Abstract

In this study, the cDNA of mature bovine interferon-α (BoIFN-α) gene was cloned and expressed in the Escherichia coli expression system. Total RNA was extracted from bovine liver cells and reverse transcribed into cDNA, and then BoIFN-α gene fragment was amplified with specific primers by PCR method before being ligated into the pET-32a (+) expression vector. Since the pET-32a (+) plasmid expresses the thioredoxin A (TrxA) tag protein, it can increase the solubility of the target protein. In order to express the target protein, the pET-32a-BoIFN-α recombinant plasmid was transformed into E. coli Rosetta (DE3) strain followed by IPTG induction. It was found that the recombinant fusion protein Trx-rBoIFN-α was finally expressed in a soluble form in the host cell, accounting for about 33.45% of the total cellular protein. The protein was purified by two-step chromatography, including Ni²⁺-chelating Sepharose affinity chromatography and DEAE anion exchange chromatography. The purity of the purified product reached 93.0%, and the cytopathic effects (CPE) inhibition method was adopted to determine the antiviral activity of the expressed fusion protein in vesicular stomatitis virus/ Madin-Darby bovine kidney cells titration system. The specific activity of the recombinant fusion protein Trx-rBoIFN-α was measured as (3.6 ± 0.25) × 10⁶ U/mg. After cutting of the Trx tag protein by enterokinase digestion, the remaining solo protein was confirmed as rBoIFN-α protein by Western blot assay. These findings will enable us to produce a large number of bioactive rBoIFN-α protein for future application.

中文翻译：

---

重组牛干扰素-α在大肠杆菌中的可溶性表达，快速纯化和抗病毒活性

摘要

在这项研究中，成熟的牛干扰素-α（BoIFN-α）基因的cDNA被克隆并在大肠杆菌表达系统中表达。从牛肝细胞中提取总RNA并反转录成cDNA，然后在连接到pET-32a（+）表达载体之前，通过PCR方法用特异性引物扩增BoIFN-α基因片段。由于pET-32a（+）质粒表达了硫氧还蛋白A（TrxA）标签蛋白，因此可以增加靶蛋白的溶解度。为了表达靶蛋白，将pET-32a-BoIFN-α重组质粒转化到大肠杆菌中。Rosetta（DE3）菌株，然后进行IPTG诱导。发现重组融合蛋白Trx-rBoIFN-α最终以可溶形式在宿主细胞中表达，约占总细胞蛋白的33.45％。通过两步色谱法纯化蛋白质，包括Ni ²⁺螯合琼脂糖亲和色谱法和DEAE阴离子交换色谱法。纯化产物的纯度达到93.0％，采用细胞病变作用（CPE）抑制法测定了表达的融合蛋白在水疱性口炎病毒/ Madin-Darby牛肾细胞滴定系统中的抗病毒活性。重组融合蛋白Trx-rBoIFN-α的比活性测定为（3.6±0.25）×10 ⁶单位 通过肠激酶消化切下Trx标签蛋白后，通过蛋白质印迹分析确认剩余的单独蛋白为rBoIFN-α蛋白。这些发现将使我们能够生产大量具有生物活性的rBoIFN-α蛋白，以备将来应用。