脊尾白虾血蓝蛋白大亚基基因的克隆及表达分析

应用RACE技术克隆脊尾白虾血蓝蛋白大亚基基因, 并通过攻毒实验揭示脊尾白虾血蓝蛋白基因的先天免疫防御作用, 为脊尾白虾(Exopalaemon carinicauda)的免疫防治研究提供依据和思路。研究成功克隆了脊尾白虾血蓝蛋白大亚基基因全长cDNA序列, 该大亚基cDNA全长 2192 bp, 开放式阅读框长 2034 bp, 5′非编码区长 21 bp, 3′非编码区长 137 bp, 将该基因命名为 EcHcL。EcHcL编码 667 个氨基酸, 前 21 个氨基酸组成信号肽, 推测成熟肽的分子量为 78.5 kD。Blast比对结果显示, 由脊尾白虾血蓝蛋白EcHcL序列推导的氨基酸序列与日本沼虾、凡纳滨对虾血蓝蛋白氨基酸序列的同源性分别达到 87%、73%, 其M结构域氨基酸序列与斑节对虾、日本对虾等物种同源性性高达 90% 左右, 由此推断该cDNA序列属于血蓝蛋白家族。组织表达分析结果显示, EcHcL基因在脊尾白虾鳃、卵巢、肝胰腺、心脏、肠、肌肉、胃、腹神经节、眼柄、血细胞中均有表达, 肝胰腺中相对表达量最高。Real-time PCR分析发现EcHcL基因在金黄色葡萄球菌、副溶血弧菌和对虾白斑综合征病毒(WSSV)感染后脊尾白虾肝胰腺和血细胞中的表达量显著增加, 并具有不同的时空表达模式, 推测脊尾白虾EcHcL基因在免疫防御中具有重要作用。     

Transcriptional regulation of extracellular copper zinc superoxide dismutase from white shrimp Litopenaeus vannamei following Vibrio alginolyticus and WSSV infection

The cDNA encoding an extracellular copper zinc superoxide dismutase (LvECSOD) was cloned from the hepatopancreas of white shrimp Litopenaeus vannamei. It consisted of 915 bp nucleotides with an open reading frame corresponding to a deduced protein of 178 amino acids. The LvECSOD contains a putative signal peptide of 16 amino acids, two potential N-linked glycosylation sites (N(115)GTA and N(135)ITG) and a copper zinc superoxide dismutase family signature sequence (G(162)NAGaRvACctI(173)). It was found that four copper binding sites, four zinc binding sites and two cysteines involving in the formation of the disulfide bridge were conserved in the protein. LvECSOD shared 33-58% identity to ECSODs from other organisms. Expression analysis revealed that LvECSOD mRNA was widely distributed in all the tissues examined. When the shrimp challenged with Vibrio alginolyticus or white spot syndrome virus (WSSV), expression of LvECSOD mRNA in the hepatopancreas and hemocytes was mediated responsively. Our results suggested that LvECSOD was implicated in the immune response induced by V. alginolyticus and WSSV.     

Blocking the large extracellular loop (LEL) domain of FcTetraspanin-3 could inhibit the infection of white spot syndrome virus (WSSV) in Chinese shrimp, Fenneropenaeus chinensis

Tetraspanins belong to the transmembrane 4 superfamily (TM(4)SF), which span the cell membrane 4 times and act as bridges or connectors. Increasing evidences have shown that tetraspanins play important role in virus infection. The large extracellular loop (LEL) of a tetraspanin is considered as a possible target of some virus. Tetraspanins are widely found in invertebrates, but the functional roles of most invertebrate tetraspanins have remained unknown. Recently, a tetraspanin, called FcTetraspanin-3, was cloned from the cDNA library of Chinese shrimp, Fenneropenaeus chinensis. The FcTetraspanin-3 constitutive expression in all examined tissues and the expression of the gene were highly induced in hepatopancreas, lymphoid organ and intestine by white spot syndrome virus (WSSV) challenge. In this study, we expressed and purified the recombinant peptide containing the LEL domain of FcTetraspanin-3, and produced the anti-LEL polyclone antibody. The expression of FcTetraspanin-3 was observed by real-time PCR and Western blot. Also, the localization of FcTetraspanin-3-positive cells in intestine and hepatopancreas were revealed by immunofluorescence. The results of anti-LEL antibody blocking experiments shown that the antibody can significantly reduce the mortality of shrimp challenged by WSSV. Additionally, dsRNA interference was utilized to examine the functional role of FcTetraspanin-3 in response to WSSV infection, and a sensible decrease of the viral copy number in the tetraspanin knockdown shrimp. These results suggested the blocking of LEL domain of FcTetraspanin-3 could inhibit the infection of WSSV. FcTetraspanin-3 might play an important role in response to WSSV infection, and the LEL domain of FcTetraspanin-3 might mediate the entry of WSSV.     

Immune response of white shrimp, Litopenaeus vannamei, after a concurrent infection with white spot syndrome virus and infectious hypodermal and hematopoietic necrosis virus

In the present study, we investigated immunological changes in viral-infected white shrimp, Litopenaeus vannamei. White shrimp were infected with white spot syndrome virus (WSSV) or co-infected with WSSV and infectious hypodermal and hematopoietic necrosis virus (IHHNV) as detected by polymerase chain reaction (PCR). The complete (100%) mortality rate of shrimp was caused by viral infection due to immune parameters being suppressed including decreases in phenoloxidase activity, total hemocyte counts, differential hemocyte counts, and the gene expressions of prophenoloxidase and peroxinectin. In addition, increases in lipopolysaccharide and beta-1,3-glucan-binding protein of hemocytes and the hepatopancreas, and respiratory bursts per cell, and a decrease in superoxide dismutase were found in viral-infected shrimp, which may have been related to the defense against viral infection.     

Susceptibility of juvenile Macrobrachium rosenbergii to different doses of high and low virulence strains of white spot syndrome virus (WSSV)

As some literature on the susceptibility of different life stages of Macrobrachium rosenbergii to white spot syndrome virus (WSSV) is conflicting, the pathogenesis, infectivity and pathogenicity of 2 WSSV strains (Thai-1 and Viet) were investigated here in juveniles using conditions standardized for Penaeus vannamei. As with P. vannamei, juvenile M. rosenbergii (2 to 5 g) injected with a low dose of WSSV-Thai-1 or a high dose of WSSV-Viet developed comparable clinical pathology and numbers of infected cells within 1 to 2 d post-infection. In contrast, a low dose of WSSV-Viet capable of causing mortality in P. vannamei resulted in no detectable infection in M. rosenbergii. Mean prawn infectious dose 50% endpoints (PID50 ml-1) determined in M. rosenbergii were in the order of 100-fold higher for WSSV-Thai-1 (105.3±0.4 PID50 ml-1) than for WSSV-Viet (103.2±0.2 PID50 ml-1), with each of these being about 20-fold and 400-fold lower, respectively, than found previously in P. vannamei. The median lethal dose (LD50 ml-1) determined in M. rosenbergii was also far higher (~1000-fold) for WSSV-Thai-1 (105.4±0.4 LD50 ml-1) than for WSSV-Viet (102.3±0.3 LD50 ml-1). Based on these data, it is clear that juvenile M. rosenbergii are susceptible to WSSV infection, disease and mortality. In comparison to P. vannamei, however, juvenile M. rosenbergii appear more capable of resisting infection and disease, particularly in the case of a WSSV strain with lower apparent virulence.     

Comparison of a novel in situ polymerase chain reaction (ISPCR) method to other methods for white spot syndrome virus (WSSV) detection in Penaeus vannamei

Penaeus vannamei were experimentally injected with white spot syndrome virus (WSSV) and tested for WSSV at different times post-injection (p.i.) by 1-step polymerase chain reaction (PCR), 2-step PCR, in situ hybridization (ISH) and in situ polymerase chain reaction (ISPCR) in order to compare sensitivity of the methods. With 1-step PCR, 4 of 15 shrimp tested positive for WSSV at 12 h p.i., and all tested positive by 24 h p.i. With 2-step PCR, 13 out of 15 samples tested positive at 2 h p.i. and all were positive by 4 h p.i. Using in situ hybridization, 1 sample tested positive at 18 h p.i. and all were positive by 36 h p.i. With ISPCR, 1 out of 5 samples was positive at 2 h p.i. and all were positive by 8 h p.i. Two-step PCR showed the highest sensitivity, followed by ISPCR, 1-step PCR and ISH. Although ISPCR revealed WSSV in 9 of 10 P. vannamei that tested positive for WSSV using 2-step PCR, none of the shrimp examined showed clinical signs of WSSV infection or detectable WSSV with 1-step PCR. The major infected organs were muscle and the hepatopancreas.     

三疣梭子蟹HMGBa基因克隆及其应答不同病原入侵的表达特征

为研究三疣梭子蟹(Portunus trituberculatus)高迁移率族蛋白B (High-mobility group box protein, HMGB)在其先天免疫中发挥的功能, 利用RACE技术首次克隆得到了三疣梭子蟹HMGBa基因, 命名为PtHMGBa。其cDNA序列全长1030 bp, 其中5′端非编码区(UTR)为94 bp, 3′端非编码区(UTR)为255 bp, 开放阅读框(ORF)为681 bp, 编码一个含有227个氨基酸, 分子量25.82 kD, 理论等电点为5.94的蛋白质。PtHMGBa蛋白包含2个HMG盒结构域和一个酸性尾部结构域。分析表明, 三疣梭子蟹HMGBa氨基酸序列与凡纳滨对虾(Litopenaeus vannamei) HMGBa相似度最高。实时荧光定量PCR结果显示, PtHMGBa基因在血细胞和肝胰腺的表达量最高, 在眼柄中表达量最低。在副溶血弧菌和WSSV感染过程中, PtHMGBa基因在肝胰腺和血细胞中均出现了表达上调。其中, 经副溶血弧菌感染后, 该基因在上述2种组织中分别于48h和6h达到表达量的峰值; 经WSSV感染后, 该基因在2种组织中均在12h达到表达量的峰值。结果表明PtHMGBa基因参与了三疣梭子蟹抵御外来病原的免疫响应, 研究为深入开展三疣梭子蟹和其他甲壳动物的免疫调控机理提供了科学依据。     

Identification of icp11, the most highly expressed gene of shrimp white spot syndrome virus (WSSV)

This study investigates white spot syndrome virus (WSSV) gene expression levels in the cells of 2 hosts (Penaeus monodon and Litopenaeus vannamei). Microarray and expressed sequence tag (EST) analysis of the mRNA profiles in WSSV-infected P. monodon cells were used to identify WSSV genes that were very highly expressed. Results showed that the mRNA of the WSSV icp11 gene consistently had the highest copy number of all (3x higher than the major envelope protein, VP28). At the protein level in WSSV-infected L. vannamei, 2-dimensional gel analysis and liquid chromatography-nano-electrospray ionization tandem mass spectrometry (LC-nanoESI-MS/MS) protein identification also showed that this WSSV non-structural protein has the highest expression levels reported to date. ICP11 is capable of self-multimerization, and it becomes located in both the cytoplasm and nucleus of the host cell. These data suggest that ICP11 plays an important, but presently unknown, role during viral infection, and that expression of the WSSV icp11 gene/WSSV ICP11 protein is potentially a good and diagnostically useful indicator of WSSV infection.     

Changes in tissue defence system in white spot syndrome virus (WSSV) infected Penaeus monodon

The present study examined the changes occurring in the pro phenoloxidase system and antioxidant defence status in haemolymph, hepatopancreas and muscle tissue of white spot syndrome virus (WSSV) infected Penaeus monodon. Tiger shrimps (P. monodon) were infected with white spot virus by intramuscular injection of the virus inoculum. Levels of lipid peroxides and the activities of phenoloxidase, glutathione-dependent antioxidant enzymes [glutathione peroxidase (GPX), glutathione-S-transferase (GST)] and antiperoxidative enzymes [superoxide dismutase (SOD) and catalase (CAT)] were determined. WSSV infection induced a significant increase in lipid peroxidation in haemolymph, muscle and hepatopancreas of experimental P. monodon compared to normal controls. This was paralleled by significant reduction in the activities of phenol oxidase, glutathione-dependent antioxidant enzymes and antiperoxidative enzymes. The results of the present study indicate that the tissue antioxidant defence system in WSSV infected P. monodon is operating at a lower rate, which ultimately resulted in the failure of counteraction of free radicals, leading to oxidative stress as evidenced by the increased level of lipid peroxidation.     

In vivo titration of white spot syndrome virus (WSSV) in specific pathogen-free Litopenaeus vannamei by intramuscular and oral routes

White spot syndrome virus (WSSV) is a devastating pathogen in shrimp aquaculture. Standardized challenge procedures using a known amount of infectious virus would assist in evaluating strategies to reduce its impact. In this study, the shrimp infectious dose 50% endpoint (SID50 ml(-1)) of a Thai isolate of WSSV was determined by intramuscular inoculation (i.m.) in 60 and 135 d old specific pathogen-free (SPF) Litopenaeus vannamei using indirect immunofluorescence (IIF) and 1-step polymerase chain reaction (PCR). Also, the lethal dose 50% endpoint (LD50 ml(-1)) was determined from the proportion of dead shrimp. The median virus infection titers in 60 and 135 d old juveniles were 10(6.8) and 10(6.5) SID50 ml(-1), respectively. These titers were not significantly different (p > or = 0.05). The titration of the WSSV stock by oral intubation in 80 d old juveniles resulted in approximately 10-fold reduction in virus titer compared to i.m. inoculation. This lower titer is probably the result of physical and chemical barriers in the digestive tract of shrimp that hinder WSSV infectivity. The titers determined by infection were identical to the titers determined by mortality in all experiments using both i.m. and oral routes at 120 h post inoculation (hpi), indicating that every infected shrimp died. The determination of WSSV titers for dilutions administered by i.m. and oral routes constitutes the first step towards the standardization of challenge procedures to evaluate strategies to reduce WSSV infection.     

Viral interference between infectious hypodermal and hematopoietic necrosis virus and white spot syndrome virus in Litopenaeus vannamei

White spot syndrome virus (WSSV) is highly virulent and has caused significant production losses to the shrimp culture industry over the last decade. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) also infects penaeid shrimp and, while being less important than WSSV, remains a major cause of significant production losses in Litopenaeus vannamei (also called Penaeus vannamei) and L. stylirostris (also called Penaeus stylirostris). These 2 viruses and their interactions were previously investigated in L. stylirostris. We report here laboratory challenge studies carried out to determine if viral interference between IHHNV and WSSV also occurs in L. vannamei, and it was found that experimental infection with IHHNV induced a significant delay in mortality following WSSV challenge. L. vannamei infected per os with IHHNV were challenged with WSSV at 0, 10, 20, 30, 40 and 50 d post-infection. Groups of na?ve shrimp infected with WSSV alone died in 3 d whereas shrimp pre-infected with IHHNV for 30, 40 or 50 d died in 5 d. Real-time PCR analysis showed that the delay correlated to the IHHNV load and that WSSV challenge induced a decrease in IHHNV load, indicating some form of competition between the 2 viruses.     

凡纳滨对虾ANT2 基因的克隆及低温表达谱分析

为研究凡纳滨对虾适应低温的分子机理, 实验对从低温处理凡纳滨对虾抑制性消减文库中筛选出的一个360 bp EST 序列进行了研究。首先, 同源比对显示该EST 片段与其他物种的ANT2 基因高度同源, 命名为凡纳滨对虾ANT2 基因(LVANT2); 其次, 通过构建凡纳滨对虾肝胰腺全长cDNA 文库, PCR 扩增获得LVANT2 基因的全长cDNA1540 bp, 其中包括1011 bp 的完整开放阅读框, 编码336 个氨基酸残基。然后, 对基因进行了不同组织和低温处理的表达谱分析: (1)组织表达谱的结果显示, 该基因在凡纳滨对虾肌肉组织中表达量最高; (2)在不同低温处理下的表达结果显示, 该基因在15℃处理下基因表达量发生显著变化, 13℃开始呈下调表达, 11℃时表达量又升高; 13℃低温处理不同时间发现该基因在12h 内表达量发生显著变化,48h 后表达量最高。LVANT2 基因的低温诱导表达模式说明其可能在凡纳滨对虾低温适应中发挥作用