重组牛α干扰素的分泌表达及抗病毒活性分析

doi:10.16431/j.cnki.1671-7236.2021.04.033

摘要/Abstract

摘要： 本研究旨在利用乳酸乳球菌构建牛α干扰素（BoIFN-α）的分泌表达系统并分析其抗病毒活性。根据乳酸乳球菌MG1363密码子使用的偏好性，对BoIFN-α基因进行优化合成。将目的基因和乳酸乳球菌表达载体质粒pAMJ399分别进行SalⅠ和BglⅡ双酶切，将胶回收产物进行连接后，转化大肠杆菌TG1感受态细胞，提取阳性质粒电转化乳酸乳球菌感受态细胞，用SDS-PAGE和Western blotting方法对表达蛋白进行分析和鉴定，同时将重组乳酸乳球菌培养28 h后取上清用PEG20000浓缩10倍左右，进行体外抗病毒试验。结果显示，试验成功构建了重组乳酸乳球菌pAMJ399-BoIFN-α/MG1363，重组菌上清和沉淀均出现约20 ku的目的条带，表明重组蛋白以分泌的形式同时存在于培养液的上清和沉淀中。浓缩后的重组BoIFN-α（rBoIFN-α）上清与水泡性口炎病毒（Vesicular stomatitis virus，VSV）同时作用，用MDBK/VSV系统以微量细胞病变抑制法测定rBoIFN-α的效价为1.02×10⁶ U/L；通过Alamer Blue法分析可知，加入rBoIFN-α后对细胞的活性影响较小；间接免疫荧光试验可见加入rBoIFN-α后细胞无绿色荧光，说明rBoIFN-α有良好的抗病毒效果；实时荧光定量PCR结果进一步证实rBoIFN-α具有一定的抗病毒效果。本试验构建了能分泌表达rBoIFN-α的乳酸乳球菌，通过一系列的体外抗病毒试验证明rBoIFN-α具有良好的抗病毒活性，试验结果为rBoIFN-α作为抗病毒药物、免疫增强剂的开发、肽类用药及临床应用提供新途径。

关键词: 牛α干扰素(BoIFN-α); 乳酸乳球菌; 分泌表达; 抗病毒活性

Abstract: The purpose of this study was to use Lactococcus lactis to construct a secretory expression system for bovine interferon-α (BoIFN-α) and analyze its antiviral activity.According to the preference of Lactococcus lactis MG1363 codon usage,the BoIFN-α gene was optimized and synthesized.The target gene and the Lactococcus lactis expression vector plasmid pAMJ399 were digested with SalⅠ and BglⅡ,respectively,and the gel recovered products were ligated and transformed into E.coli TG1 competent cells.The positive plasmid was extracted and electrotransformed into Lactococcus lactis.SDS-PAGE and Western blotting methods were used to analyze and identify the expressed protein in the mature cells.At the same time,the recombinant Lactococcus lactis was cultured for 28 h and then the supernatant was taken and concentrated with PEG20000 about 10 times for in vitro antiviral test.The results showed that the recombinant Lactococcus lactis pAMJ399-BoIFN-α/MG1363 was constructed successfully in this experiment.The supernatant and precipitate of the recombinant bacteria showed a target band of about 20 ku,indicating that the recombinant protein was secreted in both the supernatant and the precipitation.The concentrated rBoIFN-α supernatant acted simultaneously with Vesicular stomatitis virus (VSV).The titer of rBoIFN-α was determined to be 1.02×10⁶ U/L by the MDBK/VSV system by the microcytopathic inhibition method.The Alamer Blue method analysis result showed that the addition of rBoIFN-α had little effect on cell activity.Indirect immunofluorescence method showed that the cells had no green fluorescence after adding rBoIFN-α,indicating that rBoIFN-α had a good antiviral effect.Real-time PCR results further confirmed that rBoIFN-α had a certain antiviral effect.In this experiment,Lactococcus lactis could secrete and express rBoIFN-α was constructed.A series of in vitro antiviral tests proved that rBoIFN-α had good antiviral activity.The test results provided a new way for the development of rBoIFN-α as antiviral drugs,immune enhancers,peptide drugs and clinical applications.

Key words: BoIFN-α; Lactococcus lactis; secretory expression; antiviral activity

中图分类号:

S852.4

刘增素, 汪昭睿, 王雪莹, 姜艳平, 崔文, 王丽, 乔薪瑗, 周晗, 徐义刚, 李一经, 唐丽杰. 重组牛α干扰素的分泌表达及抗病毒活性分析[J]. 中国畜牧兽医, 2021, 48(4): 1449-1456.

LIU Zengsu, WANG Zhaorui, WANG Xueying, JIANG Yanping, CUI Wen, WANG Li, QIAO Xinyuan, ZHOU Han, XU Yigang, LI Yijing, TANG Lijie. Secretory Expression and Antiviral Activity Analysis of Recombinant Bovine Interferon-α[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(4): 1449-1456.

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