犬附红细胞体PGK的原核表达、纯化及其多克隆抗体的制备

doi:10.19640/j.cnki.jtau.2018.03.009

天津农学院学报 ›› 2018, Vol. 25 ›› Issue (3): 42-46.doi: 10.19640/j.cnki.jtau.2018.03.009

犬附红细胞体PGK的原核表达、纯化及其多克隆抗体的制备

宋淇淇¹, 钱丽敏¹, 苏苗¹, 于晓雪¹, 秦顺义¹, 龚岳²

1. 天津农学院动物科学与动物医学学院,天津300384;
2. 天津市滨海新区塘沽动物卫生监督所,天津 300354

收稿日期:2018-04-28 出版日期:2018-09-20 发布日期:2019-11-12
作者简介:宋淇淇（1986-）,女,讲师,博士,主要从事兽医寄生虫学教学及研究工作。E-mail：qqs0606@163.com。
基金资助:
天津农学院科学研究发展基金项目（2014N10）; 国家自然科学基金项目（31702223）; 天津农学院附属动物医院委托项目（科经第2018.96号）

Prokaryotic expression and purification of Mycoplasma hemocanis phosphoglyceric kinase and the preparation of its polyclonal antibodies

SONG Qi-qi¹, QIAN Li-min¹, SU Miao¹, YU Xiao-xue¹, QIN Shun-yi¹, GONG Yue²

1. College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300384, China;
2. Tanggu Animal Health Supervision Institute, Binhai New District, Tianjin 300354, China

Received:2018-04-28 Online:2018-09-20 Published:2019-11-12

摘要/Abstract

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。

关键词: 犬附红细胞体, 磷酸甘油酸激酶, 原核表达, 纯化, 多克隆抗体

Abstract: 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 pET32a（+）. The constructed recombinant plasmid pET32a（+）-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 pET32a（+）-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.

Key words: Mycoplasma haemocanis, phosphoglyceric kinase, prokaryotic expression, purification, polyclonal antibodies

中图分类号:

S852.62

宋淇淇, 钱丽敏, 苏苗, 于晓雪, 秦顺义, 龚岳. 犬附红细胞体PGK的原核表达、纯化及其多克隆抗体的制备[J]. 天津农学院学报, 2018, 25(3): 42-46.

SONG Qi-qi, QIAN Li-min, SU Miao, YU Xiao-xue, QIN Shun-yi, GONG Yue. Prokaryotic expression and purification of Mycoplasma hemocanis phosphoglyceric kinase and the preparation of its polyclonal antibodies[J]. Journal of Tianjin Agricultural University, 2018, 25(3): 42-46.

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犬附红细胞体PGK的原核表达、纯化及其多克隆抗体的制备

Prokaryotic expression and purification of Mycoplasma hemocanis phosphoglyceric kinase and the preparation of its polyclonal antibodies

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