猪囊尾蚴排泄分泌抗原Ts8B2基因的原核表达、纯化以及免疫反应性分析
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摘要
目的原核表达猪囊尾蚴排泄分泌抗原Ts8B2基因,分析重组蛋白的免疫反应性。方法参照GenBank中Ts8B2基因序列设计引物,以合成基因为模板获得预期DNA片段,双酶切后连接至原核表达载体pGEX-4T-1。将重组质粒转化入大肠埃希菌DH5α(E.coli DH5α),PCR、双酶切筛选阳性质粒,测序确认后转化至E.coli Rosstea,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测表达产物。尿素法提取重组蛋白,然后以脑囊虫病患者血清为一抗,Western blot分析其免疫反应性。结果 Ts8B2基因PCR扩增产物为258 bp,与理论值一致。双酶切和测序鉴定重组表达质粒构建成功,SDS-PAGE分析重组质粒转化菌表达相对分子质量为3.5×10~3,以包涵体形式存在目的蛋白,Western blot分析尿素法提取的重组蛋白能被脑囊虫患者血清识别。结论成功构建Ts8B2基因重组表达质粒表达的重组蛋白具有免疫反应性,为进一步研究该蛋白在抗体检测中的应用奠定了基础。
Objectives To express the Ts8 B2 gene of Cysticercus cellulosae in a prokaryotic expression system and to analyze the immunoreactivity of the recombinant protein. Methods Primers were designed for the Ts8 B2 gene, and the fragment was amplified with PCR using the designed primers. The PCR products were double-digested, inserted into a pGEX-4 T-1 plasmid, and transformed into Escherichia coli DH5α cells. Plasmids extracted from positive clones were verified using BamHI/XhoI double digestion and sequencing and further transformed into E. coli 21 cells. Protein expression was induced with IPTG and verified with SDS-PAGE. The expressed recombinant protein was purified and its immunoreactivity was analyzed with Western blotting. Results The amplified fragment was 258 bp in size. Double digestion and sequencing results confirmed that the recombinant plasmid was successfully constructed. SDS-PAGE indicated successful expression of the recombinant protein(Mr 35 000, respectively), in the form of inclusion bodies. Western blotting indicated that the purified recombinant protein reacted with patient sera. Conclusion The Ts8 B2 gene was successfully expressed in a prokaryotic expression system, and it displayed immunoreactivity.
Objectives To express the Ts8 B2 gene of Cysticercus cellulosae in a prokaryotic expression system and to analyze the immunoreactivity of the recombinant protein. Methods Primers were designed for the Ts8 B2 gene, and the fragment was amplified with PCR using the designed primers. The PCR products were double-digested, inserted into a pGEX-4 T-1 plasmid, and transformed into Escherichia coli DH5α cells. Plasmids extracted from positive clones were verified using BamHI/XhoI double digestion and sequencing and further transformed into E. coli 21 cells. Protein expression was induced with IPTG and verified with SDS-PAGE. The expressed recombinant protein was purified and its immunoreactivity was analyzed with Western blotting. Results The amplified fragment was 258 bp in size. Double digestion and sequencing results confirmed that the recombinant plasmid was successfully constructed. SDS-PAGE indicated successful expression of the recombinant protein(Mr 35 000, respectively), in the form of inclusion bodies. Western blotting indicated that the purified recombinant protein reacted with patient sera. Conclusion The Ts8 B2 gene was successfully expressed in a prokaryotic expression system, and it displayed immunoreactivity.
引文
[1] 江楠, 杨凤娇, 周必英. 猪带绦虫重组BCG-TSOL18疫苗的稳定性研究[J]. 中国病原生物学杂志, 2017, 12(2): 148-50.
[2] Bouwknegt M, Devleesschauwer B, Graham H, et al. Prioritisation of food-borne parasites in Europe, 2016[J]. Euro Surveil, 2016(23): 17-161.
[3] Garcia HH, Gonzalez AE, Evans CAW, et al. Taenia solium cysticercosis[J]. Lancet, 2003, 362(9383): 547-56.
[4] Torgerson PR, Devleesschauwer B, Praet N, et al. World Health Organization estimates of the global and regional disease burden of 11 foodborne parasitic diseases, 2010: a data synthesis[J]. PLoS Med, 2015, 12(12): e1001920.
[5] White Jr AC. Neurocysticercosis: updates on epidemiology, pathogenesis, diagnosis, and management[J]. Annu Re Med, 2000, 51(1): 187-206.
[6] 钟树怀, 方文. 猪囊尾蚴膜联蛋白基因的原核表达和免疫学分析[J]. 中国病原生物学杂志, 2015, 10(1): 54-6, 74.
[7] 李瑾, 肖婷, 孙慧, 等. 猪囊尾蚴排泄分泌抗原Ts8B3基因的原核表达、纯化以及免疫反应性分析[J]. 中国病原生物学杂志, 2018, 13(5): 480-3, 488.
[8] Proano-Narvaez JV, Meza-Lucas A, Mata-Ruiz O, et al. Laboratory diagnosis of human neurocysticercosis: double-blind comparison of enzyme-linked immunosorbent assay and electroimmunotransfer blot assay[J]. J Clin Microbiol, 2002, 40(6): 2115-8.
[9] Chung JY, Bahk YY, Huh S, et al. A recombinant 10-kDa protein of Taenia solium metacestodes specific to active neurocysticercosis[J]. J Infect Dis, 1999, 180(4): 1307-15.
[10] Kennedy MW. The polyprotein lipid binding proteins of nematodes[J]. Biochim Biophys Acta., 2000, 1476(2): 149-64.
[11] Greene RM, Hancock K, Wilkins PP, et al. Taenia solium: molecular cloning and serologic evaluation of 14-and 18-kDa related, diagnostic antigens[J]. J Parasitoly, 2000, 86(5): 1001-7.
[12] Sako Y, Nakao M, Ikejima T, et al. Molecular characterization and diagnostic value of Taenia solium low-molecular-weight antigen genes[J]. J Clin Microbiol, 2000, 38(12): 4439-44.
[13] 王媛媛, 杨小迪, 孙新, 等. 猪囊尾蚴病重组DNA疫苗pcDNA 3.1-TSO45W在小鼠体内诱导的体液免疫效应[J]. 中国病原生物学杂志, 2014, 9(3): 242-4, 248.
[14] Chemale G, Haag KL, Ferreira HB, et al. Echinococcus granulosus antigen B is encoded by a gene family[J]. Mol Biochem Parasitol, 2001, 116(2): 233-7.
[15] Hancock K, Khan A, Williams FB, et al. Characterization of the 8-kilodalton antigens of Taenia solium metacestodes and evaluation of their use in an enzyme-linked immunosorbent assay for serodiagnosis[J]. J Clin Microbiol, 2003, 41(6): 2577-86.
[16] Ferrer E, Bonay P, Foster-Cuevas M, et al. Molecular cloning and characterisation of Ts8B1, Ts8B2 and Ts8B3, three new members of the Taenia solium metacestode 8kDa diagnostic antigen family[J]. Mol Biochem Parasitol, 2007, 152(1): 90-100.
[17] Ferrer E, Martinez-Escribano JA, Barderas ME, et al. Evidence that peptide epitopes of the Taenia solium diagnostic antigen Ts8B2 are immunodominant in porcine cysticercosis and human neurocysticercosis[J]. Mol Biochem Parasitol, 2009(168): 168-71.
[18] Ferrer E, Sanchez J, Milano A, et al. Diagnostic epitope variability within Taenia solium 8kDa antigen family: Implications for cysticercosis immunodetection[J]. Exp Parasitol, 2012, 130(1): 78-85.
[19] Hubert K, Andriantsimahavandy A, Michault A, et al. Serological diagnosis of human cysticercosis by use of recombinant antigens from Taenia solium cysticerci[J]. Clin Diagn Lab Immunol, 1999, 6(4): 479-82.
[20] Sato MO, Sako Y, Nakao M, et al. Evaluation of purified Taenia solium glycoproteins and recombinant antigens in the serologic detection of human and swine cysticercosis[J]. J Infect Dis, 2006, 194(12): 1783-90.
[21] Scheel CM, Khan A, Hancock K, et al. Serodiagnosis of neurocysticercosis using synthetic 8-kD proteins: comparison of assay formats[J]. Am J Trop Med Hyg, 2005, 73(4): 771-6.
[22] 李瑾, 王燕, 魏庆宽, 等. 抗猪囊尾蚴IgG4抗体的特异性抗原筛选[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(5): 509-11.
[2] Bouwknegt M, Devleesschauwer B, Graham H, et al. Prioritisation of food-borne parasites in Europe, 2016[J]. Euro Surveil, 2016(23): 17-161.
[3] Garcia HH, Gonzalez AE, Evans CAW, et al. Taenia solium cysticercosis[J]. Lancet, 2003, 362(9383): 547-56.
[4] Torgerson PR, Devleesschauwer B, Praet N, et al. World Health Organization estimates of the global and regional disease burden of 11 foodborne parasitic diseases, 2010: a data synthesis[J]. PLoS Med, 2015, 12(12): e1001920.
[5] White Jr AC. Neurocysticercosis: updates on epidemiology, pathogenesis, diagnosis, and management[J]. Annu Re Med, 2000, 51(1): 187-206.
[6] 钟树怀, 方文. 猪囊尾蚴膜联蛋白基因的原核表达和免疫学分析[J]. 中国病原生物学杂志, 2015, 10(1): 54-6, 74.
[7] 李瑾, 肖婷, 孙慧, 等. 猪囊尾蚴排泄分泌抗原Ts8B3基因的原核表达、纯化以及免疫反应性分析[J]. 中国病原生物学杂志, 2018, 13(5): 480-3, 488.
[8] Proano-Narvaez JV, Meza-Lucas A, Mata-Ruiz O, et al. Laboratory diagnosis of human neurocysticercosis: double-blind comparison of enzyme-linked immunosorbent assay and electroimmunotransfer blot assay[J]. J Clin Microbiol, 2002, 40(6): 2115-8.
[9] Chung JY, Bahk YY, Huh S, et al. A recombinant 10-kDa protein of Taenia solium metacestodes specific to active neurocysticercosis[J]. J Infect Dis, 1999, 180(4): 1307-15.
[10] Kennedy MW. The polyprotein lipid binding proteins of nematodes[J]. Biochim Biophys Acta., 2000, 1476(2): 149-64.
[11] Greene RM, Hancock K, Wilkins PP, et al. Taenia solium: molecular cloning and serologic evaluation of 14-and 18-kDa related, diagnostic antigens[J]. J Parasitoly, 2000, 86(5): 1001-7.
[12] Sako Y, Nakao M, Ikejima T, et al. Molecular characterization and diagnostic value of Taenia solium low-molecular-weight antigen genes[J]. J Clin Microbiol, 2000, 38(12): 4439-44.
[13] 王媛媛, 杨小迪, 孙新, 等. 猪囊尾蚴病重组DNA疫苗pcDNA 3.1-TSO45W在小鼠体内诱导的体液免疫效应[J]. 中国病原生物学杂志, 2014, 9(3): 242-4, 248.
[14] Chemale G, Haag KL, Ferreira HB, et al. Echinococcus granulosus antigen B is encoded by a gene family[J]. Mol Biochem Parasitol, 2001, 116(2): 233-7.
[15] Hancock K, Khan A, Williams FB, et al. Characterization of the 8-kilodalton antigens of Taenia solium metacestodes and evaluation of their use in an enzyme-linked immunosorbent assay for serodiagnosis[J]. J Clin Microbiol, 2003, 41(6): 2577-86.
[16] Ferrer E, Bonay P, Foster-Cuevas M, et al. Molecular cloning and characterisation of Ts8B1, Ts8B2 and Ts8B3, three new members of the Taenia solium metacestode 8kDa diagnostic antigen family[J]. Mol Biochem Parasitol, 2007, 152(1): 90-100.
[17] Ferrer E, Martinez-Escribano JA, Barderas ME, et al. Evidence that peptide epitopes of the Taenia solium diagnostic antigen Ts8B2 are immunodominant in porcine cysticercosis and human neurocysticercosis[J]. Mol Biochem Parasitol, 2009(168): 168-71.
[18] Ferrer E, Sanchez J, Milano A, et al. Diagnostic epitope variability within Taenia solium 8kDa antigen family: Implications for cysticercosis immunodetection[J]. Exp Parasitol, 2012, 130(1): 78-85.
[19] Hubert K, Andriantsimahavandy A, Michault A, et al. Serological diagnosis of human cysticercosis by use of recombinant antigens from Taenia solium cysticerci[J]. Clin Diagn Lab Immunol, 1999, 6(4): 479-82.
[20] Sato MO, Sako Y, Nakao M, et al. Evaluation of purified Taenia solium glycoproteins and recombinant antigens in the serologic detection of human and swine cysticercosis[J]. J Infect Dis, 2006, 194(12): 1783-90.
[21] Scheel CM, Khan A, Hancock K, et al. Serodiagnosis of neurocysticercosis using synthetic 8-kD proteins: comparison of assay formats[J]. Am J Trop Med Hyg, 2005, 73(4): 771-6.
[22] 李瑾, 王燕, 魏庆宽, 等. 抗猪囊尾蚴IgG4抗体的特异性抗原筛选[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(5): 509-11.
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