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

摘要/Abstract

摘要：

为了解滑液支原体（Mycoplasma synoviae，MS）甘油磷酸二酯磷酸二酯酶（glycerophosphodoester phosphodiesterase，GDPD）的生物学功能，本试验参照GenBank中MS WVU1853株序列设计特异性引物，应用PCR技术扩增获得MS WVU1853株GDPD基因，在测序及序列分析的基础上将其克隆至pET-28a（+）质粒构建原核表达载体pET-GDPD，转化大肠杆菌BL21（DE3）感受态细胞后经IPTG诱导表达，纯化表达产物并分析其酶促活性，进而制备其多克隆抗体，应用间接ELISA和Western blotting检测其免疫原性并分析其在MS内的分布。结果表明，MS WVU1853株GDPD基因CDS全长726 bp，编码242个氨基酸，重组蛋白分子质量约为28 ku。酶促活性检测表明，重组蛋白可催化对硝基苯磷酸二钠（pNPP）转化为对硝基苯酚，且其作用的最适pH为9.0，最佳温度为37℃，Pb²⁺具有较强的抑制作用，而Ca²⁺对其具有较强的促进作用。间接ELISA及Western blotting检测结果表明，MS GDPD具有良好的免疫原性，可刺激新西兰兔产生高水平的抗体，血清效价高达1：160000；亚细胞定位结果表明，MS GDPD在细胞膜和细胞浆内均有分布，但在细胞膜的含量略高于细胞浆。本研究结果为探究MS GDPD生物学功能提供了一定的参考依据。

关键词: 滑液支原体(MS); 甘油磷酸二酯磷酸二酯酶 (GDPD); 原核表达; 酶活性测定; 亚细胞定位

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

中图分类号:

S852.62

胡荣斌, 邢小勇, 武小椿, 张阳阳, 贺健, 张生英, 包世俊. 滑液支原体GDPD基因的原核表达、酶活性分析及亚细胞定位[J]. 《中国畜牧兽医》, 2019, 46(8): 2228-2235.

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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