东北虎γ-干扰素基因的克隆、生物信息学分析及其在毕赤酵母中的表达

doi:10.16431/j.cnki.1671-7236.2021.01.008

摘要/Abstract

摘要： 本试验旨在通过克隆东北虎γ-干扰素（IFN-γ）基因，研究其分子特征并预测蛋白生物学功能，为后续研究干扰素抗病毒活性做前期准备。通过RT-PCR从ConA诱导过的东北虎血淋巴细胞中扩增东北虎IFN-γ基因并测序，应用生物信息学方法进行序列分析。结果表明：东北虎IFN-γ编码区由504个核苷酸组成，共编码167个氨基酸，蛋白相对分子质量为19.59 ku，等电点为9.03，所编码的蛋白为碱性亲水性蛋白，其中前23个氨基酸可能为信号肽，IFN-γ编码蛋白保守结构域为IFN-γ超家族，且存在跨膜结构，其中1－6位氨基酸为胞内区域，7－28位氨基酸为跨膜区域，29－167位氨基酸为胞外区域；IFN-γ编码蛋白二级结构主要以α-螺旋（58.08%）和无规则卷曲（33.53%）为主，存在5个潜在的B细胞抗原表位，3个潜在的N-糖基化位点；分子进化分析显示，东北虎IFN-γ与GenBank上发表的东北虎、非洲狮、金钱豹、美洲狮、猎豹、家猫、加拿大猞猁、野猪等的核苷酸相似性为80.4%~99.8%，氨基酸相似性为70.5%~100%，东北虎IFN-γ与非洲狮、金钱豹亲缘关系最近，美洲狮、猎豹、家猫、猞猁次之，野猪最远。通过合成改造后的东北虎IFN-γ基因，构建能表达IFN-γ蛋白的重组质粒pPIC9K-IFN-γ，将其导入高效表达系统-毕赤酵母中进行诱导表达，经SDS-PAGE分析，表达蛋白的分子量约17.8 ku，与预期大小相符，表明东北虎IFN-γ成功表达。

关键词: 东北虎; γ-干扰素(IFN-γ); 基因克隆; 生物信息学; 毕赤酵母

Abstract: The purpose of this experiment was to clone the Siberian tiger γ-interferon (IFN-γ) gene,studied its molecular characteristics and predicted the biological function of the protein,making preliminary preparations for the subsequent study of the antiviral activity of interferon.The Siberian tiger IFN-γ gene was amplified from ConA-induced Siberian tiger blood lymphocytes by RT-PCR and sequenced to obtain the sequence.Bioinformatics was used for sequence analysis.The results showed that IFN-γ gene coding region consisted of 504 nucleotides,encoding a total of 167 amino acids.The relative molecular weight of the Siberian tiger IFN-γ amino acid was 19.59 ku,the isoelectric point was 9.03.The encoded protein was a basic hydrophilic protein,of which the first 23 amino acids might be signal peptides.The conserved domain of the IFN-γ encoded protein was the IFN-γ superfamily,and there was a transmembrane structure,in which amino acids 1－6 were intracellular regions and amino acids 7－28 were transmembrane regions,amino acids 29－167 were extracellular regions.The secondary structure of IFN-γ encoded protein was mainly α-helix (58.08%) and random coil (33.53%).There were 5 potential B cell epitopes and 3 potential N-glycosylation sites.Sequence analysis showed that the Siberian tiger IFN-γ had 80.4%－99.8% nucleotide similarity to the Siberian tiger,African lion,leopard,puma,cheetah,domestic cat,Canadian lynx and wild boar published on GenBank,and the amino acid similarity was 70.5%－100%,nucleotide genetic evolution analysis showed that Siberian tiger IFN-γ had the closest relationship with African lions and leopards,followed by puma,cheetah,domestic cat and lynx,and wild boars were farthest.By synthesizing the modified Siberian tiger IFN-γ gene,a recombinant plasmid pPIC9K-IFN-γ capable of secreting and expressing IFN-γ protein was constructed,which was introduced into a highly efficient expression system-Pichia pastoris to induce expression,and analyzed by SDS-PAGE,the molecular weight of the expressed protein was about 17.8 ku,which was consistent with the expected size,indicating that the Siberian tiger IFN-γ was successfully expressed.

Key words: Siberian tiger; γ-interferon (IFN-γ); gene cloning; bioinformatics; Pichia pastoris

中图分类号:

S865.31

翟俊琼, 吴亚江, 代军威, 陈安妮, 周妞, 王晨, 杜雪晴, 罗满林, 陈武. 东北虎γ-干扰素基因的克隆、生物信息学分析及其在毕赤酵母中的表达[J]. 中国畜牧兽医, 2021, 48(1): 72-82.

ZHAI Junqiong, WU Yajiang, DAI Junwei, CHEN Anni, ZHOU Niu, WANG Chen, DU Xueqing, LUO Manlin, CHEN Wu. Cloning and Bioinformatics Analysis of the Siberian Tiger γ-interferon Gene and Its Expression in Pichia pastoris[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(1): 72-82.

参考文献

[1] WANG X J.Global number of wild tigers grows for the first time in 100 years[J].College English,2016,7:32-33.
[2] 盖春云,张灿,单虎.水貂IFN-α基因的克隆与原核表达[J].中国兽医杂志,2010,6:17-19. GAI C Y,ZHANG C,SHAN H.Cloning and prokaryotic expression of ferret IFN-α genes[J].Chinese Journal of Veterinary Medicine,2010,6:17-19.(in Chinese)
[3] ISAACS A,LINDENMANN J,VALENTINE R C.Virus interference.Ⅱ.Some properties of interferon[J].Proceedings of the Royal Society B-Biological Sciences,1957,147(927):268-273.
[4] DOLEI A,CAPOBIANCHI M R,AMEGLIO F.Human interferon-γ enhances the expression of class Ⅰ and class Ⅱ major histocompatibility complex products in neoplastic cells more effectively than interferon-α and interferon-β[J].Infection & Immunity,1983,40(1):172-176.
[5] 杨生海,殷宏,刘永生,等.干扰素-γ研究进展[J].生物技术通报,2010,8:29-34. YANG S H,YIN H,LIU Y S,et al.Advances in research of interferon-gamma[J].Biotechnology Bulletin,2010,8:29-34.(in Chinese)
[6] 邢明伟,姜广顺,孙潇,等.东北虎α干扰素及其编码基因和应用[P].中国,CN201610895330.0,2017. XING M W,JIANG G S,SUN X,et al.Amur tiger interferon alpha and its coding gene and application[P].China:CN201610895330.0,2017.(in Chinese)
[7] 刘秋云,罗樨,何康泽,等.快速制备酵母质粒和基因组DNA PCR模板[J].微生物学通报,2001,28(5):77-80. LIU Q Y,LUO X,HE K Z,et al.Rapid preparation of yeast plasmid and genomic DNA PCR template[J].Microbiology China,2001,28(5):77-80.(in Chinese)
[8] 王姝懿,赵学亮.鼠疫耶尔森菌CAF-1基因的生物信息学分析及抗原表位预测[J].中国病原生物学杂志,2019,14(10):1141-1146. WANG S Y,ZHAO X L.Bioinformatics analysis and antigen epitope prediction of Yersinia pestisCAF-1 gene[J].Journal of Pathogen Biology,2019,14(10):1141-1146.(in Chinese)
[9] 温贵兰,陈绍品,张升波,等.从江香猪γ干扰素基因克隆与序列分析[J].中国畜牧兽医,2018,45(6):1437-1446. WEN G L,CHEN S P,ZHANG S B,et al.Cloning and sequence analysis of Xiangjiang pig interferon gamma gene[J].China Animal Husbandry & Veterinary Medicine,2018,45(6):1437-1446.(in Chinese)
[10] MICHEL L V,KALMETA B,MCCREARY M,et al.Vaccine candidate P6 of nontypable Haemophilus influenzae is not a transmembrane protein based on protein structural analysis[J].Vaccine,2011,29(8):1627.
[11] 黄溢泓,谢芝勋.广西10个地方优质品种鸡γ-干扰素基因的克隆与序列分析[A].优质鸡的改良生产暨发展研讨会[C].2006. HUANG Y H,XIE Z X.Cloning and sequence analysis of interferon-γ gene of 10 local high quality chicken breeds in Guangxi[A].Workshop on Improved Production and Development of High Quality Chicken[C].2006.(in Chinese)
[12] 孙颖杰,王哲.水貂α-干扰素基因的克隆、测序和生物信息学分析[J].畜牧与兽医,2009,6:62-64. SUN Y J,WANG Z.Cloning,sequencing and bioinformatics analysis of mink interferon alpha gene[J].Animal Husbandry & Veterinary Medicine,2009,6:62-64.(in Chinese)
[13] 惠希武,陈虹,黄秉仁.N-糖基化位点突变的人IFN-λ1在毕赤酵母中的表达、纯化及表征[J].生物工程学报,2018,34(4):613-624. HUI X W,CHEN H,HUANG B R.Expression,purification and characterization of human IFN-λ1 with N-glycosylation site mutation in Pichia pastoris[J].Chinese Journal of Biotechnology,2018,34(4):613-624.(in Chinese)
[14] 黄梦秋,嵇辛勤,段志强,等.犬IFN-β的克隆及其序列分析[J].黑龙江畜牧兽医,2019,7:139-142. HUANG M Q,JI X Q,DUAN Z Q,et al.Cloning and sequence analysis of canine IFN-β[J].Heilongjiang Animal Science and Veterinary Medicine,2019,7:139-142.(in Chinese)
[15] 蔡梅红,张素芳,曹瑞兵,等.重组酵母鸡γ干扰素的抗病毒活性测定及临床初步应用[J].微生物学报,2006,46(1):115-119. CAI M H,ZHANG S F,CAO R B,et al.Determination of antiviral activity and clinical application of recombinant yeast chicken interferon gamma[J].Acta Microbiologica Sinica,2006,46(1):115-119.(in Chinese)
[16] WANG Y B,WANG Z Y,CHEN H Y,et al.Secretory expression of porcine interferon-gamma in baculovirus using HBM signal peptide and its inhibition activity on the replication of porcine reproductive and respiratory syndrome virus[J].Veterinary Immunology & Immunopathology,2009,132(2-4):317.
[17] 吴艳花,汪明,吴志光.德国牧羊犬γ干扰素基因克隆表达与抗病毒活性测定[J].中国兽医杂志,2008,44(6):3-5. WU Y H,WANG M,WU Z G.Molecular cloning,expression and antiviral activity analysis of CaIFN-γ gene of German shepherd dog[J].Chinese Journal of Veterinary Medicine,2008,44(6):3-5.(in Chinese)
[18] GRADOBOEVA A E,PADKINA M V.Bovine gamma-interferon nuclear localization sequence provides translocation of recombinant protein to yeastPichia pastoriscell nucleus[J].Cell & Tissue Biology,2010,4(6):566-571.
[19] TU Y,ZHU W Y,LU C P.Bacterial 16S rDNA sequence analysis of Siberian tiger faecal flora[J].Acta Microbiologica Sinica,2005,45(5):671-674.
[20] 朴玉仁.猪霍乱沙门氏菌孔城道夫变种感染东北虎致死的报告[J].微生物学通报,1981,8(4):23-24. PIAO Y R.Report on the death of S.choleraesuis Kongchengdorff mutant infected with Siberian tiger[J].Microbiology China,1981,8(4):23-24.(in Chinese)
[21] CAPITINI C M,HERRERO I A,ROBIN P,et al.Wound infection with Neisseria weaveri and a novel subspecies of Pasteurella multocida in a child who sustained a tiger bite[J].Clinical Infectious Diseases,2002,34(12):e74-e76.
[22] 卢岩,陈永林,潘庆山,等.东北虎(A、D)型魏氏梭菌混合感染导致猝死[J].中国兽医杂志,2006,2:60-61. LU Y,CHEN Y L,PAN Q S,et al.Sudden death due to mixed infection of Clostridium welchii type A and D of Siberian tiger[J].Chinese Journal of Veterinary Medicine,2006,2:60-61.(in Chinese)
[23] ZHANG H,SHAN F,ZHOU X,et al.Outbreak and genotyping of canine distemper virus in captive Siberian tigers and red pandas[J].Scientific Reports,2017,7(1):8132.
[24] WANG X G,LI T Y,et al.Recombinant feline parvovirus infection of immunized tigers in central China[J].Emerging Microbes & Infections,2017,6(6):e42.
[25] SUN H,LI Y,JIAO W,et al.Isolation and identification of feline herpesvirus type 1 from a South China tiger in China[J].Viruses,2014,6(3):1004-1014.
[26] WU C,PANG W,ZHAO D.Amino acid sequence of the Amur tiger prion protein[J].Virus Research,2006,121(1):96.
[27] ZHAI J Q,ZHAI S L,LIN T,et al.First complete genome sequence of parainfluenza virus 5 isolated from lesser panda[J].Archives of Virology,2017,162(5):1413-1418.
[28] RISI E,AGOULON A,ALLAIRE F,et al.Antibody response to vaccines for rhinotracheitis,caliciviral disease,panleukopenia,feline leukemia,and rabies in tigers (Panthera tigris) and lions (Panthera leo)[J].Journal of Zoo & Wildlife Medicine Official Publication of the American Association of Zoo Veterinarians,2012,43(2):248.
[29] LIU H M,YAN Q,ZHAO B,et al.Isolation,molecular characterization,and phylogenetic analysis of encephalomyocarditis virus from South China tigers in China[J].Infection Genetics & Evolution,2013,19:240-243.
[30] PATICHIMASIRI T,CHANTAMANEECHOTE T,CHUNJAI S,et al.Histology and immunohisto-chemistry of tigers and birds naturally infected with H5N1 highly pathogenic avian influenza virus in Thailand[J].Japan Agricultural Research Quarterly,2007,41(3):247-252.
[31] 王祥生,夏咸柱,范泉水,等.虎暴发锥虫病一起[J].中国兽医科学,1999,7:34-35. WANG X S,XIA X Z,FAN Q S,et al.Tiger outbreak with trypanosomiasis[J].Chinese Journal of Veterinary Science and Technology,1999,7:34-35.(in Chinese)
[32] 宋丹,刘丹,杨莲茹,等.园养东北虎等猫科动物隐孢子虫感染情况的调查[J].中国动物传染病学报,2009,17(1):72-74. SONG D,LIU D,YANG L R,et al.Investigation on the infection of cryptosporidium in cats such as Siberian tiger[J].Chinese Journal of Animal Infectious Diseases,2009,17(1):72-74.(in Chinese)
[33] 刘建丽,侯志军,华育平,等.黑龙江东北虎林园猫科动物体内寄生虫感染调查[J].野生动物学报,2007,3:28-30. LIU J L,HOU Z J,HUA Y P,et al.Survey of endoparasites infection in feline animals in the Heilongjiang Amur Tiger Park,China[J].Chinese Journal of Wildlife,2007,3:28-30.(in Chinese)
[34] DHARANESHA N K,SAMINATHAN M,MAMTA P,et al.Parasitic pneumonia caused by Paragonimus spp.in a wild royal Bengal tiger,Mysuru,South India[J].Journal of Parasitic Diseases,2019,43:528-533.