水牛IFN-γ克隆表达及其单克隆抗体的制备与应用
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摘要
水牛(Bubalus bubalus, Bb),哺乳纲牛科(Bovidae)水牛属,适宜于水田耕作。水牛奶质十分优良,所含蛋白质、氨基酸、乳脂、维生素、微量元素等均高于黑白花牛奶,其营养价值相当于黑白花牛奶的两倍,作为一类高级营养食品,水牛奶制品日渐成人们消费的“新宠”,在商业上具有潜在的巨大的价值。水牛肉,特别是小水牛肉,味香、鲜嫩,且脂肪含量少。另外,水牛角可以入中药。中国是水牛资源十分丰富的国家,据联合国粮农组织(FAO)统计资料,截止2003年,我国水牛饲养量为2275.9万头,已上升为世界第三位。
结核病(Tuberculosis)由结核分枝杆菌引起的人畜共患病。人接触患结核病的水牛或者食用未经消毒的患结核病水牛的奶,会引起人的结核病。水牛感染结核病,成为制约奶水牛发展的潜在威胁。到目前为止,我国水牛结核病检疫还没有一套完整可行的方法,一直是采用黄牛的检测标准进行检测,即结核菌素皮内试验(TST)。TST为法定的黄牛结核病检疫方法,应用该标准检测水牛结核病误差较大,不适合水牛的检测。因此,寻找一种新型的水牛结核病检测方法迫在眉睫。
干扰素是一类具有抗病毒、抗肿瘤和免疫调节作用的多种功能的免疫活性蛋白。γ-干扰素(IFN-γ)是Ⅱ型干扰素,IFN-γ体外释放法是目前检测结核病最具有应用前景的检测方法之一,它是在特异性抗IFN-γ单克隆抗体的基础上建立的对于待检样品中IFN-γ定性定量分析的一种实验方法。国外学者已经将抗原特异性IFN-γ试验的应用于多种动物疫病的检测。相对而言,水牛抗原特异性IFN-γ试验研究报道较少。
本研究克隆表达并纯化了水牛IFN-γ(BbIFN-γ),对其进行了抗病毒活性分析,制备了抗BbIFN-γ单克隆抗体与多克隆抗体,并在此基础上建立了检测BbIFN-γ的双抗体夹心ELISA方法。
1.水牛IFN-y的克隆与原核/酵母表达
通过Con A刺激,从水牛全血中提取BbIFN-γmRNA,根据GenBank上发布的广西沼泽型水牛的IFN-y mRNA序列设计引物,扩增出cDNA,克隆到原核表达载体pET-30a,转化BL21,经IPTG诱导,表达出大小为22kD左右的可溶性重组蛋白。
根据测序结果,把密码子改为毕赤酵母偏爱密码子并全基因合成,亚克隆到酵母穿梭质粒pPICZaA,转化酵母感受态GS115,经甲醇诱导,表达出18kD左右的蛋白。
2.水牛IFN-y抗VSV/IBRV病毒活性的鉴定
为了鉴定原核和酵母表达的蛋白是否具有抗病毒活性,检测了IFN-y抑制水疱性口炎病毒(VSV)和牛传染性鼻气管炎病毒(IBRV)在牛肾细胞系(MDBK)的增殖能力。结果显示,原核表达与酵母表达的两种蛋白的抗VSV活性分别为2.05×105U/mL和7.44×105U/mL,抗IBRV活性分别为5.22×104U/mL和2.61×105U/mL。所表达的两种重组BbINF-γ蛋白均具有抗病毒活性,原核表达的蛋白抗IBRV活性比酵母表达的偏低。
3.水牛IFN-γ单/多克隆抗体的制备与鉴定
本研究利用两种表达蛋白混合免疫Balb/C小鼠,并用两种蛋白分别筛选杂交瘤细胞株,获得五株高效价的单克隆抗体,分别命名为1C3、2C3、3E7、4G6、4H4,腹水效价分别为2.56×104、5.12×104、1.28×104、2.56×104、5.12×104。Western blot分析显示五株单抗均能特异性结合BbIFN-γ,表明五株单抗均为BbIFN-γ的特异性单抗。间接ELISA表明,McAb不与其他抗原反应,进一步证实其特异性良好。
利用两种表达蛋白混合免疫日本大耳白兔,获得兔高免血清,经纯化得到多克隆抗体,Western blot分析显示多抗能特异性结合BbIFN-γ
4.双抗体夹心ELISA方法的建立
用2C3株单抗、多抗及辣根过氧化物酶(HRP)标记的羊抗兔IgG,建立双抗体夹心ELISA方法,检测BbIFN-γ,结果其检测的灵敏度达到125pg/mL,灵敏度高。
该方法的建立为定量检测BbIFN-γ试剂盒的研制奠定了基础。该研究建立了高效的检测BbIFN-γ的免疫学方法,为水牛结核病的检测和防治奠定了基础。
Buffalo(Bubalus bubalus, Bb), Mammalia Bovidae Buffalo species, is suitable for paddy cultivation. Buffalo milk contains higher protein, amino acids, butterfat, vitamins, trace elements than in Holstein milk with nutrition value of it is almost twice of Holstein milk. As a class of high nutritious drink, it is gradually becoming the "new favorite" of human consumption. It has potentially of great commercial value. Buffalo meat, especially young buffalo meat, is characterized by fresh, delicious and low fat content. In addition, Buffalo horn can be used to make traditional Chinese medicines. Buffalo resources are abundant in China, according to FAO statistics, by the end of 2003, the amount of buffalo is 22.759 millions in China, has risen to the third in the world.
Tuberculosis is a zoonoses caused by Mycobacterium tuberculosis. Human may infect with TB from humans by contacting buffalo with TB, or drinking unpasteurized buffalo milk with TB. Buffalo TB has been the potential threat of the development of milking buffalo. So far, buffalo quarantine of TB by the use of tuberculin skin test(TST) which is widely used in dairy cattle is never calibrated. Therefore, development of a new method for the dection of buffalo tuberculosis is in urgent need.
Interferon is a class of proteins which have anti-virus, anti-tumor properties and immune regulation effect. IFN-γis interferon typeⅡ, IFN-y release in vitro to detect of TB is one of the most promising detection methods. It is a method for IFN-γqualitative and quantitative analysis based on the specific anti-IFN-y monoclonal antibody. Foreign scholars have applied antigen-specific IFN-y test to the detection of a variety of animal diseases. Relatively speaking, studies t of antigen-specific IFN-γtest of specific diseases for buffalo are rare.
In this study, we cloned, expressed, purified and analyzed IFN-γgene of buffalo(BbIFN-γ), prepared monoclonal antibody and polyclonal antibody of BbIFN-γ, established double-antibody sandwich ELISA method for BbIFN-γ.
1. Cloning and expression of the BbIFN-γin Escherichia coli and Yeast
Total RNA was isolated from buffalo peripheral blood lymphocytes, which were stimulated with Con A. Basing on sequence of Guangxi swamp type of bufflo IFN-y mRNA published in GenBank, a pair of primers were designed, cDNA was amplified and cloned into the prokaryotic expression pET-30a vector. Then after being transformed it into E. coli BL21 and induced by IPTG, soluble protein with the size of about 22kD was expressed.
According to sequencing results, the codons were changed based on Pichia pastoris codon usage bias and synthesized by the company, subcloned it into yeast shuttle plasmid pPICZaA, transformed it into yeast competent cell GS115, induced by methanol to express protein with the size of about 18kD.
2. Determination of anti-VSV and IBRV activity for BbIFN-γ
In order to identify whether the expressed of prokaryotic and yeast BbIFN-γhas antiviral activity or not, the ability to inhibit the replication of VSV or IBRV in MDBK cells was detected. Prokaryotic and yeast expression of two proteins anti-VSV activity was proved to be 2.05×105U/mL and 7.44×105U/mL, anti-IBRV virus activity was proved to be 5.22×104U/mL and 2.61×105U/mL. Although the two recombinant BbIFN-γhave anti-viral activity, the BbIFN-y from yeast yielded higher anti-IBRV activity than the prokaryotic protein.
3. Preparation and identification of BbIFN-γmonoclonal antibody
In this study, we mixed the two proteins and immunized Balb/C mice. The proteins were independently used to screen hybridoma cells able to produce monoclonal antibody to BbIFN-γ. Finally five monoclonal antibodies with high titer, named 1C3,2C3,3E7, 4G6,4H4 were obtained. The titers of ascites were 2.56×104,5.12×104,1.28×104, 2.56×104,5.12×104 respectively. Western blot analysis showed that five monoclonal antibodies can bind BbIFN-γspecifically. Indirect ELISA showed that McAbs have no reaction with other antigens, which further confirmed the specificity is good.
In addition, we mixed the two proteins and immunized rabbits, and rabbits hyperimmune sera were obtained. After purifying, polyclonal antibody IgG can be obtained. Western-blot analysis showed that polyclonal antibodies can bind BbIFN-γspecifically.
4. Establishment of double-antibody sandwich ELISA
By using McAb 2C3, PcAb and Goat anti Rabbit IgG-HRP, a double-antibody sandwich ELISA was established. The sensitivity of this method to detect BbIFN-γreached 125pg/mL, The establishment of this method laid the foundation of the development of the BbIFN-γdetection kit and diagnosis of buffalo TB or other diseases by using BbIFN-γdetection kit.
结核病(Tuberculosis)由结核分枝杆菌引起的人畜共患病。人接触患结核病的水牛或者食用未经消毒的患结核病水牛的奶,会引起人的结核病。水牛感染结核病,成为制约奶水牛发展的潜在威胁。到目前为止,我国水牛结核病检疫还没有一套完整可行的方法,一直是采用黄牛的检测标准进行检测,即结核菌素皮内试验(TST)。TST为法定的黄牛结核病检疫方法,应用该标准检测水牛结核病误差较大,不适合水牛的检测。因此,寻找一种新型的水牛结核病检测方法迫在眉睫。
干扰素是一类具有抗病毒、抗肿瘤和免疫调节作用的多种功能的免疫活性蛋白。γ-干扰素(IFN-γ)是Ⅱ型干扰素,IFN-γ体外释放法是目前检测结核病最具有应用前景的检测方法之一,它是在特异性抗IFN-γ单克隆抗体的基础上建立的对于待检样品中IFN-γ定性定量分析的一种实验方法。国外学者已经将抗原特异性IFN-γ试验的应用于多种动物疫病的检测。相对而言,水牛抗原特异性IFN-γ试验研究报道较少。
本研究克隆表达并纯化了水牛IFN-γ(BbIFN-γ),对其进行了抗病毒活性分析,制备了抗BbIFN-γ单克隆抗体与多克隆抗体,并在此基础上建立了检测BbIFN-γ的双抗体夹心ELISA方法。
1.水牛IFN-y的克隆与原核/酵母表达
通过Con A刺激,从水牛全血中提取BbIFN-γmRNA,根据GenBank上发布的广西沼泽型水牛的IFN-y mRNA序列设计引物,扩增出cDNA,克隆到原核表达载体pET-30a,转化BL21,经IPTG诱导,表达出大小为22kD左右的可溶性重组蛋白。
根据测序结果,把密码子改为毕赤酵母偏爱密码子并全基因合成,亚克隆到酵母穿梭质粒pPICZaA,转化酵母感受态GS115,经甲醇诱导,表达出18kD左右的蛋白。
2.水牛IFN-y抗VSV/IBRV病毒活性的鉴定
为了鉴定原核和酵母表达的蛋白是否具有抗病毒活性,检测了IFN-y抑制水疱性口炎病毒(VSV)和牛传染性鼻气管炎病毒(IBRV)在牛肾细胞系(MDBK)的增殖能力。结果显示,原核表达与酵母表达的两种蛋白的抗VSV活性分别为2.05×105U/mL和7.44×105U/mL,抗IBRV活性分别为5.22×104U/mL和2.61×105U/mL。所表达的两种重组BbINF-γ蛋白均具有抗病毒活性,原核表达的蛋白抗IBRV活性比酵母表达的偏低。
3.水牛IFN-γ单/多克隆抗体的制备与鉴定
本研究利用两种表达蛋白混合免疫Balb/C小鼠,并用两种蛋白分别筛选杂交瘤细胞株,获得五株高效价的单克隆抗体,分别命名为1C3、2C3、3E7、4G6、4H4,腹水效价分别为2.56×104、5.12×104、1.28×104、2.56×104、5.12×104。Western blot分析显示五株单抗均能特异性结合BbIFN-γ,表明五株单抗均为BbIFN-γ的特异性单抗。间接ELISA表明,McAb不与其他抗原反应,进一步证实其特异性良好。
利用两种表达蛋白混合免疫日本大耳白兔,获得兔高免血清,经纯化得到多克隆抗体,Western blot分析显示多抗能特异性结合BbIFN-γ
4.双抗体夹心ELISA方法的建立
用2C3株单抗、多抗及辣根过氧化物酶(HRP)标记的羊抗兔IgG,建立双抗体夹心ELISA方法,检测BbIFN-γ,结果其检测的灵敏度达到125pg/mL,灵敏度高。
该方法的建立为定量检测BbIFN-γ试剂盒的研制奠定了基础。该研究建立了高效的检测BbIFN-γ的免疫学方法,为水牛结核病的检测和防治奠定了基础。
Buffalo(Bubalus bubalus, Bb), Mammalia Bovidae Buffalo species, is suitable for paddy cultivation. Buffalo milk contains higher protein, amino acids, butterfat, vitamins, trace elements than in Holstein milk with nutrition value of it is almost twice of Holstein milk. As a class of high nutritious drink, it is gradually becoming the "new favorite" of human consumption. It has potentially of great commercial value. Buffalo meat, especially young buffalo meat, is characterized by fresh, delicious and low fat content. In addition, Buffalo horn can be used to make traditional Chinese medicines. Buffalo resources are abundant in China, according to FAO statistics, by the end of 2003, the amount of buffalo is 22.759 millions in China, has risen to the third in the world.
Tuberculosis is a zoonoses caused by Mycobacterium tuberculosis. Human may infect with TB from humans by contacting buffalo with TB, or drinking unpasteurized buffalo milk with TB. Buffalo TB has been the potential threat of the development of milking buffalo. So far, buffalo quarantine of TB by the use of tuberculin skin test(TST) which is widely used in dairy cattle is never calibrated. Therefore, development of a new method for the dection of buffalo tuberculosis is in urgent need.
Interferon is a class of proteins which have anti-virus, anti-tumor properties and immune regulation effect. IFN-γis interferon typeⅡ, IFN-y release in vitro to detect of TB is one of the most promising detection methods. It is a method for IFN-γqualitative and quantitative analysis based on the specific anti-IFN-y monoclonal antibody. Foreign scholars have applied antigen-specific IFN-y test to the detection of a variety of animal diseases. Relatively speaking, studies t of antigen-specific IFN-γtest of specific diseases for buffalo are rare.
In this study, we cloned, expressed, purified and analyzed IFN-γgene of buffalo(BbIFN-γ), prepared monoclonal antibody and polyclonal antibody of BbIFN-γ, established double-antibody sandwich ELISA method for BbIFN-γ.
1. Cloning and expression of the BbIFN-γin Escherichia coli and Yeast
Total RNA was isolated from buffalo peripheral blood lymphocytes, which were stimulated with Con A. Basing on sequence of Guangxi swamp type of bufflo IFN-y mRNA published in GenBank, a pair of primers were designed, cDNA was amplified and cloned into the prokaryotic expression pET-30a vector. Then after being transformed it into E. coli BL21 and induced by IPTG, soluble protein with the size of about 22kD was expressed.
According to sequencing results, the codons were changed based on Pichia pastoris codon usage bias and synthesized by the company, subcloned it into yeast shuttle plasmid pPICZaA, transformed it into yeast competent cell GS115, induced by methanol to express protein with the size of about 18kD.
2. Determination of anti-VSV and IBRV activity for BbIFN-γ
In order to identify whether the expressed of prokaryotic and yeast BbIFN-γhas antiviral activity or not, the ability to inhibit the replication of VSV or IBRV in MDBK cells was detected. Prokaryotic and yeast expression of two proteins anti-VSV activity was proved to be 2.05×105U/mL and 7.44×105U/mL, anti-IBRV virus activity was proved to be 5.22×104U/mL and 2.61×105U/mL. Although the two recombinant BbIFN-γhave anti-viral activity, the BbIFN-y from yeast yielded higher anti-IBRV activity than the prokaryotic protein.
3. Preparation and identification of BbIFN-γmonoclonal antibody
In this study, we mixed the two proteins and immunized Balb/C mice. The proteins were independently used to screen hybridoma cells able to produce monoclonal antibody to BbIFN-γ. Finally five monoclonal antibodies with high titer, named 1C3,2C3,3E7, 4G6,4H4 were obtained. The titers of ascites were 2.56×104,5.12×104,1.28×104, 2.56×104,5.12×104 respectively. Western blot analysis showed that five monoclonal antibodies can bind BbIFN-γspecifically. Indirect ELISA showed that McAbs have no reaction with other antigens, which further confirmed the specificity is good.
In addition, we mixed the two proteins and immunized rabbits, and rabbits hyperimmune sera were obtained. After purifying, polyclonal antibody IgG can be obtained. Western-blot analysis showed that polyclonal antibodies can bind BbIFN-γspecifically.
4. Establishment of double-antibody sandwich ELISA
By using McAb 2C3, PcAb and Goat anti Rabbit IgG-HRP, a double-antibody sandwich ELISA was established. The sensitivity of this method to detect BbIFN-γreached 125pg/mL, The establishment of this method laid the foundation of the development of the BbIFN-γdetection kit and diagnosis of buffalo TB or other diseases by using BbIFN-γdetection kit.
引文
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28.赵翔,霍克克,李育阳.毕赤酵母的密码子用法分析.生物工程学报,2000,16(3)：308-311.
29.周鹏,郭安平,沈文涛,黎小瑛,梁国栋.毕赤氏酵母SMD1168/HuIFNα-2b分泌型表达的研究.药物生物技术,2003,10(5)：287-291.
30.朱冬冬.牛IFN-γ单克隆抗体的研制与抗体夹心ELISA检测牛结核方法的建立及初步应用.[硕士学位论文].扬州：扬州大学图书馆,2007
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24.余大海,韦一然,蔡宏,朱玉贤.牛结核病鉴别诊断的方法研究.中国人兽共患病学报,2006,22(10)：932-935.
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26.张桂荣.牛结核病特异抗体间接ELISA检测方法的建立及初步应用.[硕士学位论文].武汉：华中农业大学图书馆,2006
27.张君,于三科..干扰素-γ介导的抗弓形虫作用及其机理.动物医学进展,2000,1(1)：25-27.
28.赵翔,霍克克,李育阳.毕赤酵母的密码子用法分析.生物工程学报,2000,16(3)：308-311.
29.周鹏,郭安平,沈文涛,黎小瑛,梁国栋.毕赤氏酵母SMD1168/HuIFNα-2b分泌型表达的研究.药物生物技术,2003,10(5)：287-291.
30.朱冬冬.牛IFN-γ单克隆抗体的研制与抗体夹心ELISA检测牛结核方法的建立及初步应用.[硕士学位论文].扬州：扬州大学图书馆,2007
31. Adah SA, Bayly SF, Cramer H, Silverman RH, Tonence PF. Chemistry and biochemistry of 2',5'-oligoadenylate-based antisense strategy. Curr Med Chem,2001, 8(10):1189-1212.
32. Aderem A, Underhill D M. Annu Rev Immunol,1999,17:593-623.
33. Amadori M, Tameni S, Scaccaglia P, et al. Antibody Tests for Identification of Mycobacterium Bovis-Infected Bovine Herds. J Clin Microbiol.1998,36(2): 566-568.
34. Amieosante M, Houde M, Guaraldi G, Saltini C. Sensitivity and specificity of a multi-antigen ELISA test for the serological diagnosis of tuberculosis. IntJ Tuberc Lung Dis.1999,3(8):736-740.
35. Andersen P, Munk ME, Polloek JM, Doherty TM. Specific immune-based diagnosis of tuberculosis. Lancet,2000,335(9235):1099-1104.
36. Basler CF, Garcia-Sastre A. Viruses and the type I interferon antiviral system: induction and Int Rev Immunol,2002,21(4-5):305-337.
37. Becker DM, Guarente L. High-efficiency transformation of yeast by electroporation. Methods Enzymol,1991,194:182.
38. Bengis RG, Kriek NP, Keet DF, Raath JP, de Vos V, Huchzermeyer HF. An outbreak of bovine tuberculosis in a free-living African buffalo(Syncerus caffer-sparrman) population in the Kruger National Park:a preliminary report. Onderstepoort J Vet Res.1996,63(1):15-18.
39. Bonjardim CA. Interferons (IFIVs) are immune responses-and viruses cytokines in both innate and adaptive antiviral action. Microbes Infect,2005,7(3):569-578.
40. Cerretti DP, Mckereghan K, Larsen A, Cosman D, Gillis S, Baker PE. Cloning, sequence and expression of bovine interferon-gamma. Immunol,1986,136(12): 4561-4564.
41. Cho YS, Jung SC, Kim JM, Yoo HS. Enzyme-linked immunosorbent assay of bovine tuberculosis by crude mycobacterial protein 70. J Immunoassay Immunochem.2007, 28(4):409-418.
42. Clarke CJ, Trapani JA, Johnstone RW. Mechanisms of interferon mediated anti-viral resistance. Curr Drug Targets Immune Endocr Metabol Disord,2001,1(2):117-130.
43. Colarow L, Turini M, Teneberg S, Berger A. Characterization and Biological Activity of Gangliosides in buffalo Milk. Biochimica et Biophysica Acta,2003, 1631:94-106.
44. Cregg JM, Tschopp JF, Stillman C, et al. Biotechnolog,2001,5(4):479-485.
45. Dockrell HM, Weir RE. Whole blood cytokine assay s-a new generation of diagnostic tests for tuberculosis?. Int J Tuberc Lung Dis,1998,2(6):441-442.
46. Dolin PJ, Raviglione MC, Kochi A. Global tuberculosis incidence and mortality during 1990-2000. Bull World Health Organ,1994,72(2):213-220.
47. Dominguez CM. The interferon and the antiviral defense. An R Acad Nac Med (Madr),2006,123(2):321-339.
48. Dye C, Scheele S, Dolin P, Pathania V, Raviglione MC. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. Journal of American Medical Association,1999,282(7):677-686.
49. Gao Q, Kripk K, Arinc Z, Voskuil M, Small P. Comparative expression studies of a complex phenotype:Cord formation in mycobacterium tubeculosis. Tuberculosis, 2004,84:188-196.
50. Giuseppina A, Enrico T, Andrea M. Characterization of Buffalo Milk by Pnuclear Magnetic Resonance Spectroscopy. Journal of Food Composition and Analysis,2006, 19:843-849.
51. Gormley E, Doyle MB, Fitzsimon T, McGill K, Collins JD. Diagnosis of Mycobacterium bovis infection in cattle by use of the gamma-interferon(Bovigam) assay. Vet Microbiol,2006,112(24):171-179.
52. Gosling JP. Immunoassays A Practical Approach.America:Oxford University Press. 2000:41.
53. Han BZ, Meng Y, Li M, Yang YX, Ren FZ, Zeng QK, Nout MJR A Survey on the Microbiological and Chemical Composition of Buffalo Milk in China. Food Control, 2007,18:742-746.
54. Harrington NP, Surujballi OP, Prescott JF, Duncan JR, Waters WR, Lyashchenko K, Greenwald R. Antibody responses of cervids(Cervus elaphus) following experimental Mycobacterium bovis infection and the implications for immunodiagnosis. Clin Vaccine Immunol.2008,15(11):1650-1658.
55. Haus O. The genes of interferons and interferon-related factors:localization and relationships with chromosome aberrations in cancer. Arch Immunol Ther Exp (Warsz),2000,48(2):95-100.
56. Heim MH. Intracellular signalling and antiviral effects of interferons. Dig Liver Dis, 2000,32(3):257-263.
57. Hein WR, Tomasovic AA. An abattoir survey of tuberculosis in feral buffalo. Australian Veterinary Journal,1981,57(12):543-547.
58. Hurlock EC. Interferons:ntial roles in affect. Med Hypotheses,2001,56(5):558-566.
59. Issacs A, Lindenmann J. Virus interference:I. The interferon. Proc R Soc Lond Ser, 1957,147(927):258-263.
60. Jasmer RM, Nahid P, Hopewell PC. Clinical practice Latent tuberculosis infection. N Engl J Med,2002,347(23):1860-1866.
61. Kanameda M, Ekgatat M. Isolation of mycobacterium bovis from the water buffalo(Bubalus bubalis). Tropical Animal Health and Production,1995,27(4): 227-228.
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