犬附红细胞体PGK的原核表达、纯化及其多克隆抗体的制备
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摘要
为了原核表达、纯化犬附红细胞体磷酸甘油酸激酶(PGK),并制备多克隆抗体,本试验从NCBI中已公布的犬附红细胞体(Mycoplasma haemocanis)全基因组序列中,调取其磷酸甘油酸激酶(PGK)的基因序列,并选用原核生物偏爱的密码子优化基因序列,化学合成全新的基因序列pgk,插入质粒pET32a(+)中,构建重组表达质粒pET32a(+)-pgk,转化E. coli BL21(DE3)感受态细胞,用IPTG诱导表达,利用Western-blot方法检测表达产物,表达产物经镍离子亲和层析纯化后免疫昆明小鼠,获得鼠抗犬附红细胞体PGK多克隆抗体,并利用间接ELISA法检测血清抗体效价。结果表明:成功构建了pET32a(+)-pgk原核表达载体,并在BL21中得以诱导表达,重组蛋白相对分子质量约为60 ku,在包涵体和上清中均有表达,经镍离子亲和层析获得了纯度较高的目的蛋白,利用纯化后的蛋白所制备的鼠抗血清效价可达1∶51 200。
In order to express and purify phosphoglyceric kinase(PGK)of Mycoplasma haemocanis, and prepare its polyclonal antibodies, the information of PGK gene sequence was obtained from the published Mycoplasma haemocanis genome on NCBI.The modified PGK gene sequence was synthesized chemically according to the prokaryotic cells-preferred codon, and inserted into pET32 a(+). The constructed recombinant plasmid pET32 a(+)-pgk was transformed to E. coli BL21(DE3)competent cells, and induced with IPTG. The expression product was detected by Western-blot, and then purified by Ni-affinity purification column. The purified recombinant protein was used to immune Kunming mice and to produce the polyclonal antibodies. The serum antibody titer was determined by indirect ELISA. The results showed that recombinant plasmid pET32 a(+)-pgk was constructed correctly, and successfully expressed in BL21. The relative molecular mass of recombinant protein was about 60 ku.And the protein was existed in both inclusion bodies and supernatant. The high-purified recombinant protein was obtained by Ni-affinity purification column, and the prepared mice antiserum reached a titer of 1∶51 200.
In order to express and purify phosphoglyceric kinase(PGK)of Mycoplasma haemocanis, and prepare its polyclonal antibodies, the information of PGK gene sequence was obtained from the published Mycoplasma haemocanis genome on NCBI.The modified PGK gene sequence was synthesized chemically according to the prokaryotic cells-preferred codon, and inserted into pET32 a(+). The constructed recombinant plasmid pET32 a(+)-pgk was transformed to E. coli BL21(DE3)competent cells, and induced with IPTG. The expression product was detected by Western-blot, and then purified by Ni-affinity purification column. The purified recombinant protein was used to immune Kunming mice and to produce the polyclonal antibodies. The serum antibody titer was determined by indirect ELISA. The results showed that recombinant plasmid pET32 a(+)-pgk was constructed correctly, and successfully expressed in BL21. The relative molecular mass of recombinant protein was about 60 ku.And the protein was existed in both inclusion bodies and supernatant. The high-purified recombinant protein was obtained by Ni-affinity purification column, and the prepared mice antiserum reached a titer of 1∶51 200.
引文
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[15]夏晓辉,丁晓双,张晓轩,等.犬附红细胞体PCR检测方法的建立及应用[J].畜牧与兽医,2013,45(3):81-84.
[16]张伟,刁有祥,臧大鹏,等.犬附红细胞体病PCR检测方法的建立及应用[J].西南师范大学学报(自然科学版),2009,34(1):67-71.
[17]周勇志,李庄,石磊,等.犬附红细胞体PCR检测方法的建立[J].中国兽医寄生虫病,2008,16(4):1-4.
[18]车京波.犬附红细胞体病形态学研究及PCR快速诊断方法的建立与应用[D].泰安:山东农业大学,2007.
[19]薛慧亮.犬附红细胞体形态学观察及PCR诊断方法的建立[D].长春:吉林大学,2014.
[20] Neimark H,Johansson K E,Rikihisa Y,et al. Proposal to transfer some members of the genera Haemobartonella and Eperythrozoon to the genus Mycoplasma with description of‘Candidatus Mycoplasma haemofelis’,‘Candidatus Mycoplasma heamomuris’,‘Candidatus Mycoplasma haemosuis’and‘Candidatus Mycoplasma wenyonii’[J]. Int J Syst Evol Microbiol,2001,51:891-899.
[2] Sykes J E,Ball L M,Bailiff N L,et al.‘Candidatus Mycoplasma haematoparvum’,a novel small haemotropic mycoplasma from a dog[J]. Int J Syst Evol Microbiol,2005,55(1):27-30.
[3]全炳昭,谢才丰.犬的附红细胞体病及其诊疗报告[J].中国实验动物学杂志,1991,1(2):119-120.
[4]华修国,张秀根,李中夏.犬附红细胞体病[J].中国动物检疫,1992,9(3):30-31.
[5] do Nascimento N C,Guimaraes A M S,Santos A P,et al.Complete genome sequence of Mycoplasma haemocanis strain Illinois[J]. Journal of Bacteriology,2012,194(6):1605-1606.
[6] Santos A P,Guimaraes A M S,do Nascimento N C,et al.Genome of Mycoplasma haemofelis,unraveling its strategies for survival and persistence[J]. Veterinary Research,2011,42:102-117.
[7] Opperdoes F R. Compartmentation of carbohydrate metabolism in trypanosomes[J]. Annu Rev Microbiol,1987,41:127-151.
[8] Hong S J,Seong K Y. Molecular cloning and immunological characterization of phosphoglycerate kinase from Clonorchis sinensis[J]. Mol Biochem Parasitol,2000,108:207-216.
[9]黄莉妮.日本血吸虫磷酸甘油酸激酶SjPGK基因的克隆表达及生物学特性的初步探索[D].南京:南京农业大学,2012.
[10] Barker E N,Tasker S,Day M J,et al. Development and use of real-time PCR to detect and quantify Mycoplasma haemocanis and“Candidatus Mycoplasma haematoparvum”in dogs[J]. Veterinary Microbiology,2010,140(1):167-170.
[11] Compton S M,Maggi R G,Breitschwerdt E B. Candidatus Mycoplasma haematoparvum and Mycoplasma haemocanis infections in dogs from the United States[J]. Comparative Immunology,Microbiology and Infectious diseases,2012,35(6):557-562.
[12] Novacco M,Meli M L,Gentilini F,et al. Prevalence and geographical distribution of canine hemotropic mycoplasma infections in Mediterranean countries and analysis of risk factors for infection[J]. Veterinary Microbiology,2010,142(3):276-284.
[13] Tennant K V,Barker E N,Polizopoulou Z,et al. Real-time quantitative polymerase chain reaction detection of haemoplasmas in healthy and unhealthy dogs from Central Macedonia,Greece[J]. Journal of Small Animal Practice,2011, 52(12):645-649.
[14] Wengi N,Willi B,Boretti F S,et al. Real-time PCRbased prevalence study,infection follow-up and molecular characterization of canine hemotropic mycoplasmas[J].Veterinary Microbiology,2008,126(1):132-141.
[15]夏晓辉,丁晓双,张晓轩,等.犬附红细胞体PCR检测方法的建立及应用[J].畜牧与兽医,2013,45(3):81-84.
[16]张伟,刁有祥,臧大鹏,等.犬附红细胞体病PCR检测方法的建立及应用[J].西南师范大学学报(自然科学版),2009,34(1):67-71.
[17]周勇志,李庄,石磊,等.犬附红细胞体PCR检测方法的建立[J].中国兽医寄生虫病,2008,16(4):1-4.
[18]车京波.犬附红细胞体病形态学研究及PCR快速诊断方法的建立与应用[D].泰安:山东农业大学,2007.
[19]薛慧亮.犬附红细胞体形态学观察及PCR诊断方法的建立[D].长春:吉林大学,2014.
[20] Neimark H,Johansson K E,Rikihisa Y,et al. Proposal to transfer some members of the genera Haemobartonella and Eperythrozoon to the genus Mycoplasma with description of‘Candidatus Mycoplasma haemofelis’,‘Candidatus Mycoplasma heamomuris’,‘Candidatus Mycoplasma haemosuis’and‘Candidatus Mycoplasma wenyonii’[J]. Int J Syst Evol Microbiol,2001,51:891-899.