猪β干扰素在毕赤酵母中的分泌表达及其对伪狂犬病毒的抑制作用

为了研制高活性的重组猪β干扰素,对PoIFN-β成熟蛋白第3、7和164位的3个氨基酸密码子进行毕赤酵母偏嗜性改造并构建了酵母表达载体pPICZαA-PIB。pPICZαA-PIB经SacⅠ酶切线性化后电转化导入毕赤酵母菌株X-33。多株PCR鉴定为阳性的酵母转化子经甲醇诱导发酵分泌表达了PoIFN-β,其中B1株酵母的PoIFN-β产量最高,约为2.5×105U/mL,其表达量约为60μg/mL,比活为4.17×106U/mg。将发酵上清液用PEG20000浓缩后进行SDS-PAGE和Western blot检测,结果表明表达产物是分子量约为28kDa和25kDa蛋白的混合物,两者均可与PoIFN-β阳性抗血清发生特异反应。表达产物比PoIFN-β理论推导分子量(约20.8kDa)大,推测可能是表达产物发生了不同程度的糖基化。重组PoIFN-β对伪狂犬病毒在细胞中增殖可呈现抑制作用,并且rPoIFN-β对伪狂犬病毒在MDBK细胞上早期增殖的抑制效果最为明显。

Secreted expression of porcine interferon beta in Pichia pastoris and its inhibition effect on the replication of pseudorabies virus

In order to develop recombinant porcine interferon beta with high bioactivity, the rare codes that encoded 3th, 7th and 164th amino acids of porcine interferon beta mature protein were mutant into bias codes of Pichia pastoris and then the modified gene was introduced to yeast secreted expression vector pPICZ alphaA which resulted in pPICZalphaA-PIB. The SacI linearized plasmid pPICZalphaA-PIB was transformed into Pichia pastoris X-33 by electroporation. The transformants were identified by PCR using PoIFN-beta and AOX1 specific primers. The expression of PoIFN-beta was induced with methanol. Several positive clones were obtained and the one namely B1 produced the highest level of PoIFN-beta. The B1 was further fermented in shake-flask in larger volume. The concentration of the secreted PoIFN-beta was about 60 microg/mL and its antiviral activity is about 2.5 x 10(5) U/mL, so the specific activity of porcine interferon beta produced by the Pichia pastoris is approximately 4.17 x 10(6) U/mg. The expressed supernatant was concentrated and identified by SDS-PAGE and Western blot. There are two major proteins with respective molecular mass of approximately 25 kDa and 28 kDa in the supernatant. The results of Western blot indicated that the two proteins were positively reacted and manifested well PoIFN-beta antigenicity. In contrast with the deduced theoretical molecular mass value of PoIFN-beta, the expressed two major proteins were larger which maybe due to the difference of glycosylation. The antiviral effect of recombinant porcine interferon beta (rPoIFN-beta) on Pseudorabies virus (PrV) was studied in the present experiment. The result indicated that rPoIFN-beta could effectively inhibit the replication of PrV in MDBK cells, especially during the early phage of the virus replication.