扬子鳄α型干扰素基因的克隆分析及表达量的测定
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摘要
为研究扬子鳄IFN-α基因的序列、结构及生物学功能,对其进行了基因克隆、序列分析及表达量的测定。根据GenBank中登录的扬子鳄IFN-α基因序列(XM_006026085.1)设计合成特异性引物,通过RTPCR从成年扬子鳄外周血中扩增及克隆IFN-α基因CDS区,对其进行测序分析、进化树构建、同源性分析及结构和功能预测,并对扬子鳄不同组织中IFN-α的表达水平进行测定和分析。结果显示,扬子鳄IFN-α基因的完整ORF序列为672 bp,编码氨基酸223个,其中前26个氨基酸为信号肽。成熟蛋白的分子质量为26ku,等电点为9.51。三级结构预测显示该蛋白含有5段α螺旋,符合I型干扰素结构特征。同源性和进化树分析结果显示,扬子鳄IFN-α基因与密西西比鳄的同源性最高,在进化树中处于同一分支。IFN-α表达量在血液中最高,肌肉组织中表达量最低,非冬眠期的表达量高于冬眠期的表达量。上述研究结果为进一步研究扬子鳄IFN-α基因表达调控机制和基因功能奠定了基础。
In order to study the sequence structure and biological functions of IFN-α gene,a pair of specific primers were designed to amplify the CDS region of Chinese alligator IFN-α gene.The sequence was analyzed by phylogenetic tree analysis,homology analysis and structural prediction.The differential expression of IFN-α protein in different tissues were semi quantitatively analyzed during hibernation and non-hibernation.The results showed that the complete ORF of Chinese alligator IFN-α gene was 672 bp,encoding 223 amino acids and the first 26 amino acids were signal peptides.In mature protein,the molecular weight was 26 ku and the isoelectric point was 9.51.The 3 D structure showed that the protein consisted of 5 alpha helixes. The IFN-α gene of Chinese alligator was closest to that of Alligator mississippiensis.It was found that the highest expression level of IFN-α was in the blood and the lowest was in the muscle.The results laid the foundation for further studies on the mechanism of IFN-α gene expression regulation and functions of the gene.
In order to study the sequence structure and biological functions of IFN-α gene,a pair of specific primers were designed to amplify the CDS region of Chinese alligator IFN-α gene.The sequence was analyzed by phylogenetic tree analysis,homology analysis and structural prediction.The differential expression of IFN-α protein in different tissues were semi quantitatively analyzed during hibernation and non-hibernation.The results showed that the complete ORF of Chinese alligator IFN-α gene was 672 bp,encoding 223 amino acids and the first 26 amino acids were signal peptides.In mature protein,the molecular weight was 26 ku and the isoelectric point was 9.51.The 3 D structure showed that the protein consisted of 5 alpha helixes. The IFN-α gene of Chinese alligator was closest to that of Alligator mississippiensis.It was found that the highest expression level of IFN-α was in the blood and the lowest was in the muscle.The results laid the foundation for further studies on the mechanism of IFN-α gene expression regulation and functions of the gene.
引文
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[19] SONG C S,RAO T R,DEMYAN W F,et al.Androgen receptor messenger ribonucleic acid(mRNA)in the rat liver:changes in mRNA levels during maturation,aging and calorie restriction[J].Endocrinology,1991,128(1):349-356.
[20] ZIMMERMAN L M,VOGEL L A,BOWDEN R M.Understanding the vertebrate immune system:insights from the reptilian perspective[J].The Journal of Experimental Biology,2010,213(5):661-671.
[2]吴荣,周永康.安徽扬子鳄国家级自然保护区现状分析[J].安徽林业科技,2015,41(5):37-41.WU Rong,ZHOU Yong-kang.Analysis on the actualities of anhui Chinese alligator national nature reserve[J].Anhui Forestry Science and Technology,2015,41(5):37-41.(in Chinese)
[3] BARBALAT R,LAU L,LOCKSLEY R M,et al.Toll-like receptor 2 on inflammatory monocytes induces typeⅠinterferon in response to viral but not bacterial ligands[J].Nature Immunology,2009,10(11):1200-1207.
[4] MILLER D L,MAUEL M J,BALDWIN C,et al.West Nile virus in farmed alligators[J].Emerg Infect Disease,2003,9(7):794-799.
[5] HUCHZERMEYER F W,HUCHZERMEYER F W.Crocodiles:biology,husbandry and diseases[J].Journal of the South African Veterinary Association,2012,74(4):98-138.
[6] SAMUEL C E.Protein-nucleic acid interactions and cellular responses to interferon[J].Methods(San Diego,Calif.),1998,15(3):161-165.
[7]甘霖,赵俊,陈敬贤,等.干扰素抗病毒效力影响因素的研究进展[J].微生物与感染,2018,13(1):43-48.GAN Lin,ZHAO Jun,CHEN Jin-xian,et al.Research progress on the influencing factors of antiviral efficacy of interferon[J].Journal of Microbes and Infections,2018,13(1):43-48.(in Chinese)
[8]刘洋,刘永亮,葛胜祥.干扰素刺激基因及临床意义研究进展[J].中国免疫学杂志,2018,34(3):454-459.LIU Yang,LIU Yong-liang,GE Sheng-xiang.Research progress towards interferon stimulated genes and its clinical significance[J].Chinese Journal of Immunology,2018,34(3):454-459.(in Chinese)
[9] SCHMITTGEN T D,LIVAK K J.Analyzing real-time PCR data by the comparative C(T)method[J].Nature Protocols,2008,3(6):1101-1108.
[10] VISCOMI G C.Structure-activity of typeⅠinterferons[J].Biotherapy(Dordrecht,Netherlands),1997,10(1):59-86.
[11] CONKLIN D.Recognition of the helical cytokine fold[J].Journal of Computational Biology(Print),2005,11(6):1189-1200.
[12] KARPUSAS M,NOLTE M,BENTON C B,et al.The crystal structure of human interferon beta at 2.2-A resolution[J].Proceedings of the National Academy of Sciences of the United States of America,1997,94(22):11813-11818.
[13] LIS H,SHARON N.Protein glycosylation structure and functional aspects[J].European Journal of Biochemistry,1993,218(1):1-27.
[14] ROBERTSEN B.The interferon system of teleost fish[J].Fish Shellfish Immunol,2005,20(2):172-191.
[15] ZOU J,TAFALLA C,TRUCKLE J,et al.Identification of a second group of typeⅠIFNs in fish sheds light on IFN evolution in vertebrates[J].Journal of Immunology,2007,179(6):3859-3871.
[16]秘晶玮,王君伟,许丽娜,等.鹅α和γ干扰素基因的克隆及序列分析[J].中国预防兽医学报,2005,27(5):359-362.MI Jing-wei,WANG Jun-wei,XU Li-na,et al.Cloning and sequencing of goose typeαandγinterferon[J].Chinese Journal of Preventive Veterinary Medicine,2005,27(5):359-362.(in Chinese)
[17]胡智博,李林俐,费荣梅.扬子鳄Viperin基因的遗传衍化分析及表达量的测定[J].中国兽医科学,2018,48(2):182-188.HU Zhi-bo,LI Lin-li,FEI Rong-mei.Cloning and evolution research and tissue expression detection of Chinese alligator Viperin gene[J].Chinese Veterinary Science,2018,48(2):182-188.(in Chinese)
[18]周永康,谢万树.扬子鳄的越冬管理[J].四川动物,2000(1):43-44.ZHOU Yong-kang,XIE Wan-shu.The management of Chinese alligator in hibernation[J].Sichuan Journal of Zoology,2000(1):43-44.(in Chinese)
[19] SONG C S,RAO T R,DEMYAN W F,et al.Androgen receptor messenger ribonucleic acid(mRNA)in the rat liver:changes in mRNA levels during maturation,aging and calorie restriction[J].Endocrinology,1991,128(1):349-356.
[20] ZIMMERMAN L M,VOGEL L A,BOWDEN R M.Understanding the vertebrate immune system:insights from the reptilian perspective[J].The Journal of Experimental Biology,2010,213(5):661-671.