Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Antiviral Res. 2014 Aug;108:173-80. doi: 10.1016/j.antiviral.2014.05.019. Epub 2014 Jun 12.
Chikungunya virus (CHIKV) outbreaks have led to a serious economic burden, as the available treatment strategies can only alleviate disease symptoms, and no effective therapeutics or vaccines are currently available for human use. Here, we report the use of a new cost-effective approach involving production of a recombinant antiviral peptide-fusion protein that is scalable for the treatment of CHIKV infection. A peptide-fusion recombinant protein LATA-PAP1-THAN that was generated by joining Latarcin (LATA) peptide with the N-terminus of the PAP1 antiviral protein, and the Thanatin (THAN) peptide to the C-terminus, was produced in Escherichia coli as inclusion bodies. The antiviral LATA-PAP1-THAN protein showed 89.0% reduction of viral plaque formation compared with PAP1 (46.0%), LATA (67.0%) or THAN (79.3%) peptides alone. The LATA-PAP1-THAN protein reduced the viral RNA load that was 0.89-fold compared with the untreated control cells. We also showed that PAP1 resulted in 0.44-fold reduction, and THAN and LATA resulting in 0.78-fold and 0.73-fold reductions, respectively. The LATA-PAP1-THAN protein inhibited CHIKV replication in the Vero cells at an EC50 of 11.2μg/ml, which is approximately half of the EC50 of PAP1 (23.7μg/ml) and protected the CHIKV-infected mice at the dose of 0.75mg/ml. We concluded that production of antiviral peptide-fusion protein in E. coli as inclusion bodies could accentuate antiviral activities, enhance cellular internalisation, and could reduce product toxicity to host cells and is scalable to epidemic response quantities.
基孔肯雅病毒 (CHIKV) 爆发导致了严重的经济负担，因为现有的治疗策略只能缓解疾病症状，目前尚无针对人类的有效治疗方法或疫苗。在这里，我们报告了一种新的具有成本效益的方法的使用，该方法涉及生产一种可扩展用于治疗 CHIKV 感染的重组抗病毒肽融合蛋白。通过将 Latarcin (LATA) 肽与 PAP1 抗病毒蛋白的 N 端连接，并将 Thanatin (THAN) 肽与 C 端连接，生成了抗病毒肽融合重组蛋白 LATA-PAP1-THAN，该蛋白在大肠杆菌中作为包涵体产生。与 PAP1（46.0%）、LATA（67.0%）或 THAN（79.3%）肽单独相比，抗病毒 LATA-PAP1-THAN 蛋白使病毒斑形成减少了 89.0%。LATA-PAP1-THAN 蛋白使病毒 RNA 载量减少了 0.89 倍与未经处理的对照细胞相比。我们还表明，PAP1 导致减少了 0.44 倍，而 THAN 和 LATA 分别导致减少了 0.78 倍和 0.73 倍。LATA-PAP1-THAN 蛋白在 Vero 细胞中以 EC50 为 11.2μg/ml 抑制 CHIKV 复制，这大约是 PAP1（23.7μg/ml）的 EC50 的一半，并且以 0.75mg/ml 的剂量保护感染 CHIKV 的小鼠。我们得出结论，作为包涵体在大肠杆菌中生产抗病毒肽融合蛋白可以增强抗病毒活性，增强细胞内化作用，并降低产品对宿主细胞的毒性，并且可以扩展到应对疫情的数量。
