Generation and evaluation of a recombinant myxomavirus expressing the VP60 protein of rabbit haemorrhagic disease virus / 生物工程学报

Rabbit haemorrhagic disease virus (RHDV) and myxoma virus (MYXV), are two pathogens that have harmful effect on rabbit breeding and population decline of European rabbits in their native range, causing rabbit haemorrhagic disease (rabbit fever) and myxomatosis, respectively. The capsid protein VP60 of the RHDV represents the major antigenic protein. To develop a recombinant bivalent vaccine candidate that can simultaneously prevent these two diseases, we used the nonessential gene TK (thymidine kinase) of MYXV as the insertion site to construct a recombinant shuttle vector p7.5-VP60-GFP expressing the RHDV major capsid protein (VP60) and the selectable marker GFP. Then the shuttle vector p7.5-VP60-GFP was transfected into rabbit kidney cell line RK13 which was previously infected with MYXV. After homologous recombination, the recombinant virus expressing GFP was screened under a fluorescence microscope and named as rMV-VP60-GFP. Finally, the specific gene-knock in and expression verification of the vp60 and gfp genes of the recombinant virus was confirmed by PCR and Western blotting. The results showed that these two genes were readily knocked into the MYXV genome and also successfully expressed, indicating that the recombinant MYXV expressing the vp60 of RHDV was generated. Protection against MYXV challenge showed that the recombinant virus induced detectable antibodies against MYXV which would shed light on development of the effective vaccine.

Effect of myxomavirus on the proliferation of ovarian cancer cells and its Mechanism / 肿瘤

Objective: To investigate the effect of myxomavirus (MV) on the proliferation of human ovarian cancer SKOV3 cells, and its molecular mechanism. Methods: The human ovarian cancer SKOV3 cells were cultured in vitro and infected with MV. At the same time, SKOV3 cells infected with inactivated virus or only cultured with RPMI 1640 medium were used as the negative control group or the blank group, respectively. The proliferation of SKOV3 cells in the three groups was determined by CCK-8 assay. The mRNA levels of Bcl-2 and survivin were detected by real-Time fluorescent quantitative PCR. The cell cycle distribution was analyzed by FCM. The expressions of total extracellular regulated protein kinase 1/2 (ERK1/2), phosphorylated ERK1/2 (p-ERK1/2), Akt, p-Akt, Bcl-2 and survivin proteins were measured by Western blotting. The activities of caspase-3 and caspase-8 were also quantified by colorimetric method. Results: Compared with the negative control group and the blank group, MV significantly inhibited the proliferation and cell cycle progression of human ovarian cancer SKOV3 cells (all P < 0.05). After SKOV3 cells were infected with MV for 96 h, the mRNA and protein expressions of Bcl-2 and survivin were significantly down-regulated (both P < 0.05), while the phosphorylation levels of ERK1/2 and Akt were significantly decreased (both P < 0.05), but the activities of caspase-3 and caspase-8 were obviously enhanced (both P < 0.05). Conclusion: MV can inhibit the proliferation of ovarian cancer cells, and its mechanism may be related to blocking cell cycle progression, down-regulating the expressions of anti-Apoptotic proteins Bcl-2 and survivin, increasing the activation of caspase-3 and caspase-8, and inhibiting the phosphorylation of ERK and Akt in proliferation-related signal pathway.