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首页> 外文期刊>Revue de Medecine Veterinaire >Modifications of the porcine interferon-alpha gene and its antiviral activity in vitro and in vivo
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Modifications of the porcine interferon-alpha gene and its antiviral activity in vitro and in vivo

机译：猪干扰素α基因的修饰及其在体内外的抗病毒活性

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摘要

In order to up-regulate the expression of the porcine interferon-alpha (IFN-alpha) in Pichia pastoris, some rare codons encoding for 6 amino acids were modified to biased codons of P. pastoris. The modified gene was cloned into pPICZ alpha C electro-transformed X-33 cells for screening high Zeocin-resis-tant colonies. The recombinant modified or original porcine IFN-a proteins were detected by SDS-PAGE and Western blot. The codon modifications have enhanced by a factor 5 the protein expression. Usingculture superna-tants containing IFN-a on different cell lines infected with the VSV, the TGEV or the PPRSV, it was observed that the modified protein exhibited higher antiviral activities magnified 10-100 times for the TGEV and the PPRSV and even 300 times for the VSV compared to the original cytokine. Using the same IFN-a concentration, it was also noted that the anti-VSV activity of the modified cytokine was multiplied by 9. In an in vivo model of pigs infected with PRRSV-infected pigs, co-treatmentwith the IFN-a (modified or not) and antibiotics has led to significantly higher cure rates than treatment with antibiotics alone, and the modified protein was again significantly more efficient (P < 0.05). These results demonstrate the interest to genetically modify some rare codons in the porcine IFN-a gene for increasing the protein expression and its antiviral activities in vitro and in vivo.
机译：为了上调巴斯德毕赤酵母中猪干扰素-α（IFN-α）的表达，一些编码6个氨基酸的稀有密码子被修饰为巴斯德毕赤酵母的偏性密码子。将修饰的基因克隆到pPICZαC电转化的X-33细胞中，以筛选高Zeocin抗性克隆。通过SDS-PAGE和蛋白质印迹检测重组重组或原始猪IFN-α蛋白。密码子修饰使蛋白质表达增强了5倍。在感染了VSV，TGEV或PPRSV的不同细胞系上使用含有IFN-α的培养上清液，观察到修饰的蛋白显示出更高的抗病毒活性，对于TGEV和PPRSV放大了10-100倍，甚至是300倍。 VSV与原始细胞因子相比。使用相同的IFN-α浓度，还注意到修饰的细胞因子的抗VSV活性乘以9。在感染PRRSV感染的猪的体内模型中，与IFN-α共同治疗（修饰与否）和抗生素相比，与单独使用抗生素相比，抗生素的治愈率显着更高，而且修饰的蛋白质再次显着更有效（P <0.05）。这些结果证明了对猪IFN-α基因中一些稀有密码子进行遗传修饰以增加蛋白质表达及其体外和体内抗病毒活性的兴趣。

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