reduced, plaque size was decreased to 62% of that of the wild-type virus. Ultrastructural analysis revealed large accumulations of unenveloped nucleocapsids in the cytoplasm, but incorporation of the tegument protein pUL37 was not abolished. After intranasal infection with PrV-UL36(C(26)S) mice showed survival times two times longer than those of mice infected with wild-type or rescued virus. Thus, the DUB activity is important for PrV replication in vitro and for neuroinvasion in mice.     

Analysis of the interaction between the essential herpes simplex virus 1 tegument proteins VP16 and VP1/2

The incorporation of tegument proteins into the herpes simplex virus 1 (HSV-1) virion during virion assembly is thought to be a complex, multistage process occurring via numerous interactions between the tegument and the capsid, within the tegument, and between the tegument and the envelope. Here, we set out to examine if the direct interaction between two essential tegument proteins VP1/2 and VP16 is required for connecting the inner tegument with the outer tegument. By using glutathione S-transferase (GST) pulldowns, we identified an essential role of lysine 343 in VP16, mutation of which to a neutral amino acid abrogated the interaction between VP1/2 and VP16. When the K343A substitution was inserted into the gene encoding VP16 (UL48) of the viral genome, HSV-1 replicated successfully although its growth was delayed, and final titers were reduced compared to titers of wild-type virus. Surprisingly, the mutated VP16 was incorporated into virions at levels similar to those of wild-type VP16. However, the analysis of VP16 on cytoplasmic capsids by fluorescence microscopy showed that VP16 associated with cytoplasmic capsids less efficiently when the VP16-VP1/2 interaction was inhibited. This implies that the direct interaction between VP1/2 and VP16 is important for the efficiency/timing of viral assembly but is not essential for HSV-1 replication in cell culture. These data also support the notion that the incorporation of tegument proteins into the herpesviruses is a very complex process with significant redundancy.     

The Pattern of Tegument-Capsid Interaction in the Herpes Simplex Virus Type 1 Virion Is Not Influenced by the Small Hexon-Associated Protein VP26

Examination of the three-dimensional structure of intact herpes simplex virus type 1 (HSV-1) virions had revealed that the icosahedrally symmetrical interaction between the tegument and capsid involves the pentons but not the hexons (Z. H. Zhou, D. H. Chen, J. Jakana, F. J. Rixon, and W. Chiu, J. Virol. 73:3210-3218, 1999). To account for this, we postulated that the presence of the small capsid protein, VP26, on top of the hexons was masking potential binding sites and preventing tegument attachment. We have now tested this hypothesis by determining the structure of virions lacking VP26. Apart from the obvious absence of VP26 from the capsids, the structures of the VP26 minus and wild-type virions were essentially identical. Notably, they showed the same tegument attachment patterns, thereby demonstrating that VP26 is not responsible for the divergent tegument binding properties of pentons and hexons.     

Gene/protein expression level, immunolocalization and binding characteristics of fatty acid binding protein from Clonorchis sinensis (CsFABP)

Clonorchis sinensis fatty acid-binding protein (CsFABP) belongs to a multigene family of lipid-binding proteins and is considered to be a promising vaccine candidate for human clonorchiasis. In this study, binding characteristics of CsFABP have been examined for the first time. The recombinant CsFABP (rCsFABP) was found to bind 11-(dansylamino) undecanoic acid (DAUDA), causing a blue shift in the fluorescence emission from 543 to 531 nm with an excitation wavelength of 345 nm and a substantial increase in fluorescence intensity. Fluorimetric titration of rCsFABP with DAUDA exhibited an apparent dissociation constant (K (d)) of 1.58 ± 0.14 μM. In the competitive experiment, the rCsFABP efficiently bound saturated C(10)-C(18) fatty acids and unsaturated fatty acids (oleic acid and linoleic acid), and the latter presented the higher affinity. Furthermore, quantitative RT-PCR and western blotting analysis revealed that CsFABP mRNA and protein were differentially expressed throughout the developmental cycle stages of the parasite, which occur in the definitive host (metacercariae, adult worms, and eggs). In addition, immunolocalization assay showed that CsFABP was localized on the vitelline gland, tegument, intestine, seminal vesicle, eggs in uterus, ovary, and testicle of C. sinensis adult worm, as well as on the vitelline gland of metacercaria. Intriguingly, the surface tissue of the bile duct where C. sinensis resided in the infected Sprague-Dawley rat was also strongly labeled, implying that CsFABP may possibly mediate direct interactions with host cells as a component of excretory/secretory products.     

UL26-deficient human cytomegalovirus produces virions with hypophosphorylated pp28 tegument protein that is unstable within newly infected cells

The human cytomegalovirus UL26 open reading frame encodes proteins of 21 and 27 kDa that result from the use of two different in-frame initiation codons. The UL26 protein is a constituent of the virion and thus is delivered to cells upon viral entry. We have characterized a mutant of human cytomegalovirus in which the UL26 open reading frame has been deleted. The UL26 deletion mutant has a profound growth defect, the magnitude of which is dependent on the multiplicity of infection. Two very early defects were discovered. First, even though they were present in normal amounts within mutant virions, the UL99-coded pp28 and UL83-coded pp65 tegument proteins were present in reduced amounts at the earliest times assayed within newly infected cells; second, there was a delay in immediate-early mRNA and protein accumulation. Further analysis revealed that although wild-type levels of the pp28 tegument protein were present in UL26 deletion mutant virions, the protein was hypophosphorylated. We conclude that the UL26 protein influences the normal phosphorylation of at least pp28 in virions and possibly additional tegument proteins. We propose that the hypophosphorylation of tegument proteins causes their destabilization within newly infected cells, perhaps disrupting the normal detegumentation process and leading to a delay in the onset of immediate-early gene expression.     

Thermal‐ and urea‐induced unfolding processes of glutathione S‐transferase by molecular dynamics simulation

The Schistosoma juponicum 26 kDa glutathione S‐transferase (sj26GST) consists of the N‐terminal domain (N‐domain), containing three alpha‐helices (named H1‐H3) and four anti‐parallel beta‐strands (S1‐S4), and the C‐terminal domain (C‐domain), comprising five alpha‐helices (named H4‐H8). In present work, molecular dynamics simulations and fluorescence spectroscopic were used to gain insights into the unfolding process of sj26GST. The molecular dynamics simulations on sj26GST subunit both in water and in 8 M urea were carried out at 300 K, 400 K and 500 K, respectively. Spectroscopic measurements were employed to monitor structural changes. Molecular dynamics simulations of sj26GST subunit induced by urea and temperature showed that the initial unfolding step of sj26GST both in water and urea occurred on N‐domain, involving the disruption of helices H2, H3 and strands S3 and S4, whereas H6 was the last region exposed to solution and was the last helix to unfold. Moreover, simulations analyses combining with fluorescence and circular dichroism spectra indicated that N‐domain could not fold independent, suggesting that correct folding of N‐domain depended on its interactions with C‐domain. We further proposed that the folding of GSTs could begin with the hydrophobic collapse of C‐domain whose H4, H5, H6 and H7 could move close to each other and form a hydrophobic core, especially H6 wrapped in the hydrophobic center and beginning spontaneous formation of the helix. S3, S4, H3, and H2 could form in the wake of the interaction between C‐domain and N‐domain. The paper can offer insights into the molecular mechanism of GSTs unfolding. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 247–259, 2015.     

日本血吸虫26kD抗原基因在BCG中的表达

研究了外源基因日本血吸虫２６ｋＤ抗原（Ｓｊ２６ＧＳＴ）在卡介苗（ｂａｃｉｌｕｓＣａｌｍｅｔｅ－Ｇｕｅｒｉｎ,ＢＣＧ）、耻垢分枝杆菌（Ｍ．ｓｍｅｇｍａｔｉｓ）和大肠杆菌（Ｅ．ｃｏｌｉ）中的表达．运用重组ＤＮＡ和聚合酶链反应（ＰＣＲ）等分子生物学技术,以表达Ｓｊ２６ＧＳＴ的Ｅ．ｃｏｌｉｐＧＥＸ衍生质粒为模板,经ＰＣＲ得到编码Ｓｊ２６ＧＳＴ的全长ｃＤＮＡ片段．将其按正确的阅读框顺序,克隆到人结核杆菌热休克蛋白（ｈｅａｔｓｈｏｃｋｐｒｏｔｅｉｎ,ＨＳＰ）７０的启动子下游,再将ＨＳＰ７０启动子和Ｓｊ２６ＧＳＴ基因一起亚克隆到Ｅ．ｃｏｌｉ－分枝杆菌穿梭质粒ｐＢＣＧ－２０００中,得到Ｅ．ｃｏｌｉ－分枝杆菌穿梭表达质粒ｐＢＣＧ－Ｓｊ２６．ｐＢＣＧ－Ｓｊ２６电转化入ＢＣＧ和Ｍ．ｓｍｅｇｍａｔｉｓｍｃ２１５５中表达Ｓｊ２６ＧＳＴ抗原,所表达的天然重组Ｓｊ２６ＧＳＴ（ｒＳｊ２６ＧＳＴ）为可溶性蛋白,在ＳＤＳ－ＰＡＧＥ上分子量为２６ｋＤ处可见明显的表达蛋白带．其表达量分别占ＢＣＧ和Ｍ．ｓｍｅｇｍａｔｉｓ菌体总蛋白的１５％和１０％．可见,Ｓｊ２６ＧＳＴ基因能在ＢＣＧ中高效表达．     

至少6个突变基因型冬虫夏草菌在冬虫夏草子座中表达的动态变化

本研究检测了在冬虫夏草成熟过程中突变基因型冬虫夏草菌在子座中表达的动态变化。根据冬虫夏草菌基因内转录间隔区(ITS)序列中大量散在的点突变,设计了8个单核苷酸多态性(SNP)延伸引物,采用SNP质谱基因分型法测定各个SNP位点的单核苷酸延伸反应,观察冬虫夏草子座中转换突变和颠换突变基因型菌在冬虫夏草成熟过程中表达的动态变化。其中5个SNP延伸引物(067721-211,067721-240,067721-477,067721-531和067721-581)位于rDNA ITS1和ITS2段,用于区分GC和AT偏倚基因型,但不区分2个AT偏倚基因型。另外3个延伸引物(067740-324,067740-328和067740-360)位于rDNA 5.8S段,用于区分2个AT基因型。采用PCR+EcoRⅠ限制性酶切法检验2个GC偏倚基因型冬虫夏草菌在冬虫夏草成熟过程中表达的动态变化。结果表明:冬虫夏草子座rDNA ITS1-5.8S-ITS2片段的8个SNP位点的质谱图谱显示冬虫夏草子座中存在至少5个冬虫夏草菌转换突变和颠换突变等位基因。它们的表达在冬虫夏草成熟过程中呈现动态变化。067721-211和067721-477位点的AT偏倚型等位基因的峰高明显高于GC偏倚型;随着冬虫夏草的成熟,这2个位点的AT型等位基因峰高大幅度下降。SNP质谱图不仅显示转换突变的等位基因,颠换突变等位基因也有很高的检出率,它们的峰高在一些位点甚至超过GC和AT基因型等位基因;随着冬虫夏草的成熟,颠换突变等位基因的检出率和峰高趋于下降。在区分2个AT偏倚基因型的研究中,AB067744和AB067740 2个基因型的等位基因同时存在于未成熟冬虫夏草子座中。随着冬虫夏草的成熟,AB067744基因型等位基因的峰高明显降低,而AB067740基因型等位基因的峰高明显升高。PCR产物EcoRⅠ酶切试验发现子座中存在2组GC偏倚型菌,具有完全不同的成熟模式。综上,冬虫夏草子座中存在至少6个突变基因型冬虫夏草菌,其表达随着冬虫夏草的成熟呈现动态变化。这些突变基因型菌表达的动态变化对于冬虫夏草子座的萌发和成熟具有重要菌物学意义。     

中华绒螯蟹不同生理阶段腹肢的结构变化及粘液腺的发育特征

用显微及亚显微方法研究了中华绒螯蟹雌体不同生理阶段(幼蟹、未成熟蟹、成熟蟹、抱卵蟹和流产蟹)腹肢的体壁结构变化和粘液腺发育特征。体壁的上皮细胞层常与粘液腺相连,粘液腺分泌物经导管穿过各层角膜排出。不同生理状况下中华绒螯蟹的腹肢体壁各角膜层结构的比例及致密度有明显的差异,粘液腺细胞及导管的数量不同,粘液腺与上皮细胞的连接程度也不同。粘液腺是否正常分泌、刚毛囊的开闭均与中华绒螯蟹的胚胎附着有关。比较研究发现,太湖抱卵蟹腹肢体腔内腺体比温州本地抱卵蟹腹肢内腺体发达,分泌的粘液也特别多。分析认为中华绒螯蟹腹肢组织结构、粘液腺的发育和分泌状况与胚胎流产有密切的关系。     

Identification of the nucleocapsid, tegument, and envelope proteins of the shrimp white spot syndrome virus virion

The protein components of the white spot syndrome virus (WSSV) virion have been well established by proteomic methods, and at least 39 structural proteins are currently known. However, several details of the virus structure and assembly remain controversial, including the role of one of the major structural proteins, VP26. In this study, Triton X-100 was used in combination with various concentrations of NaCl to separate intact WSSV virions into distinct fractions such that each fraction contained envelope and tegument proteins, tegument and nucleocapsid proteins, or nucleocapsid proteins only. From the protein profiles and Western blotting results, VP26, VP36A, VP39A, and VP95 were all identified as tegument proteins distinct from the envelope proteins (VP19, VP28, VP31, VP36B, VP38A, VP51B, VP53A) and nucleocapsid proteins (VP664, VP51C, VP60B, VP15). We also found that VP15 dissociated from the nucleocapsid at high salt concentrations, even though DNA was still present. These results were confirmed by CsCl isopycnic centrifugation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry, by a trypsin sensitivity assay, and by an immunogold assay. Finally, we propose an assembly process for the WSSV virion.