滑液支原体GDPD基因的原核表达、酶活性分析及亚细胞定位

doi:10.16431/j.cnki.1671-7236.2019.08.005

Abstract

Abstract:

To understand the biological function of Mycoplasma synoviae (MS) glycerophosphodoester phosphodiesterase (GDPD), specific primers were designed according to the sequence of MS WVU1853 strains in GenBank, the GDPD gene of MS WVU1853 strain was amplified by PCR.On the basis of sequencing and sequence analysis, it was cloned into pET-28a (+) plasmid to construct prokaryotic expression vector pET-GDPD.After transforming the competent cells of Escherichia coli BL21 (DE3), the expression was induced by IPTG.The expressed products were purified and their enzymatic activities were analyzed.Then the polyclonal antibody was prepared, and its immunogenicity was detected by indirect ELISA and Western blotting, and its distribution in MS was analyzed.The results showed that the full length of GDPD gene CDS of MS WVU1853 strain was 726 bp, encoding 242 amino acids, and the molecular weight of the recombinant protein was about 28 ku.Enzymatic activity assay showed that the recombinant protein could catalyze the conversion of p-nitrophenyl disodium phosphate (pNPP) to p-nitrophenol, and the optimum pH and temperature were 9.0 and 37℃, respectively.Pb²⁺ had strong inhibition effect, while Ca²⁺ had strong promotion effect.The results of indirect ELISA and Western blotting showed that MS GDPD had good immunogenicity and could stimulate New Zealand rabbits to produce high levels of antibodies with serum titer as high as 1:160 000.Subcellular localization results showed that MS GDPD was distributed in cell membrane and cytoplasm, but the content of MS GDPD in cell membrane was slightly higher than that in cytoplasm.The results of this study provided some reference for exploring the biological function of MS GDPD.

Key words: Mycoplasma synoviae (MS); glycerophosphodoester phosphodiesterase (GDPD); prokaryotic expression; enzyme activity assay; subcellular localization

CLC Number:

S852.62

HU Rongbin, XING Xiaoyong, WU Xiaochun, ZHANG Yangyang, HE Jian, ZHANG Shengying, BAO Shijun. Prokaryotic Expression,Enzyme Activity Analysis and Subcellular Localization of Mycoplasma synoviae GDPD Gene[J]. , 2019, 46(8): 2228-2235.

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Prokaryotic Expression,Enzyme Activity Analysis and Subcellular Localization of Mycoplasma synoviae GDPD Gene

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