A thioredoxin response to the WSSV challenge on the Chinese white shrimp, Fenneropenaeus chinensis

Thioredoxin (TRX) is involved in cell redox homeostasis. In addition, it is responsible for maintaining proteins in their reduced state. In our study, a Fenneropenaeus chinensis thioredoxin (FcTRX) gene was identified from the Chinese white shrimp. The full length of FcTRX was 777 bp, including a 60 bp 5′ untranslated region (UTR), a 318 bp open reading frame (ORF) encoding a 105 amino acids protein, and a 399 bp 3′ UTR. FcTRX contained a TRX domain with a conserved motif of Cys-Gly-Pro-Cys (CGPC). No signal peptide was predicted by SMART analysis. The molecular mass and pI of FcTRX were 12 kDa and 4.62, respectively. FcTRX is a widely distributed gene, and its mRNA is detected in hemocytes, hearts, hepatopancreas, gills, stomach, and intestine from an unchallenged shrimp. The expression level of FcTRX was the highest in hepatopancreas, where it was down-regulated to the lowest level at 12 h white spot syndrome virus (WSSV) challenge. In the gills, it went up to the highest level at 6 h. Western blot showed that FcTRX protein in hepatopancreas challenged with WSSV was down-regulated from 2 h to 12 h and then restored to the level similar to that of unchallenged shrimp at 24 h. In the gills challenged with WSSV, the FcTRX protein was up-regulated from 6 h to 24 h. Our research indicated its possible role in the anti-WSSV innate immunity of shrimps.     

White spot syndrome virus (WSSV) interaction with crayfish haemocytes

WSSV particles were detected in separated granular cells (GCs) and semigranular cells (SGCs) by in situ hybridisation from WSSV-infected crayfish and the prevalence of WSSV-infected GCs was 5%, whereas it was 22% in SGCs. This indicates that SGCs are more susceptible to WSSV and that this virus replicated more rapidly in SGCs than in GCs and as a result the number of SGCs gradually decreased from the blood circulation. The effect of haemocyte lysate supernatant (HLS), containing the degranulation factor (peroxinectin), phorbol 12-myristate 13-acetate (PMA), the Ca(2+) ionophore A23187 on GCs from WSSV-infected and sham-injected crayfish was studied. The results showed that the percentage of degranulated GCs of WSSV-infected crayfish treated with HLS or PMA was significantly lower than that in the control, whereas no significant difference was observed when treated with the Ca(2+) ionophore. It was previously shown that peroxinectin and PMA have a degranulation effect via intracellular signalling involving protein kinase C (PKC), whereas the Ca(2+) ionophore uses an alternative pathway. HLS treatment of GCs and SGCs from WSSV-infected crayfish results in three different morphological types: non-spread, spread and degranulated cells. The non-spread cell group from both GCs and SGCs after treatment with HLS had more WSSV positive cells than degranulated cells, when detected by in situ hybridisation. Taken together, it is reasonable to speculate that the PKC pathway might be affected during WSSV infection. Another interesting phenomenon was that GCs from non-infected crayfish exhibited melanisation, when incubated in L-15 medium, while no melanisation was found in GCs of WSSV-infected crayfish. However, the phenoloxidase activities of both sham- and WSSV-injected crayfish in HLS were the same as well as proPO expression as detected by RT-PCR. This suggests that the WSSV inhibits the proPO system upstream of phenoloxidase or simply consumes the native substrate for the enzyme so that no activity is shown. The percentage of apoptotic haemocytes in WSSV-infected crayfish was very low, but it was significantly higher than that in the sham-injected crayfish on day 3 or 5 post-infection. The TEM observation in haematopoietic cells (hpt cells) suggests that WSSV infect specific cell types in haematopoietic tissue and non-granular hpt cells seem more favourable to WSSV infection.     

The function and structural influence of selective relaxed constraint at functional intracellular loop3 of 5-HT(1A) serotonin-1 receptor family

Superoxide dismutases (SODs) are metalloenzymes that represent one important line of defense against reactive oxygen species (ROS). In this paper, two novel SOD genes, MdSOD1 and MdSOD2, which putatively encode 261 and 214 amino acid residues respectively were identified and characterized from the housefly Musca domestica. The high similarity of MdSOD1 and MdSOD2 with SODs from other organisms indicated that they should be two new members of the SOD family. qPCR exhibited a universal expression of MdSOD1 and MdSOD2 detected in various tissues of housefly larva, including the fat body, gut, hemocyte and epidermis. Expression profiling reveals that MdSOD1 and MdSOD2 can be induced significantly via not only heat shock and cadmium (Cd) stress but also Escherichia coli and Staphylococcus aureus challenge. The two genes were cloned into the prokaryotic expression vector pET-28a to obtain the fusion proteins rMdSOD1 and rMdSOD2. Between them, the activity of rMdSOD2 was found by visual assay methods. ESI-LC-MS/MS analysis showed that three peptide fragments of the protein rMdSOD2 were identical to the corresponding sequence of M. domestica MdSOD2. MdSOD1 and MdSOD2 in housefly larvae were abrogated by feeding bacteria expressing dsRNA. High mortalities were observed in the larvae treated with dsRNA of SODs at heat shock, Cd stress and bacterial invasion. This phenomenon indicated that MdSOD1 and MdSOD2 are related to the survival of M. domestica under stress. This may provide new insights into the role of the two SOD genes in protecting M. domestica against both stress and bacterial invasion.     

白斑综合症病毒囊膜蛋白VP19、VP28的融合表达及中和抗体的制备

根据GenBank上WSSV囊膜蛋白基因vp19和vp28的序列,设计并合成两对引物,PCR扩增得到vp19和vp28两基因,大小分别为370bp和630bp。通过EcoRI位点连接两基因,再按正确的阅读框插入表达载体pET-22b( )中,构建出重组表达载体pET-vp(19 28)并转化大肠杆菌BL21(DE3)。基因工程菌株35℃IPTG诱导,表达产物经SDS-PAGE检测显示有与预期大小41kDa相吻合的融合蛋白带。用Ni^2 -柱纯化的基因工程蛋白免疫新西兰大白兔制备抗血清,进行螯虾活体中和病毒实验,结果表明抗血清对WSSV的中和效率达到了100％。     

一种筛选对虾白斑综合症病毒中和抗体的新方法

用对虾淋巴组织的原代培养细胞与对虾白斑综合症病毒进行相互作用,洗去不能结合的病毒,然后用甲醛固定,再用ELISA法检测所吸附的病毒。当病毒与抗血清保温后,抑制病毒与细胞的吸附能力的抗血清中应含有中和抗体。此方法可以在缺少细胞的情况下,简便地筛选到中和抗体。     

一种改良的对虾白斑综合征病毒的提纯技术

The envelope proteins of White spot syndrome virus (WSSV) are very fragile and easy to be destroyed during purification. It was difficult to obtain a large quantity of intact virions by routine sucrose gradient centrifugation. After modifying the sucrose gradient by adding citrate sodium, we can obtain a large quantity of intact virions and nucleocapsids. This purified virions and nucleocapsids were subsequently used for analyzing viral structural proteins and DNA extraction. The result showed that this modified techniaue is very efficient for virus purification.