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

Journal of Tianjin Agricultural University ›› 2018, Vol. 25 ›› Issue (3): 42-46.doi: 10.19640/j.cnki.jtau.2018.03.009

• Researches and Scientific Notes • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S852.62

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

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