A cytokine receptor domeless promotes white spot syndrome virus infection via JAK/STAT signaling pathway in red claw crayfish Cherax quadricarinatus.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway is pivotal in immune responses for a variety of pathogens in both vertebrates and invertebrates. Domeless (Dome), as a unique cytokine receptor, involves in the upstream JAK/STAT pathway in invertebrates. In this study, the full-length cDNA sequence of a cytokine receptor Dome was identified from red claw crayfish Cherax quadricarinatus (named as CqDome), which contained an open reading frame of 4251 bp, encoding 1416 amino acids. The CqDome contained extracellular conservative domains of a signal peptide, two cytokine binding modules (CBM), three fibronectin-type-III-like (FN3) domains and a transmembrane region. Tissue distribution analysis showed that CqDome generally expressed in all the tissues selected with a high expression in hemocyte. The gene expression of both the viral immediately early gene (IE1) and a late gene envelope protein VP28 of white spot syndrome virus (WSSV) were significantly decreased after gene silencing of CqDome in crayfish haematopoietic tissue (Hpt) cells, indicating a key role of CqDome in promoting WSSV infection. Furthermore, the phosphorylation level of CqSTAT was significantly inhibited by gene silencing of CqDome in Hpt cells, indicating that CqDome participated in signal transduction of JAK/STAT pathway in red claw crayfish. These data together suggest that CqDome is likely to promote WSSV infection via JAK/STAT pathway, which sheds new light on further elucidation of the pathogenesis of WSSV.

A CqFerritin protein inhibits white spot syndrome virus infection via regulating iron ions in red claw crayfish Cherax quadricarinatus.

It is well known that iron is an essential element for all living organism. The intracellular iron availability is also important for the host's innate immune response to various pathogens, in which the iron homeostasis can be regulated by ferritin due to its iron storage property. In this study, a full-length cDNA sequence of ferritin (named as CqFerritin) was identified with 1410 bp from red claw crayfish Cherax quadricarinatus, which contained an open reading frame of 513 bp, encoding 170 amino acids with a conserved ferritin domain. Tissue distribution analysis demonstrated that CqFerritin was widely expressed in various tissues with high presence in haemocyte, haematopoietic tissue (Hpt) and heart, while lowest expression in hepatopancreas. In addition, loss-of-function of CqFerritin by gene silencing resulted in significantly higher expression of an envelope protein VP28 of white spot syndrome virus (WSSV) in red claw crayfish Hpt cell cultures, indicating the potential antiviral response of CqFerritin. To further explore the effect on WSSV replication by CqFerritin, recombinant CqFerritin protein (rCqFerritin) was transfected into Hpt cells followed by WSSV infection. Importantly, the replication of WSSV was obviously decreased in Hpt cells if transfected with rCqFerritin protein, suggesting that CqFerritin had clearly negative effect on WSSV infection. Furthermore, intracellular accumulation of iron ions was found to promote the WSSV replication in a dose-dependent manner, illustrating that the iron level regulated by CqFerritin was likely to be vital for WSSV infection in red claw crayfish. Taken together, these data suggest that CqFerritin plays an important role in immune defense against WSSV infection in a crustacean C. quadricarinatus.

[Preparation of recombinant HIV-TATm-survivin (T34A) protein and its pro-apoptosis activity to cancer cells in vitro].

As a novel member of the IAP (Inhibitor of apoptosis protein) family, survivin was observed to be expressed in most human cancerous cells. Fusion protein TATm-survivin (T34A) has drawn considerable attention because it is a potential anti-tumor protein that can be transduced into cancer cell with the help of HIV-TAT domain. In this study, the cDNA encoding survivin was cloned by RT-PCR from human breast cancer cell lines B-Cap-37. An expression vector of pRSET-B-HIV-tatm-survivin (T34A) was constructed by PCR after survivin (T34A) was mutated by site-directed mutagenesis. Subsequently, the resultant plasmid was transformed into E. coli BL21 (DE3). Recombinant HIV-TATm-Survivin (T34A) protein was expressed efficiently with 0.5mM IPTG as inducer, reaching a yield of 650mg/liter (as inclusion body) in fermentation culture. The inclusion bodies were solubilized, refolded and purified to a purity of 96% by ion exchange chromatograghy and size-exclusion chromatography. Remarkable effects of the purified recombinant HIV-TATm-Survivin (T34A) on the morphology of cell line SW1990 and B-Cap-37 were observed after being administrated for 4h. MTT assay showed recombinant HIV-TATm-survivin (T34A) protein could inhibit significantly cell proliferation of SW1990 and B-Cap-37 and SSMC-7721 in vitro. Apoptosis rate and cell circle of SW1990 and B-Cap-37 that had been treated with target protein (final concentration 30 microg/mL) were detected with flow cytometry. Results revealed that more than 65% cancer cells were arrested at G1 phase. The study suggested that TATm-survivin (T34A) protein was a hopeful protein drug in the treatment of cancers by facilitating apoptosis of cancer cells. Key words recombinant HIV-TATm-Survivin (T34A), expression and purification, pro-apoptosis bioactivity, SW1990 and B-Cap-37 cancer cell lines