重组鸡γ-干扰素在家蚕中的表达及抗病毒活性
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摘要
鸡γ-干扰素(Ch IFN-γ)是一类具有免疫调节及抗病毒活性的细胞因子。利用家蚕杆状病毒表达系统表达具有高生物活性的鸡γ-干扰素。首先,对Ch IFN-γ基因序列进行优化与合成,再将其克隆到杆状病毒转移载体p VL1393多克隆位点中,与缺少orf1629基因的家蚕杆状病毒re Bm Bac共转染Bm N细胞获得重组病毒re Bm-ChIFN-γ。将re Bm-ChIFN-γ注射感染家蚕5龄幼虫,检测感染后96～120 h之间发病蚕体的血淋巴。PCR扩增结果显示成功构建了重组杆状病毒re Bm-ChIFN-γ,Western blot检测重组杆状病毒在家蚕体内表达了目的干扰素,微量细胞病变抑制法检测表达产物具有明显的抗病毒活性,效价约为(3. 24±0. 32)×10~6U/mL。试验结果表明,利用家蚕杆状病毒表达系统成功地表达出具有较高抗病毒活性的鸡γ-干扰素,为鸡γ-干扰素相关生物制剂的研发奠定了基础。
Chicken gamma interferon( Ch IFN-γ) is a kind of cytokine which plays a role in immune regulation and antiviral activity. Here,we expressed Ch IFN-γ with high biological activity by silkworm baculovirus expression system.First of all,the codons of Ch IFN-γ were optimized and the gene was synthesized and cloned into multiple cloning site of baculovirus transfer vector pVL1393. Then, we co-transfected Bm N cells with both the recombinant plasmid and re Bm Bac bacmid without orf1629 gene,obtained and purified the recombinant virus re Bm-ChIFN-γ. Subsequently,we injected 5 th instar larvae of Bombyx mori with re Bm-ChIFN-γ,collected the hemolymph of silkworm 96-120 h after injection and preserved for further detection. PCR results suggested that we constructed the recombinant baculovirus re BmCh IFN-γ successfully and target protein expression was confirmed by Western blot. In addition,biological potency of the protein was approximately( 3. 24± 0. 32) × 10~6 U/mL as determined by cytopathic effect inhibition assay. We conclude that,the Ch IFN-γ with high biological activity was expressed successfully in silkworm bioreactor through silkworm baculovirus expression system,which sets the good stage for subsequent biological agents related to Ch IFN-γ.
Chicken gamma interferon( Ch IFN-γ) is a kind of cytokine which plays a role in immune regulation and antiviral activity. Here,we expressed Ch IFN-γ with high biological activity by silkworm baculovirus expression system.First of all,the codons of Ch IFN-γ were optimized and the gene was synthesized and cloned into multiple cloning site of baculovirus transfer vector pVL1393. Then, we co-transfected Bm N cells with both the recombinant plasmid and re Bm Bac bacmid without orf1629 gene,obtained and purified the recombinant virus re Bm-ChIFN-γ. Subsequently,we injected 5 th instar larvae of Bombyx mori with re Bm-ChIFN-γ,collected the hemolymph of silkworm 96-120 h after injection and preserved for further detection. PCR results suggested that we constructed the recombinant baculovirus re BmCh IFN-γ successfully and target protein expression was confirmed by Western blot. In addition,biological potency of the protein was approximately( 3. 24± 0. 32) × 10~6 U/mL as determined by cytopathic effect inhibition assay. We conclude that,the Ch IFN-γ with high biological activity was expressed successfully in silkworm bioreactor through silkworm baculovirus expression system,which sets the good stage for subsequent biological agents related to Ch IFN-γ.
引文
[1]葛忠源,张秀云.鸡干扰素研究进展[J].家禽科学,2010(5):46-48
[2] BAZER F W,SPENCER T E,OTT T L. Interferon tau:a novel pregnancy recognition signal[J].Am J Reprod Immunol Microbiol,1997,37(6):412-420
[3] LEVY D E,MARI I J,DURBIN J E.Induction and function of typeⅠandⅢinterferon in response to viral infection[J].Curr Opin Virol,2011,1(6):476-486
[4] BACH E A,AGUET M,SCHREIBER R D.The IFN-γreceptor:a paradigm for cytokine receptor signaling[J]. Annu Rev Immunol,15(1):563-591
[5] JONASCH E,HALUSKA F G.Interferon in oncological practice:review of interferon biology,clinical applications,and toxicities[J].Oncologist,2001,6(1):34-55
[6] KAISER P,POH T Y,ROTHWELL L,et al.A genomic analysis of chicken cytokines and chemokines[J]. J Interferon Cytokine Res,2005,25(8):467-484
[7] SCHIJNS V E,SCHOLTES N C,ZUILEKOM H I,et al.Facilitation of antibody forming responses to viral vaccine antigens in young cats by recombinant baculovirus-expressed feline IFN-γ[J]. Vaccine,2002,20(13/14):1718-1724
[8]程坚,刘秀梵,彭大新,等.表达H9亚型禽流感病毒血凝素基因的重组鸡痘病毒及其免疫效力[J].病毒学报,2002,19(4):52-58
[9] PROKUNINAOLSSON L,MUCHMORE B,TANG W,et al. A variant upstream of IFNL3(IL28B)creating a novel interferon gene IFNL4 is associated with impaired clearance of hepatitis C virus[J].Nat Genet,2013,45(2):164-171
[10]冯丽光,刘维藩.新型干扰素——IFN-λs的研究概况[J].中国现代普通外科进展,2008,11(2):136-138
[11] SEKELLICK M J,FERRANDINO A F,HOPKINS D A,et al.Chicken interferon gene:cloning,expression,and analysis[J].J Interferon Res,1994,14(2):71-79
[12] DIGBY M R,LOWENTHAL J W. Cloning and expression of the chicken interferon-gamma gene[J]. J Interferon Cytokine Res,1995,15(11):939-945
[13]叶秀华,蔡建平,吴志光,等.重组鸡γ干扰素的抗球虫作用[J].中国兽医杂志,2004,40(7):3-5
[14] DIMIER I H,QU R P,NACIRI M,et al. Inhibition of Eimeria tenella development in vitro mediated by chicken macrophages and fibroblasts treated with chicken cell supernatants with IFN-γactivity[J].Avian Dis,1998,42(2):239-247
[15] LIU X,WEI Y,LI Y,et al. A highly efficient and simple construction strategy for producing recombinant baculovirus Bombyx mori nucleopolyhedrovirus[J/OL]. PLoS ONE,2016,11(3):e0152140[2018-04-15]. https://www. ncbi. nlm.nih. gov/pubmed/27008267.DOI:10.1371/journal.pone.0152140
[16] POSSEE R D,SUN T P,HOWARD S C,et al.Nucleotide sequence of the Autographa californica nuclear polyhedrosis 9.4 kbp EcoRI-I and-R(polyhedrin gene)region[J]. Virology,1991,185(1):229-241
[17] JE Y H,CHANG J H,KIM M H,et al.The use of defective Bombyx mori nucleopolyhedrovirus genomes maintained in Escherichia coli for the rapid generation of occlusion-positive and occlusion-negative expression vectors[J].Biotechnol Lett,2001,23(21):1809-1817
[18]张韵.口蹄疫病毒组合基因在家蚕杆状病毒表达系统中的表达及差异分析[D].兰州:甘肃农业大学,2008
[19]斯佩克特D L,戈德曼R D,莱因万德L A.细胞实验指南[M].北京:科学出版社,2001:32-35
[20]涂纳新,张志芳,郭锡杰,等.应用PCR技术诊断家蚕核型多角体病毒病[J].蚕业科学,1994,20(2):124-125
[21]郑晓灵,刘艳芬,陈绍红,等.重组狮头鹅α干扰素的制备及其抗病毒活性[J].中国兽医科学,2010(7):733-737
[22] HUANG L,CAO R B,WANG N,et al.The design and recombinant protein expression of a consensus porcine interferon:CoPoIFN-α[J].Cytokine,2012,57(1):37-45
[23] GUSTAFSSON C,GOVINDARAJAN S,MINSHULL J.Codon bias and heterologous protein expression[J]. Trends Biotechnol,2004,22(7):346-353
[24] ZHOU J,LIU W J,PENG S W,et al.Papillomavirus capsid protein expression level depends on the match between codon usage and tRNA availability[J].J Virol,1999,73(6):4972-4982
[25] DENG T.Bacterial expression and purification of biologically active mouse c-Fos proteins by selective codon optimization[J]. FEBS Lett,1997,409(2):269-272
[26] LI A,KATO Z,OHNISHI H,et al. Optimized gene synthesis and high expression of human interleukin-18[J]. Protein Expr Purif,2003,32(1):110-118
[27]刘新文,王秀丽,郭东春,等.鸡γ-干扰素在毕赤酵母中的分泌表达及其抗病毒活性研究[J].中国家禽,2017,39(1):626-629
[28] ARAKAWA T,HSU Y R,CHANG D,et al.Structure and activity of glycosylated human interferon-γ[J].J Interferon Res,1986,6(6):687-695
[29] SONG K D,LILLEHOJ H S,CHOI K D,et al.Expression and functional characterization of recombinant chicken interferon-gamma[J].Vet Immunol Immunopathol,1997,58(3/4):321-333
[30]蔡梅红,曹瑞兵,周斌,等.鸡γ干扰素成熟蛋白基因的表达及其产物抗病毒活性测定[J].中国病毒学,2004,19(1):32-35
[2] BAZER F W,SPENCER T E,OTT T L. Interferon tau:a novel pregnancy recognition signal[J].Am J Reprod Immunol Microbiol,1997,37(6):412-420
[3] LEVY D E,MARI I J,DURBIN J E.Induction and function of typeⅠandⅢinterferon in response to viral infection[J].Curr Opin Virol,2011,1(6):476-486
[4] BACH E A,AGUET M,SCHREIBER R D.The IFN-γreceptor:a paradigm for cytokine receptor signaling[J]. Annu Rev Immunol,15(1):563-591
[5] JONASCH E,HALUSKA F G.Interferon in oncological practice:review of interferon biology,clinical applications,and toxicities[J].Oncologist,2001,6(1):34-55
[6] KAISER P,POH T Y,ROTHWELL L,et al.A genomic analysis of chicken cytokines and chemokines[J]. J Interferon Cytokine Res,2005,25(8):467-484
[7] SCHIJNS V E,SCHOLTES N C,ZUILEKOM H I,et al.Facilitation of antibody forming responses to viral vaccine antigens in young cats by recombinant baculovirus-expressed feline IFN-γ[J]. Vaccine,2002,20(13/14):1718-1724
[8]程坚,刘秀梵,彭大新,等.表达H9亚型禽流感病毒血凝素基因的重组鸡痘病毒及其免疫效力[J].病毒学报,2002,19(4):52-58
[9] PROKUNINAOLSSON L,MUCHMORE B,TANG W,et al. A variant upstream of IFNL3(IL28B)creating a novel interferon gene IFNL4 is associated with impaired clearance of hepatitis C virus[J].Nat Genet,2013,45(2):164-171
[10]冯丽光,刘维藩.新型干扰素——IFN-λs的研究概况[J].中国现代普通外科进展,2008,11(2):136-138
[11] SEKELLICK M J,FERRANDINO A F,HOPKINS D A,et al.Chicken interferon gene:cloning,expression,and analysis[J].J Interferon Res,1994,14(2):71-79
[12] DIGBY M R,LOWENTHAL J W. Cloning and expression of the chicken interferon-gamma gene[J]. J Interferon Cytokine Res,1995,15(11):939-945
[13]叶秀华,蔡建平,吴志光,等.重组鸡γ干扰素的抗球虫作用[J].中国兽医杂志,2004,40(7):3-5
[14] DIMIER I H,QU R P,NACIRI M,et al. Inhibition of Eimeria tenella development in vitro mediated by chicken macrophages and fibroblasts treated with chicken cell supernatants with IFN-γactivity[J].Avian Dis,1998,42(2):239-247
[15] LIU X,WEI Y,LI Y,et al. A highly efficient and simple construction strategy for producing recombinant baculovirus Bombyx mori nucleopolyhedrovirus[J/OL]. PLoS ONE,2016,11(3):e0152140[2018-04-15]. https://www. ncbi. nlm.nih. gov/pubmed/27008267.DOI:10.1371/journal.pone.0152140
[16] POSSEE R D,SUN T P,HOWARD S C,et al.Nucleotide sequence of the Autographa californica nuclear polyhedrosis 9.4 kbp EcoRI-I and-R(polyhedrin gene)region[J]. Virology,1991,185(1):229-241
[17] JE Y H,CHANG J H,KIM M H,et al.The use of defective Bombyx mori nucleopolyhedrovirus genomes maintained in Escherichia coli for the rapid generation of occlusion-positive and occlusion-negative expression vectors[J].Biotechnol Lett,2001,23(21):1809-1817
[18]张韵.口蹄疫病毒组合基因在家蚕杆状病毒表达系统中的表达及差异分析[D].兰州:甘肃农业大学,2008
[19]斯佩克特D L,戈德曼R D,莱因万德L A.细胞实验指南[M].北京:科学出版社,2001:32-35
[20]涂纳新,张志芳,郭锡杰,等.应用PCR技术诊断家蚕核型多角体病毒病[J].蚕业科学,1994,20(2):124-125
[21]郑晓灵,刘艳芬,陈绍红,等.重组狮头鹅α干扰素的制备及其抗病毒活性[J].中国兽医科学,2010(7):733-737
[22] HUANG L,CAO R B,WANG N,et al.The design and recombinant protein expression of a consensus porcine interferon:CoPoIFN-α[J].Cytokine,2012,57(1):37-45
[23] GUSTAFSSON C,GOVINDARAJAN S,MINSHULL J.Codon bias and heterologous protein expression[J]. Trends Biotechnol,2004,22(7):346-353
[24] ZHOU J,LIU W J,PENG S W,et al.Papillomavirus capsid protein expression level depends on the match between codon usage and tRNA availability[J].J Virol,1999,73(6):4972-4982
[25] DENG T.Bacterial expression and purification of biologically active mouse c-Fos proteins by selective codon optimization[J]. FEBS Lett,1997,409(2):269-272
[26] LI A,KATO Z,OHNISHI H,et al. Optimized gene synthesis and high expression of human interleukin-18[J]. Protein Expr Purif,2003,32(1):110-118
[27]刘新文,王秀丽,郭东春,等.鸡γ-干扰素在毕赤酵母中的分泌表达及其抗病毒活性研究[J].中国家禽,2017,39(1):626-629
[28] ARAKAWA T,HSU Y R,CHANG D,et al.Structure and activity of glycosylated human interferon-γ[J].J Interferon Res,1986,6(6):687-695
[29] SONG K D,LILLEHOJ H S,CHOI K D,et al.Expression and functional characterization of recombinant chicken interferon-gamma[J].Vet Immunol Immunopathol,1997,58(3/4):321-333
[30]蔡梅红,曹瑞兵,周斌,等.鸡γ干扰素成熟蛋白基因的表达及其产物抗病毒活性测定[J].中国病毒学,2004,19(1):32-35
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