牛白细胞介素2基因克隆、表达及多克隆抗体的制备
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摘要
牛白细胞介素2主要是由辅助性T淋巴细胞产生的一种淋巴因子,通过免疫网络调控机体免疫功能,在机体免疫反应中起着极为重要的作用。因其具有免疫增强剂、抗肿瘤和抗感染的特点是理想的免疫佐剂和免疫治疗剂。但正常情况下,机体产生的BoIL-2的量非常有限,一定程度上限制了对BoIL-2的研究和应用。本实验主要应用重组DNA和基因表达技术,通过RT-PCR方法克隆BoIL-2基因,并对其基因序列进行分析和表达,旨在为大量制备和生产高效价廉的BoIL-2,为其生物学活性及应用研究奠定基础。
根据Genbank发表的BoIL-2基因序列,设计了一对引物。以ConA刺激的外周血淋巴细胞(PMN)为材料,提取总RNA,应用RT-PCR的方法,扩增BoIL-2基因序列。琼脂糖凝胶电泳显示目的片段约为477bp。测序结果显示,克隆的BoIL-2基因与Genbank发表的BoIL-2基因序列同源性为98.7%,氨基酸同源性为99.4%。
将测序正确的pMD-18-T-IL-2重组质粒及pET30a(+)载体,用EcoRI及SalI进行双酶切,分别回收后T4连接酶进行连接,转化、提取重组质粒。通过双酶切及PCR鉴定阳性的重组质粒进行序列测定后在大肠杆菌的表达,分子量约为23.49ku;表达产物主要分布在包涵体中。Western Blot证实所得到的重组蛋白为BoIL-2重组蛋白。通过MTT法测定BoIL-2重组蛋白的活性,可知重组BoIL-2(rBoIL-2)对牛及山羊淋巴细胞有明显作用,对猪淋巴细胞作用不明显。用镍离子亲和树脂对所得的BoIL-2重组蛋白进行纯化,并免疫新西兰兔成功制备兔抗rBoIL-2多克隆抗体,为下阶段的研究提供了重要的实验材料。
IVBovine Interleukin-2 (BoIL-2), which has the ability to regulate the immune response through immune network, is mainly secreted by T helper cell. BoIL-2 is an usful adjuvant and therapeutics in control of diseases. As these features, it was extensively used in treatment of malignant tumors and infectious diseases. Naturally, the production of BoIL-2 in animal body is limited in amount and thus confined its study and application. With modern genetic engineering technique, BoIL-2 gene was cloned using reverse transcription polymerase chain reaction (RT-PCR) method, expressed with prokaryotic plasmid and prepared its polyclonal antibody. This would provide solid basis for the studying of its biological activities and application.
Specific primers for bovine IL-2 cDNA were designed and synthesized according to the previously published sequence in Genebank. The total cell RNA, isolated from ConA-stimulated peripheral blood lymphocyte of Bovine, was used as template to generate complementary DNA by reverse transcription. The 477bp DNA fragments were amplified by polymerase chain reaction (PCR), and cloned into the pMD-18-T vector. The fragment was confirmed bovine interleukin-2 by DNA sequencing. The sequence analysis showed that it was similar to the sequence of bovine IL-2 publised in Genbank, the homology was 98.7% in nucleotide acid and 99.4% in amino acid.
Then the prokaryotic expression plasmid pET30a/Rcap IL-2 was constructed and transformed into BL21 cell. The recombinant rBoIL-2 was expressed efficiently in forms of inclusion body induced by IPTG. SDS-PAGE and western blot analysis showed that the recombinant fusion protein had a molecular weight 23ku. The inclusion body was solubilized by 8M Urea and purified by QIAGEN Ni-NTA resin. After annealing treatment of the recombinant protein with urea, its biological activity was tested with MTT assay. The results demonstrated that it had the biological activity to sustain the proliferation of bovine and goat peripheral blood lymphocytes stimulated with ConA preliminary, however no effect to that of swine. The protein was purified and used to immunize rabbit, and polyconal antibody was prepared and identificated by AGP and western blot.
In general, the cloning, expression of bovine interleukin 2 gene and the preparation of its polyclonal antibody will provide solid basis for the biological activity study and application of bovine interleukin 2.
根据Genbank发表的BoIL-2基因序列,设计了一对引物。以ConA刺激的外周血淋巴细胞(PMN)为材料,提取总RNA,应用RT-PCR的方法,扩增BoIL-2基因序列。琼脂糖凝胶电泳显示目的片段约为477bp。测序结果显示,克隆的BoIL-2基因与Genbank发表的BoIL-2基因序列同源性为98.7%,氨基酸同源性为99.4%。
将测序正确的pMD-18-T-IL-2重组质粒及pET30a(+)载体,用EcoRI及SalI进行双酶切,分别回收后T4连接酶进行连接,转化、提取重组质粒。通过双酶切及PCR鉴定阳性的重组质粒进行序列测定后在大肠杆菌的表达,分子量约为23.49ku;表达产物主要分布在包涵体中。Western Blot证实所得到的重组蛋白为BoIL-2重组蛋白。通过MTT法测定BoIL-2重组蛋白的活性,可知重组BoIL-2(rBoIL-2)对牛及山羊淋巴细胞有明显作用,对猪淋巴细胞作用不明显。用镍离子亲和树脂对所得的BoIL-2重组蛋白进行纯化,并免疫新西兰兔成功制备兔抗rBoIL-2多克隆抗体,为下阶段的研究提供了重要的实验材料。
IVBovine Interleukin-2 (BoIL-2), which has the ability to regulate the immune response through immune network, is mainly secreted by T helper cell. BoIL-2 is an usful adjuvant and therapeutics in control of diseases. As these features, it was extensively used in treatment of malignant tumors and infectious diseases. Naturally, the production of BoIL-2 in animal body is limited in amount and thus confined its study and application. With modern genetic engineering technique, BoIL-2 gene was cloned using reverse transcription polymerase chain reaction (RT-PCR) method, expressed with prokaryotic plasmid and prepared its polyclonal antibody. This would provide solid basis for the studying of its biological activities and application.
Specific primers for bovine IL-2 cDNA were designed and synthesized according to the previously published sequence in Genebank. The total cell RNA, isolated from ConA-stimulated peripheral blood lymphocyte of Bovine, was used as template to generate complementary DNA by reverse transcription. The 477bp DNA fragments were amplified by polymerase chain reaction (PCR), and cloned into the pMD-18-T vector. The fragment was confirmed bovine interleukin-2 by DNA sequencing. The sequence analysis showed that it was similar to the sequence of bovine IL-2 publised in Genbank, the homology was 98.7% in nucleotide acid and 99.4% in amino acid.
Then the prokaryotic expression plasmid pET30a/Rcap IL-2 was constructed and transformed into BL21 cell. The recombinant rBoIL-2 was expressed efficiently in forms of inclusion body induced by IPTG. SDS-PAGE and western blot analysis showed that the recombinant fusion protein had a molecular weight 23ku. The inclusion body was solubilized by 8M Urea and purified by QIAGEN Ni-NTA resin. After annealing treatment of the recombinant protein with urea, its biological activity was tested with MTT assay. The results demonstrated that it had the biological activity to sustain the proliferation of bovine and goat peripheral blood lymphocytes stimulated with ConA preliminary, however no effect to that of swine. The protein was purified and used to immunize rabbit, and polyconal antibody was prepared and identificated by AGP and western blot.
In general, the cloning, expression of bovine interleukin 2 gene and the preparation of its polyclonal antibody will provide solid basis for the biological activity study and application of bovine interleukin 2.
引文
陈吉刚,陈维虎,周继勇,王金勇,齐静,郑肖娟. 2005.重组鸡白细胞介素2的诱导表达与生物学活性测定[J].中国农业科学. 38 (5):1034~1039.
耿建军,赫晓燕. 2002.细胞因子与兽医学.畜牧兽医杂志. 21 (1):21~23.
龚非力. 2000.医学免疫学[M].科学出版社.71~73
洪云平,文心田. 2000.细胞因子及其在兽医上的应用[J].预防兽医学进展. 26(5):32~34.
胡慧琼,王红宁,袁斌. 2006.鸡IL-2和IL-15基因真核表达质粒对禽流感疫苗的免疫增强作用[J].中国兽医科学. 36 (10):805~810.
金伯泉.2001.细胞和分子免疫学[M].科学出版社. 125~130
李祥瑞,金红. 1999.鸡IL-2cDNA克隆,南京农业大学学报. 17(1):16~19
刘胜旺,孔宪刚. 2002.新发现的几种禽细胞因子及其研究进展[J].中国预防兽医学. 24(2):153~155.
潘洪波. 1995.用牛γ-干扰素实验检测皮内试验阴性牛的牛分枝杆菌感染[J].畜禽传染病. 15 (2):43~44.
彭贵勇,庞政. 2001.白细胞介素2对人肝癌细胞侵袭力及转移力的调节作用[J].中华肝脏病杂志. 5:303～305.
万建青,吴文学,夏春. 2002.毕赤酵母表达猪干扰素-γ基因及其抑制蓝耳病病毒效果[J]. 生物工程学报. 18(6):683～686.
许万祥. 1996.细胞因子在牛病防治上的应用前景[J].甘肃畜牧兽医. (6):27～30.
徐前明,李培英,查日华. 2005.鸡白介素2对柔嫩艾美耳球虫亚单位疫苗免疫增强作用的研究[J].安徽农业科学. 33 (2):290~293.
叶秀华,蔡建平,吴志光,汪明. 2004.重组鸡γ干扰素的抗球虫作用[J].中国兽医杂志. 40 (7):3~5.
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Arunachalam B, Subba Rao M V, Ram G C, et al. 1989. Bovine interleukin 2 biochemical and biological characterization[J]. Vet Immunol Immunopathol . 23(3-4):377~383
Alice K P, Jorge A T. 2002. Therapeutic use of interleukin-2 in HIV-infected patients[J]. Current Opinion In Pharmacology. 2: 433~439.
Babiak L A. 1985. Effect of bovine a interferon on bovine herpes virus type1-induce respiratory disease[J]. Gen Virol. 66(8):2382~2384.
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Chinsangaram J, Moraes M P, Koster M, Grubman M J. 2003. Novel viral disease control strategy: adeno virus expressing alpha interferon rapidly protects swine from foot and mouth disease[J]. Virol. 77(2):1621~1625.
Dubaniewicz A, Trzonkowski P, Dubaniewicz-Wybieralska M, Dubaniewicz A, Singh M, My?liwski A. 2007. Mycobacterial heat shock protein-induced blood T lymphocytes subsets and cytokine pattern: Comparison of sarcoidosis with tuberculosis and healthy controls[J]. Respirology. 12(3):346~354.
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龚非力. 2000.医学免疫学[M].科学出版社.71~73
洪云平,文心田. 2000.细胞因子及其在兽医上的应用[J].预防兽医学进展. 26(5):32~34.
胡慧琼,王红宁,袁斌. 2006.鸡IL-2和IL-15基因真核表达质粒对禽流感疫苗的免疫增强作用[J].中国兽医科学. 36 (10):805~810.
金伯泉.2001.细胞和分子免疫学[M].科学出版社. 125~130
李祥瑞,金红. 1999.鸡IL-2cDNA克隆,南京农业大学学报. 17(1):16~19
刘胜旺,孔宪刚. 2002.新发现的几种禽细胞因子及其研究进展[J].中国预防兽医学. 24(2):153~155.
潘洪波. 1995.用牛γ-干扰素实验检测皮内试验阴性牛的牛分枝杆菌感染[J].畜禽传染病. 15 (2):43~44.
彭贵勇,庞政. 2001.白细胞介素2对人肝癌细胞侵袭力及转移力的调节作用[J].中华肝脏病杂志. 5:303～305.
万建青,吴文学,夏春. 2002.毕赤酵母表达猪干扰素-γ基因及其抑制蓝耳病病毒效果[J]. 生物工程学报. 18(6):683～686.
许万祥. 1996.细胞因子在牛病防治上的应用前景[J].甘肃畜牧兽医. (6):27～30.
徐前明,李培英,查日华. 2005.鸡白介素2对柔嫩艾美耳球虫亚单位疫苗免疫增强作用的研究[J].安徽农业科学. 33 (2):290~293.
叶秀华,蔡建平,吴志光,汪明. 2004.重组鸡γ干扰素的抗球虫作用[J].中国兽医杂志. 40 (7):3~5.
于康震,卢景良,谷守林. 1989.牛外周血T, B和单核细胞的分离、鉴定及扫描电镜观察[J].中国兽医科技. 12:5~7.
余传霖,叶天星,陆德源. 1998.现代医学免疫学[M].上海医科大学出版社. 132~133.
郑乃智. 1997.癫痫的白细胞介素-2研究进展[J].医学研究通讯. 26: 5~6.
Avendano S C. 2006. Interferons: types and actions. Gastroenterol Hepatol Oct; 29(2):125~128
Arunachalam B, Subba Rao M V, Ram G C, et al. 1989. Bovine interleukin 2 biochemical and biological characterization[J]. Vet Immunol Immunopathol . 23(3-4):377~383
Alice K P, Jorge A T. 2002. Therapeutic use of interleukin-2 in HIV-infected patients[J]. Current Opinion In Pharmacology. 2: 433~439.
Babiak L A. 1985. Effect of bovine a interferon on bovine herpes virus type1-induce respiratory disease[J]. Gen Virol. 66(8):2382~2384.
Collins R A, Tayton H K, Gelder K I, et al. 1994. Cloning and expression of bovine and porcine interleukin-2 in baculovirus and analysis of species cross-reactivity[J]. Vet Immunol Immunopathol. 40(4):313~24.
Choi K D, Lillehoj H S. 2000. Role of chicken IL-2 on gamma delta T-cells and Eimeria acervulina-induced changes in intestinal IL-2 mRNA expression and gamma delta T-cells[J]. Veterinary Immunology and Immunopathology. 73(3-4):309~321.
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