重组犬α6干扰素的酵母表达及其抗病毒活性评价
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摘要
试验旨在构建犬α6干扰素毕赤酵母表达系统,并对其进行优化和筛选,以期获得高活性的重组犬α6干扰素(CaIFN-α6)。根据CaIFN-α6基因序列,按毕赤酵母菌密码子偏好性对CaIFN-α6全基因序列进行优化与合成,用XhoⅠ和NotⅠ双酶切将其连接至载体pPICZαA中,构建pPICZαA-CaIFN-α6重组表达质粒,转化大肠杆菌DH5α感受态细胞。提取质粒pPICZαA-CaIFN-α6并线性化,电转入酵母感受态细胞X33中制备重组菌。采用甲醇进行诱导表达,收集上清,超滤浓缩,最终获得纯化的重组CaIFN-α6。利用BCA法测得纯化后的CaIFN-α6蛋白浓度为1.5 mg/mL,Western blotting分析表明CaIFN-α6蛋白具有良好的反应原性,SDS-PAGE显示其纯度约在95%以上,MDCK/VSV法检测其效价为2.37×10~7 IU/mL,比活性为1.58×10~7 IU/mg。结果表明犬α6干扰素在毕赤酵母pPICZαA表达载体系统中成功表达,且具有较高的生物活性,为后期的犬病毒病的临床预防与治疗提供了良好的支撑。
The aim of the experiment was to construct the expression system of canine interferon alpha 6(CaIFN-α6) in Pichia pastoris,optimize and screen the expression system in order to obtain recombinant canine interferon alpha 6 with high activity.According to the sequence of CaIFN-α6 gene and the codon preference of Pichia pastoris,the whole gene sequence of CaIFN-α6 was optimized and synthesized.The recombinant expression plasmid of pPICZαA-CaIFN-α6 was constructed using Xho Ⅰ and Not Ⅰ and linked to the vector pPICZαA.The recombinant expression plasmid was transformed into E.coli DH5α competent cells.The plasmid pPICZαA-CaIFN-α6 was extracted and linearized.The recombinant strain was prepared by electroporation into yeast competent cell X33.The recombinant CaIFN-α6 was purified by methanol induction,supernatant collection and ultrafiltration concentration.The concentration of purified CaIFN-α6 protein was 1.5 mg/mL by BCA.Western blotting analysis showed that CaIFN-α6 protein had good reactivity.SDS-PAGE showed that the purity of CaIFN-α6 protein was above 95%.The titer of CaIFN-α6 protein was 2.37×10~7 IU/mL by MDCK/VSV detection and the specific activity was 1.58×10~7 IU/mg.The results showed that CaIFN-α6 was successfully expressed in Pichia pastoris pPICZαA expression vector system with high biological activity,which provided a good support for the clinical prevention and treatment of canine viral disease in the later stage.
The aim of the experiment was to construct the expression system of canine interferon alpha 6(CaIFN-α6) in Pichia pastoris,optimize and screen the expression system in order to obtain recombinant canine interferon alpha 6 with high activity.According to the sequence of CaIFN-α6 gene and the codon preference of Pichia pastoris,the whole gene sequence of CaIFN-α6 was optimized and synthesized.The recombinant expression plasmid of pPICZαA-CaIFN-α6 was constructed using Xho Ⅰ and Not Ⅰ and linked to the vector pPICZαA.The recombinant expression plasmid was transformed into E.coli DH5α competent cells.The plasmid pPICZαA-CaIFN-α6 was extracted and linearized.The recombinant strain was prepared by electroporation into yeast competent cell X33.The recombinant CaIFN-α6 was purified by methanol induction,supernatant collection and ultrafiltration concentration.The concentration of purified CaIFN-α6 protein was 1.5 mg/mL by BCA.Western blotting analysis showed that CaIFN-α6 protein had good reactivity.SDS-PAGE showed that the purity of CaIFN-α6 protein was above 95%.The titer of CaIFN-α6 protein was 2.37×10~7 IU/mL by MDCK/VSV detection and the specific activity was 1.58×10~7 IU/mg.The results showed that CaIFN-α6 was successfully expressed in Pichia pastoris pPICZαA expression vector system with high biological activity,which provided a good support for the clinical prevention and treatment of canine viral disease in the later stage.
引文
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[15] 李哲,李俊峰,刘千辉,等.梅花鹿α干扰素基因在MDBK细胞中表达及抗BVDV活性检测[J].中国兽医学报,2016,36(12):2049-2053.LI Z,LI J F,LIU Q H,et al.Expression of Sika deer interferon alpha gene in MDBK cells and detection of its anti BVDV activity[J].Chinese Journal of Veterinary Science,2016,36(12):2049-2053.(in Chinese)
[16] 谭成成.2种不同分泌信号对犬干扰素在毕赤酵母中分泌表达的影响[D].长春:吉林大学,2016.TAN C C.Effects of 2 different secretory signals on secretory expression of canine interferon in Pichia pastoris[D].Changchun:Jilin University,2016.(in Chinese)
[17] 乌日勒,任宇,喻桃,等.干扰素治疗带状疱疹的疗效及对患者细胞免疫功能的影响[J].河北医学,2017,23(4):600-603.WU R L,REN Y,YU T,et al.Effect of interferon on herpes zoster and its effect on cellular immune function[J].Hebei Medicine,2017,23(4):600-603.(in Chinese)
[18] 邓桂馨,梁果义,鲁琳,等.绵羊干扰素tau在毕赤酵母中的表达[J].中国草食动物科学,2016,36(4):5-10.DENG G X,LIANG G Y,LU L,et al.Secretory expression of ovine interferon tau in Pichia pastoris[J].Chinese herbivore Science,2016,36(4):5-10.(in Chinese)
[19] SOADIUT O,HERRWIG C.Dynamics in bioprocess development for Pichia pastoris[J].Bioengineered,2014,5(6):401-405.
[20] 郑华斌,张中旺,吕建亮,等.口蹄疫病毒多基因及猪α干扰素共表达载体的构建及其免疫豚鼠效果研究[J].中国畜牧兽医,2017,44(5):1452-1461.ZHENG H B,ZHANG Z W,LYU J L,et al.Construction of coexpression vector containing multigene of FMDV and porcine IFN-α and its immunogenicity effect in guinea pig[J].China Animal Husbandry & Veterinary Medicine,2017,44(5):1452-1461.(in Chinese)
[21] HAAGMANS B L,KUIKEN T,MARTINA B E,et al.Pegylated interferon-alpha protects typeⅠ pneumocytes against SARS coronavirus infection in macaques[J].Nature Medicine,2004,10(3):290-293.
[22] DAMASCENO L M,HUANG C J,BATT C A,et al.Protein secretion in Pichia pastoris and advances in protein production[J].Applied Microbiology and Biotechnology,2012,93(1):31-39.
[23] ENSERINK M.SARS treatment.Interferon shows promise in monkeys[J].Science,2004,303(5662):1273-1275.
[24] NORDSTROM E M,BIGGINS S W,CRALLA J,et al.Consensus interferon for recurrent hepatitis C infection in nonresponders to peginterferon and ribavirin after liver transplant[J].Experimental and Clinical Transplantation,2015,13(6):543-549.
[25] 杨晓璐,闫若潜,李勤楠,等.重组鸡α干扰素/白细胞介素-2融合蛋白体外活性评价方法研究[J].中国畜牧兽医,2015,42(12):3153-3159.YANG X L,YAN R Q,LI Q N,et al.Study on the activity estimation methods of recombinant chicken interferon-α/interleukin-2 fusion protein in vitro[J].China Animal Husbandry & Veterinary Medicine,2015,42(12):3153-3159.(in Chinese)
[2] DING Y,ZHANG H,CHEN H,et al.Association of immune response parameters with virological response in hepatitis C virus patients treated with pegylated consensus interferon[J].International Journal of Clinical Pharmacology,2016,54(3):163-171.
[3] 刘欢,金红岩,侯强,等.犬α干扰素在不同表达载体中的生物学活性比较[J].中国畜牧兽医,2016,43(10):2560-2565.LIU H,JIN H Y,HOU Q,et al.The comparison of canine α-interferon antiviral activity in different expression vectors[J].China Animal Husbandry & Veterinary Medicine,2016,43(10):2560-2565.(in Chinese)
[4] 杨生海.干扰素-γ研究进展[J].生物技术通报,2010,8:29-34.YANG S H.Research progress of interferon gamma[J].Biotechnology Bulletin,2010,8:29-34.(in Chinese)
[5] BARNES B J,MOORE P A,PITHA P M.Virus specific activation of a novel interferon regulatory factor,IRF-5,results in the induction of distinct interferon alpha genes[J].Journal of Biological Chemistry,2001,276(26):23382-23390.
[6] 邓成刚.犬IFN-α基因的克隆、表达及其生物学活性的研究[D].扬州:扬州大学,2008.DENG C G.Cloning,expression and biological activity of IFN-α gene in dogs[D].Yangzhou:Yangzhou University,2008.(in Chinese)
[7] 汪恭富,冯杰,马妍.干扰素在宠物病毒性疾病治疗上的使用效果分析[J].畜牧与兽医,2015,47(9):87-88.WANG G F,FENG J,MA Y.Analysis on the effect of interferon in the treatment of pet viral diseases[J].Animal Husbandry & Veterinary Medicine,2015,47(9):87-88.(in Chinese)
[8] 尚淑芬,郭小红.干扰素在治疗犬病毒性疾病中的应用[J].中国畜牧兽医文摘,2014,30(7):196.SHANG S F,GUO X H.Application of interferon in the treatment of viral diseases in canine[J].Chinese Journal of Animal Husbandry & Veterinary Medicine Abstract,2014,30(7):196.(in Chinese)
[9] 田春玲,马玉忠,秦建华,等.犬干扰素α1和胸腺肽α1融合蛋白在毕赤酵母GS115中的表达[J].河北农业大学学报,2018,41(1):88-91.TIAN C L,MA Y Z,QIN J H,et al.Expression of canine IFNα1-THYα1 fusion protein in Pichia pastoris GS115[J].Journal of Hebei Agricultural University,2018,41(1):88-91.(in Chinese)
[10] 董亚青,朱文斗,王琳琳,等.鸡α-干扰素在毕赤酵母中的表达及其表达条件优化[J].江苏农业科学,2015,43(11):275-277.DONG Y Q,ZHU W D,WANG L L,et al.Expression of chicken interferon alpha in Pichia pastoris and optimization of expression conditions[J].Jiangsu Agriculture Science,2015,43(11):275-277.(in Chinese)
[11] 张德芳.重组干扰素α-2b合成研究进展[J].海峡药学,2017,29(7):57-59.ZHANG D F.Research progress in synthesis of recombinant interferon α-2b[J].Strait Pharmaceutical Journal,2017,29(7):57-59.(in Chinese)
[12] 彭统全,邵建伟,张润祥,等.牛α0干扰素在毕赤酵母中的分泌表达及抗病毒活性鉴定[J].畜牧兽医学报,2015,46(1):124-129.PENG T Q,SHAO J W,ZHANG R X,et al.Expression of the bovine interferon alpha0 with antiviral activity in Pichia pastoris[J].Acta Veterinaria et Zootechnica Sinica,2015,46(1):124-129.(in Chinese)
[13] 刘新文,王秀丽,郭东春,等.鸡β干扰素在毕赤酵母中的分泌表达及其抗病毒活性测定[J].中国畜牧兽医,2017,44(7):2203-2208.LIU X W,WANG X L,GUO D C,et al.Expression of chicken interferon-β in Pichia pastoris and its antiviral effect[J].China Animal Husbandry & Veterinary Medicine,2017,44(7):2203-2208.(in Chinese)
[14] DRAGOSITS M,SPADIUT O,DIETZSCH C.Determination of a dynamic feeding strategy forrecombinant Pichia pastoris strains[J].Methods in Molecular Biology,2014,1152:185-194.
[15] 李哲,李俊峰,刘千辉,等.梅花鹿α干扰素基因在MDBK细胞中表达及抗BVDV活性检测[J].中国兽医学报,2016,36(12):2049-2053.LI Z,LI J F,LIU Q H,et al.Expression of Sika deer interferon alpha gene in MDBK cells and detection of its anti BVDV activity[J].Chinese Journal of Veterinary Science,2016,36(12):2049-2053.(in Chinese)
[16] 谭成成.2种不同分泌信号对犬干扰素在毕赤酵母中分泌表达的影响[D].长春:吉林大学,2016.TAN C C.Effects of 2 different secretory signals on secretory expression of canine interferon in Pichia pastoris[D].Changchun:Jilin University,2016.(in Chinese)
[17] 乌日勒,任宇,喻桃,等.干扰素治疗带状疱疹的疗效及对患者细胞免疫功能的影响[J].河北医学,2017,23(4):600-603.WU R L,REN Y,YU T,et al.Effect of interferon on herpes zoster and its effect on cellular immune function[J].Hebei Medicine,2017,23(4):600-603.(in Chinese)
[18] 邓桂馨,梁果义,鲁琳,等.绵羊干扰素tau在毕赤酵母中的表达[J].中国草食动物科学,2016,36(4):5-10.DENG G X,LIANG G Y,LU L,et al.Secretory expression of ovine interferon tau in Pichia pastoris[J].Chinese herbivore Science,2016,36(4):5-10.(in Chinese)
[19] SOADIUT O,HERRWIG C.Dynamics in bioprocess development for Pichia pastoris[J].Bioengineered,2014,5(6):401-405.
[20] 郑华斌,张中旺,吕建亮,等.口蹄疫病毒多基因及猪α干扰素共表达载体的构建及其免疫豚鼠效果研究[J].中国畜牧兽医,2017,44(5):1452-1461.ZHENG H B,ZHANG Z W,LYU J L,et al.Construction of coexpression vector containing multigene of FMDV and porcine IFN-α and its immunogenicity effect in guinea pig[J].China Animal Husbandry & Veterinary Medicine,2017,44(5):1452-1461.(in Chinese)
[21] HAAGMANS B L,KUIKEN T,MARTINA B E,et al.Pegylated interferon-alpha protects typeⅠ pneumocytes against SARS coronavirus infection in macaques[J].Nature Medicine,2004,10(3):290-293.
[22] DAMASCENO L M,HUANG C J,BATT C A,et al.Protein secretion in Pichia pastoris and advances in protein production[J].Applied Microbiology and Biotechnology,2012,93(1):31-39.
[23] ENSERINK M.SARS treatment.Interferon shows promise in monkeys[J].Science,2004,303(5662):1273-1275.
[24] NORDSTROM E M,BIGGINS S W,CRALLA J,et al.Consensus interferon for recurrent hepatitis C infection in nonresponders to peginterferon and ribavirin after liver transplant[J].Experimental and Clinical Transplantation,2015,13(6):543-549.
[25] 杨晓璐,闫若潜,李勤楠,等.重组鸡α干扰素/白细胞介素-2融合蛋白体外活性评价方法研究[J].中国畜牧兽医,2015,42(12):3153-3159.YANG X L,YAN R Q,LI Q N,et al.Study on the activity estimation methods of recombinant chicken interferon-α/interleukin-2 fusion protein in vitro[J].China Animal Husbandry & Veterinary Medicine,2015,42(12):3153-3159.(in Chinese)