刚地弓形虫磷酸丙糖异构酶多克隆抗体的制备及鉴定分析
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摘要
目的研制刚地弓形虫磷酸丙糖异构酶(TPI)兔多克隆抗体(多抗)并分析其诊断价值。方法在大肠埃希菌BL21重组表达菌表达TPI,镍柱亲和层析法纯化后与弗氏佐剂等体积混合,颈背部多点注射免疫新西兰大白兔(TPI 200μg/次,共3次)。末次免疫后2周收集兔血清, Protein A亲和层析纯化多抗。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析多抗纯度, ELISA分析多抗效价,蛋白质印迹(Western blotting)及ELISA分析多抗识别弓形虫排泄分泌抗原及纯化TPI的效果。利用多抗偶联磁珠,富集11份弓形虫感染小鼠和11份健康小鼠血清后, Dot-blot分析多抗的诊断价值,并与弓形虫循环抗原ELISA试剂盒的检测结果作比较。结果表达并纯化获得条带单一的TPI蛋白。SDS-PAGE分析结果显示,免疫兔血清经Protein A亲和纯化后获得的多抗,重链与轻链清晰可见。第3次免疫后兔血清抗体效价可达1∶280 000以上;纯化后的多抗纯度高,效价可达1∶100 000以上; Western blotting分析结果显示,纯化的多抗能识别弓形虫排泄分泌抗原中的TPI及纯化的TPI,在相对分子质量(Mr) 28 000处均出现一特异性条带; ELISA分析结果显示,纯化的多抗能结合纯化的TPI,其A450值与TPI蛋白的包被浓度呈正相关。Dot-blot结果显示, 11份感染鼠血清中10份鉴定为阳性, 1份阴性; 11份健康小鼠血清中10份鉴定为阴性, 1份假阳性。弓形虫循环抗原ELISA试剂盒检测结果显示, 11份感染小鼠血清中6份鉴定为阳性, 5份阴性; 11份健康小鼠血清均鉴定为阴性。Dot-blot与ELISA试剂盒检测结果差异无统计学意义(P> 0.05)。结论制备并纯化了弓形虫TPI多抗,该多抗具有潜在诊断价值,可用于开发免疫诊断试剂以检测弓形虫感染。
Objective Triosephosphate isomerase(TPI) of Toxoplasma gondii is an immunodominant antigen secreted by T. gondii during infection and therefore an important diagnostic antigen. To explore its potential for diagnostic purpose, the polyclonal antibody anti-TPI was made in rabbit and its application to detect T. gondii infection was explored. Methods A New Zealand white rabbit was immunized with 200 μg recombinant TPI protein expressed in Escherichia coli and emulsified with Freund 's adjuvant for three times. The serum was collected from the immunized rabbit 2 weeks after the last immunization. The total IgG in immunized-rabbit serum was purified with Protein A affinity purification column. SDS-PAGE was used to detect the purity of the purified IgG. The anti-TPI antibody titers in the purified IgG and in the immune sera was measured by ELISA and its specific recognition of recombinant TPI and native TPI in T. gondii excretory/secretory(ES) products was detected by Western blotting.The anti-TPI IgG was conjugated on magnet beats to pull down the TPI in T. gondii-infected mouse sera and the pull-downed TPI was applied on NC membrane to be detected by the anti-TPI IgG, and the results were compared with that detected by ELISA kit. Results The recombinant TPI protein was expressed in E. coli and the purified TPI protein was used to immunize a rabbit. The rabbit anti-TPI serum was obtained and the total IgG in the serum was purified with Protein-A affinity column. SDS-PAGE analysis identified that the purified IgG contained typical IgG heavy chain and light chain. The anti-TPI specific antibody titers were 1 ∶ 128 000 in immunized rabbit serum and in the purified IgG measured by ELISA. Western blotting showed that the recombinant TPI protein and native TPI in T. gondii ES products could be specifically recognized by the purified IgG. The purified rabbit IgG was able to pull down the TPI antigen in 10 of 11 sera of mice infected with T. gondii, however, one of the 11 normal mouse sera also showed positive. The results detected by ELISA kit showed that 6 of the 11 infected mice were positive and 5 were negative; 11 healthy mice were all negative. There was no significant difference in the results between Dot blot and ELISA detection. Conclusion The anti-TPI polyclonal antibody was prepared in immunized rabbit. An anti-TPI IgG-based immunological assay was potentially established to detect T. gondii infection. This TPI-specific IgG could be used to develop an immunodiagnostic assay to detect T. gondii infection.
Objective Triosephosphate isomerase(TPI) of Toxoplasma gondii is an immunodominant antigen secreted by T. gondii during infection and therefore an important diagnostic antigen. To explore its potential for diagnostic purpose, the polyclonal antibody anti-TPI was made in rabbit and its application to detect T. gondii infection was explored. Methods A New Zealand white rabbit was immunized with 200 μg recombinant TPI protein expressed in Escherichia coli and emulsified with Freund 's adjuvant for three times. The serum was collected from the immunized rabbit 2 weeks after the last immunization. The total IgG in immunized-rabbit serum was purified with Protein A affinity purification column. SDS-PAGE was used to detect the purity of the purified IgG. The anti-TPI antibody titers in the purified IgG and in the immune sera was measured by ELISA and its specific recognition of recombinant TPI and native TPI in T. gondii excretory/secretory(ES) products was detected by Western blotting.The anti-TPI IgG was conjugated on magnet beats to pull down the TPI in T. gondii-infected mouse sera and the pull-downed TPI was applied on NC membrane to be detected by the anti-TPI IgG, and the results were compared with that detected by ELISA kit. Results The recombinant TPI protein was expressed in E. coli and the purified TPI protein was used to immunize a rabbit. The rabbit anti-TPI serum was obtained and the total IgG in the serum was purified with Protein-A affinity column. SDS-PAGE analysis identified that the purified IgG contained typical IgG heavy chain and light chain. The anti-TPI specific antibody titers were 1 ∶ 128 000 in immunized rabbit serum and in the purified IgG measured by ELISA. Western blotting showed that the recombinant TPI protein and native TPI in T. gondii ES products could be specifically recognized by the purified IgG. The purified rabbit IgG was able to pull down the TPI antigen in 10 of 11 sera of mice infected with T. gondii, however, one of the 11 normal mouse sera also showed positive. The results detected by ELISA kit showed that 6 of the 11 infected mice were positive and 5 were negative; 11 healthy mice were all negative. There was no significant difference in the results between Dot blot and ELISA detection. Conclusion The anti-TPI polyclonal antibody was prepared in immunized rabbit. An anti-TPI IgG-based immunological assay was potentially established to detect T. gondii infection. This TPI-specific IgG could be used to develop an immunodiagnostic assay to detect T. gondii infection.
引文
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[14] Bringaud F,Rivière L,Coustou V. Energy metabolism of trypanosomatids:adaptation to available carbon sources[J].Mol Biochem Parasitol,2006,149(1):1-9.
[15] Abrahamsen MS,Templeton TJ,Enomoto S,et al. Complete genome sequence of the apicomplexan,Cryptosporidium parvum[J]. Science,2004,304(5669):441-445.
[16] Bhowmick IP,Kumar N,Sharma S,et al. Plasmodium falciparum enolase:stage-specific expression and sub-cellular localization[J]. Malar J,2009,8:179.
[17] Ginger ML,McFadden GI,Michels PAM. Rewiring and regulation of cross-compartmentalized metabolism in protists[J]. Phil Trans R Soc B,2010,365(1541):831-845.
[18] Rider SD Jr,Zhu G. Cryptosporidium:genomic and biochemical features[J]. Exp Parasitol,2010,124(1):2-9.
[19]孙铭飞,吴彩艳,戚南山,等.顶复门原虫糖酵解代谢及其关键酶:己糖激酶的研究进展[J].畜牧与兽医,2011,43(11):89-92.
[20] Kotresha D,Noordin R. Recombinant proteins in the diagnosis of toxoplasmosis[J]. APMIS,2010,118(8):529-542.
[21] Liesenfeld O,Montoya JG,Kinney S,et al. Effect of testing for IgG avidity in the diagnosis of Toxoplasma gondii infection in pregnant women:experience in a US reference laboratory[J]. J Infect Dis,2001,183(8):1248-1253.
[2] Pereira KS,Franco RM,Leal DA. Transmission of toxoplasmosis(Toxoplasma gondii)by foods[J]. Adv Food Nutr Res,2010,60:1-19.
[3] Dubey JP. The history of Toxoplasma gondii:the first 100years[J]. J Eukaryot Microbiol,2008,55(6):467-475.
[4]马远林,吕芳丽.Ⅰ型干扰素在原虫感染中的作用[J].中国寄生虫学与寄生虫病杂志,2018,36(4):399-404.
[5] Nissapatorn V. Toxoplasmosis in HIV/AIDS:a living legacy[J].Southeast Asian J Trop Med Public Health,2009,40(6):1158-1178.
[6] Gao XJ,Zhao ZJ,He ZH,et al. Toxoplasma gondii infection in pregnant women in China[J]. Parasitology,2012,139(2):139-147.
[7] Wang H,Wang T,Luo QL,et al. Prevalence and genotypes of Toxoplasma gondii in pork from retail meat stores in Eastern China[J]. Int J Food Microbiol,2012,157(3):393-397.
[8] Garcia JL,Navarro IT,Vidotto O,et al. Toxoplasma gondii:comparison of a rhoptry-ELISA with IFAT and MAT for antibody detection in sera of experimentally infected pigs[J].Exp Parasitol,2006,113(2):100-105.
[9] Rostami A,Karanis P,Fallahi S. Advances in serological,imaging techniques and molecular diagnosis of Toxoplasma gondii infection[J]. Infection,2018,46(3):303-315.
[10] Nam HW. GRA proteins of Toxoplasma gondii:maintenance of host-parasite interactions across the parasitophorous vacuolar membrane[J]. Korean J Parasitol,2009,47(Suppl):S29-S37.
[11] Shwab EK,Jiang TT,Pena HF,et al. The ROP18 and ROP5 gene allele types are highly predictive of virulence in mice across globally distributed strains of Toxoplasma gondii[J]. Int J Parasitol,2016,46(2):141-146.
[12]沈双,肖迪,宋丽君,等.应用免疫蛋白质组学方法鉴定弓形虫病诊断抗原分子[J].中国病原生物学杂志,2015,10(2):152-158,164.
[13]沈双,殷旭仁,宋丽君,等.弓形虫磷酸丙糖异构酶原核表达及免疫保护的初步研究[J].中国血吸虫病防治杂志,2017,29(6):780-783.
[14] Bringaud F,Rivière L,Coustou V. Energy metabolism of trypanosomatids:adaptation to available carbon sources[J].Mol Biochem Parasitol,2006,149(1):1-9.
[15] Abrahamsen MS,Templeton TJ,Enomoto S,et al. Complete genome sequence of the apicomplexan,Cryptosporidium parvum[J]. Science,2004,304(5669):441-445.
[16] Bhowmick IP,Kumar N,Sharma S,et al. Plasmodium falciparum enolase:stage-specific expression and sub-cellular localization[J]. Malar J,2009,8:179.
[17] Ginger ML,McFadden GI,Michels PAM. Rewiring and regulation of cross-compartmentalized metabolism in protists[J]. Phil Trans R Soc B,2010,365(1541):831-845.
[18] Rider SD Jr,Zhu G. Cryptosporidium:genomic and biochemical features[J]. Exp Parasitol,2010,124(1):2-9.
[19]孙铭飞,吴彩艳,戚南山,等.顶复门原虫糖酵解代谢及其关键酶:己糖激酶的研究进展[J].畜牧与兽医,2011,43(11):89-92.
[20] Kotresha D,Noordin R. Recombinant proteins in the diagnosis of toxoplasmosis[J]. APMIS,2010,118(8):529-542.
[21] Liesenfeld O,Montoya JG,Kinney S,et al. Effect of testing for IgG avidity in the diagnosis of Toxoplasma gondii infection in pregnant women:experience in a US reference laboratory[J]. J Infect Dis,2001,183(8):1248-1253.