猪囊尾蚴抗原基因GP50的克隆、表达及鉴定
|
摘要
目的：从猪囊尾蚴cDNA中扩增出GP50编码基因，亚克隆至真核表达载体，利用所得的重组蛋白，为建立一种灵敏检测猪囊尾蚴感染的实验方法作准备。方法：设计并合成引物，从猪囊尾蚴cDNA文库中PCR扩增GP50编码基因，通过与线性克隆载体pGEM-T连接，经酶切和PCR鉴定及DNA测序证实后，将目的基因亚克隆入双表达载体pBK-CMV，将获得的pBK-CMV-GP50阳性重组子转化宿主菌E．coli BL21，IPTG诱导表达，Western blot鉴定。结果：PCR法扩增出了一897bp左右的DNA片断，将重组质粒pGEM-T-GP50、pBK-CMV-GP50分别作BamH Ⅰ和Xho Ⅰ双酶切，均能得到一个大小与PCR扩增产物一致的插入片断，对插入片断的测序结果表明，GP50具有一个长度为897bp的完整开放阅读框(ORF)，编码298个氨基酸，理论分子量33kDa，与GenBank收录(编号为AY212945)的猪囊尾蚴GP50基因具有高度的同源性(99％)。表明GP50基因的克隆和亚克隆均获成功，用IPTG诱导带有重组质粒pBK-CMV-GP50的大肠杆菌，产物行SDS-PAGE，可得到一约36kDa融合蛋白，Western-blot法鉴定该蛋白能被猪囊尾蚴感染的人血清所识别。
Objective: To genernate a quick and accurate detection of Cysticercosis by use of recombinant protein, GP50 was amplified from Cysticercus cellulosae and subcoloned into the expression vector pBK-CMV. Methods: The specific primers were designed and synthesized. The DNA fragment encoding GP50 was amplified by PCR from cDNA of C.cellulosae. The PCR products were cloned into pGEM-T vector and recombinants were screened by PCR, risfriction digestion followed by sequencing. Then the GP50 was subcloned into pBK-CMV expression vector and the recombinant plasmid was transformed into E. coli BL21,IPTG was added to induce fusion expression of fused GP50. The expression was identified by Western blot. Results: The size of pGEM-T-GP50,pBK-CMV-GP50 digested with BamH I and Xho I was identical to the length of the PCR generated products. DNA sequencing indicated that the GP50 gene has an open reading frame of 897bp,which encodes 298 amino acids with an approximate molecular weight of 33kDa and has high homology with the G
P50 gene submitted to GenBank (AY212945). As consequence GP50 gene was successfully cloned and subcloned into the vectors. With the positive clones induced by IPTG, a fusion protein was expressed in E.coli BL21. The fusion protein could be recognized by the sera of patients infected with Cysticercus cellulosae.
Objective: To genernate a quick and accurate detection of Cysticercosis by use of recombinant protein, GP50 was amplified from Cysticercus cellulosae and subcoloned into the expression vector pBK-CMV. Methods: The specific primers were designed and synthesized. The DNA fragment encoding GP50 was amplified by PCR from cDNA of C.cellulosae. The PCR products were cloned into pGEM-T vector and recombinants were screened by PCR, risfriction digestion followed by sequencing. Then the GP50 was subcloned into pBK-CMV expression vector and the recombinant plasmid was transformed into E. coli BL21,IPTG was added to induce fusion expression of fused GP50. The expression was identified by Western blot. Results: The size of pGEM-T-GP50,pBK-CMV-GP50 digested with BamH I and Xho I was identical to the length of the PCR generated products. DNA sequencing indicated that the GP50 gene has an open reading frame of 897bp,which encodes 298 amino acids with an approximate molecular weight of 33kDa and has high homology with the G
P50 gene submitted to GenBank (AY212945). As consequence GP50 gene was successfully cloned and subcloned into the vectors. With the positive clones induced by IPTG, a fusion protein was expressed in E.coli BL21. The fusion protein could be recognized by the sera of patients infected with Cysticercus cellulosae.
引文
[1] 周开学 我国猪带绦／猪囊尾蚴病的流行病学及防 中国寄生虫病防治杂志，1998，11(4)：344-345
[2] 吕广，韩梅，孟庆宝，等 猪囊尾蚴病的流行与治疗现状．内蒙古民族大学学报(自然版)，2002，17(1)：79-80
[3] 王敏，徐之杰，李雅杰，等 猪囊尾蚴重组体的培养及其诱导表达 哈尔滨医科大学学报，1998，32(1)：15-17
[4] 苏彩霞，才学鹏，韩学清，等 猪囊尾蚴磷蛋白在毕赤酵母中的表达及初步应用．生物工程学报，2003，19(4)：424-427
[5] 张莉，刘殿武 猪囊尾蚴线粒体NADH脱氢酶的基因克隆 中国寄生虫学与寄生虫病杂志，2003，21(1)34-36
[6] 沈斌，高荣，孟民杰，等 猪带绦虫抗原基因 TS76的克隆及原核表达 中国人兽共患病杂志，2002，18(2)：61-63
[7] 彭郁葱，陈蕊雯，孙树汉，等 以融合蛋白为抗原的简化Westernblot法诊断囊虫病[J] 中国寄生虫学与寄生虫病杂志，1998，16(5)360-363
[8] Chang JY, Young YB, Sun H, et al. A recombinant 10-KD of Taenia solium metacestode specific to active neurocydticercosis [J].J Infect Dis, 1999, 180:1307-15
[9] Fleury A, Beltran C, Ferrer E, et al. Application of synthetic peptides to the diagnosis of neurocysticercosis. Trop Med Int Health. 2003, 8(12): 1124-1130
[10] Tsang VC, Brand JA,boyer AE .An enzyme-linked immunoelectrotransfer blot assay and glycoprotein for diagnosis human cysticercosis (taenea solium).J Infect Dis, 1989, 159(1):50-9
[11] Tsang VC, Pilcher JA, Zhou W, et al Efficacy of the immunoblot assay for cysticercosis pigs and modulated expression of distinct IgM/IgG activities to Taenia solium antigens in experiment infection.Vet Immunopathol 1991 Aug;29(1-2):69-78
[12] Kathy Hancock, Sowmya Pattabhi, Kyan M, et al, Characterization and clong of GP50, a Taenia solium antigen diagnostic for cysticercusis, Molecular &Biochemical Parasitology, 2004, 133:115-124
[13] Tachado SD, Mazhari-Tabrizi R, Schofield L, Specificity in signal transduction among glycosylphosphatidylinositols of Plasmodium falciparum,Trypanosoma brucei, Trypanosoma cruzi and leishmania spp.Parasite Immunol. 1999 Dec;21 (12):609-17
[14] Lesbron-Delauw MF et al.J Bio Chem, 1994;268(23): 16217-22
[15] 顾善兰 糖基化磷脂酰肌醇特异性磷脂酶 D 的研究进展 国外医学分子生物学分册，2002，24(1)：56-58
[16] 郑福英，刁有祥，李庆宫 我国猪囊虫病的研究进展 中国兽医寄生虫病，2003，11(2)：55-57
[17] 孙树汉，王俊霞，陈蕊雯，等 囊虫病诊断用抗原编码cDNA的分子克隆 中国寄生虫学与寄生虫病杂志，1997，15(1)：15-20
[18] Yasuhito Sako, Minoru Nakao, Takashi Ikejima, et al. Molecular Characterization and Diagnostic Value of Taenia solium Low-Molecular-Weight Antigen Genes. Journal of Clinical Microbiology, 2000, 38(12):4439-4444
[19] 陈颖，刘淼，薛海筹，等 猪囊尾蚴特异抗原的基因克隆和表达 中国血吸虫病防治杂志，2000，12(5)284-285
[20] 陈蕊雯，林懿，孙树汉 猪囊尾蚴抗原CC1在大肠杆菌中的克隆及高效表达 中国寄生虫学与寄生虫病杂志，2000，18(1)37-39
[21] 杨湘越，孙树汉，陈蕊雯，等 猪囊尾蚴抗原CC1在巴斯德毕赤酵母中的表达中国人兽共患病杂志，2002，18(3)31-33
[22] 吴丹，孙树汉，，郭瀛军，等 猪囊尾蚴抗原与γ干扰素融合蛋白在大肠杆菌中的表达 中国寄生虫学与寄生虫病杂志，1999，17(4)：215—217
[23] 王庆敏，戴建新，张平武，等 猪囊尾蚴抗原B基因cDNA编码区的克隆 中国寄生虫学与寄生虫病杂志，2000，18(2)73-75
[24] 刘新月 膜蛋白的糖脂锚及其临床 武汉医学杂志，1996，20(1)：49-50
[25] 刘永全 整和素与信号转导 国外医学免疫分册，2000，23(1)：34-37
[26] Breathnach R, Chambon P. Organization and expression of eukaryotic split genes coding for proteins. Annu Rev Biochem 1981 ;50:349-83.
[27] Levine MZ, Sanchez CC, Wilkins PP, et al. Characterization, cloning and
expression of two diagnostic antigens for Taenia solium tapeworm infection. J Parasitol, in press.
[28] Restrepo BI, Obregon-Henao A, Mesa M, et al. Characterization of the carbohydrate components of Taenia solium metacestode glycoprotein antigens. Int J Parasitol 2000;30:689-96.
[29] Obregon-Henao A, Gil DL, Gomez DI, et al The role of N-linked carbohydrates in the antigenicity of Taenia solium metacestode glycoproteins of 12, 16 and 18 kDa. Mol Biochem Parasitol 2001; 114:209-15.
[30] Butikofer P, Malherbe T, Boschung M, Roditi I. GPI-anchored proteins: now you see' em, now you don't. FASEB J 2001;15:545-8.
[31] Kukulansky T , Abramovitch S , Hollander N. Cleavage of the glycosylphosphatidylinositol anchor affects the reactivity of Thy-1 with antibodies. J Immunol 1999; 162:5993-7.
[32] Barboni E, Rivero BP, George AJT, et al. The glycophosphatidylinositol anchor affects the conformation of Thy-1 protein. J Cell Sci, 1995;108:487-97.
[33] Martinez AP, Margos G, Barker G, et al. The roles of the glycosylphosphatidylinositol anchor on the production and immunogenicity of recombinant ookinete surface antigen Pbs21 of Plasmodium berghei when prepared in a baculovirus expression system. Parasite Immunol 2000;22:493-500.
[34] Striepen B, Zinecker CF, Damm JBL, et al. Molecular structure of the "low molecular weight antigen" of Toxoplasma gondii: a glucose alpha 1-4 N-acetlygalactosamine makes free glycosyl-phosphatidylinositols highly immunogenic. J Mol Biol 1997;266:797-813.
[2] 吕广，韩梅，孟庆宝，等 猪囊尾蚴病的流行与治疗现状．内蒙古民族大学学报(自然版)，2002，17(1)：79-80
[3] 王敏，徐之杰，李雅杰，等 猪囊尾蚴重组体的培养及其诱导表达 哈尔滨医科大学学报，1998，32(1)：15-17
[4] 苏彩霞，才学鹏，韩学清，等 猪囊尾蚴磷蛋白在毕赤酵母中的表达及初步应用．生物工程学报，2003，19(4)：424-427
[5] 张莉，刘殿武 猪囊尾蚴线粒体NADH脱氢酶的基因克隆 中国寄生虫学与寄生虫病杂志，2003，21(1)34-36
[6] 沈斌，高荣，孟民杰，等 猪带绦虫抗原基因 TS76的克隆及原核表达 中国人兽共患病杂志，2002，18(2)：61-63
[7] 彭郁葱，陈蕊雯，孙树汉，等 以融合蛋白为抗原的简化Westernblot法诊断囊虫病[J] 中国寄生虫学与寄生虫病杂志，1998，16(5)360-363
[8] Chang JY, Young YB, Sun H, et al. A recombinant 10-KD of Taenia solium metacestode specific to active neurocydticercosis [J].J Infect Dis, 1999, 180:1307-15
[9] Fleury A, Beltran C, Ferrer E, et al. Application of synthetic peptides to the diagnosis of neurocysticercosis. Trop Med Int Health. 2003, 8(12): 1124-1130
[10] Tsang VC, Brand JA,boyer AE .An enzyme-linked immunoelectrotransfer blot assay and glycoprotein for diagnosis human cysticercosis (taenea solium).J Infect Dis, 1989, 159(1):50-9
[11] Tsang VC, Pilcher JA, Zhou W, et al Efficacy of the immunoblot assay for cysticercosis pigs and modulated expression of distinct IgM/IgG activities to Taenia solium antigens in experiment infection.Vet Immunopathol 1991 Aug;29(1-2):69-78
[12] Kathy Hancock, Sowmya Pattabhi, Kyan M, et al, Characterization and clong of GP50, a Taenia solium antigen diagnostic for cysticercusis, Molecular &Biochemical Parasitology, 2004, 133:115-124
[13] Tachado SD, Mazhari-Tabrizi R, Schofield L, Specificity in signal transduction among glycosylphosphatidylinositols of Plasmodium falciparum,Trypanosoma brucei, Trypanosoma cruzi and leishmania spp.Parasite Immunol. 1999 Dec;21 (12):609-17
[14] Lesbron-Delauw MF et al.J Bio Chem, 1994;268(23): 16217-22
[15] 顾善兰 糖基化磷脂酰肌醇特异性磷脂酶 D 的研究进展 国外医学分子生物学分册，2002，24(1)：56-58
[16] 郑福英，刁有祥，李庆宫 我国猪囊虫病的研究进展 中国兽医寄生虫病，2003，11(2)：55-57
[17] 孙树汉，王俊霞，陈蕊雯，等 囊虫病诊断用抗原编码cDNA的分子克隆 中国寄生虫学与寄生虫病杂志，1997，15(1)：15-20
[18] Yasuhito Sako, Minoru Nakao, Takashi Ikejima, et al. Molecular Characterization and Diagnostic Value of Taenia solium Low-Molecular-Weight Antigen Genes. Journal of Clinical Microbiology, 2000, 38(12):4439-4444
[19] 陈颖，刘淼，薛海筹，等 猪囊尾蚴特异抗原的基因克隆和表达 中国血吸虫病防治杂志，2000，12(5)284-285
[20] 陈蕊雯，林懿，孙树汉 猪囊尾蚴抗原CC1在大肠杆菌中的克隆及高效表达 中国寄生虫学与寄生虫病杂志，2000，18(1)37-39
[21] 杨湘越，孙树汉，陈蕊雯，等 猪囊尾蚴抗原CC1在巴斯德毕赤酵母中的表达中国人兽共患病杂志，2002，18(3)31-33
[22] 吴丹，孙树汉，，郭瀛军，等 猪囊尾蚴抗原与γ干扰素融合蛋白在大肠杆菌中的表达 中国寄生虫学与寄生虫病杂志，1999，17(4)：215—217
[23] 王庆敏，戴建新，张平武，等 猪囊尾蚴抗原B基因cDNA编码区的克隆 中国寄生虫学与寄生虫病杂志，2000，18(2)73-75
[24] 刘新月 膜蛋白的糖脂锚及其临床 武汉医学杂志，1996，20(1)：49-50
[25] 刘永全 整和素与信号转导 国外医学免疫分册，2000，23(1)：34-37
[26] Breathnach R, Chambon P. Organization and expression of eukaryotic split genes coding for proteins. Annu Rev Biochem 1981 ;50:349-83.
[27] Levine MZ, Sanchez CC, Wilkins PP, et al. Characterization, cloning and
expression of two diagnostic antigens for Taenia solium tapeworm infection. J Parasitol, in press.
[28] Restrepo BI, Obregon-Henao A, Mesa M, et al. Characterization of the carbohydrate components of Taenia solium metacestode glycoprotein antigens. Int J Parasitol 2000;30:689-96.
[29] Obregon-Henao A, Gil DL, Gomez DI, et al The role of N-linked carbohydrates in the antigenicity of Taenia solium metacestode glycoproteins of 12, 16 and 18 kDa. Mol Biochem Parasitol 2001; 114:209-15.
[30] Butikofer P, Malherbe T, Boschung M, Roditi I. GPI-anchored proteins: now you see' em, now you don't. FASEB J 2001;15:545-8.
[31] Kukulansky T , Abramovitch S , Hollander N. Cleavage of the glycosylphosphatidylinositol anchor affects the reactivity of Thy-1 with antibodies. J Immunol 1999; 162:5993-7.
[32] Barboni E, Rivero BP, George AJT, et al. The glycophosphatidylinositol anchor affects the conformation of Thy-1 protein. J Cell Sci, 1995;108:487-97.
[33] Martinez AP, Margos G, Barker G, et al. The roles of the glycosylphosphatidylinositol anchor on the production and immunogenicity of recombinant ookinete surface antigen Pbs21 of Plasmodium berghei when prepared in a baculovirus expression system. Parasite Immunol 2000;22:493-500.
[34] Striepen B, Zinecker CF, Damm JBL, et al. Molecular structure of the "low molecular weight antigen" of Toxoplasma gondii: a glucose alpha 1-4 N-acetlygalactosamine makes free glycosyl-phosphatidylinositols highly immunogenic. J Mol Biol 1997;266:797-813.