【摘要】 牛支原体(Mycoplasma bovis,M.b)是牛(Bos taurus)的一种重要致病性支原体,感染后可引起牛多种疾病。丙酮酸脱氢酶复合体E1(pyruvate dehydrogenase E1,PDHc E1)是参与生物体糖代谢过程的一个限速酶,广泛存在于微生物、哺乳动物及高等植物中,其直接影响丙酮酸向乙酰辅酶A的转化。本研究参照Gen Bank中M.b PG45株的PDHc E1α亚基基因pdhα和PDHc E1β亚基基因pdhβ序列设计引物,通过PCR扩增获得M.b武威株的pdhα(Gen Bank登录号:KU355295)及pdhβ基因(Gen Bank登录号:KU355296),在序列测定的基础上应用重叠延伸PCR(Overlap PCR)技术完成基因优化并构建原核表达质粒p ET-pdhα和p ET-pdhβ。表达质粒分别转化大肠杆菌(Escherichia coli)BL21(DE3)后经异丙基硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导表达重组PDHc E1α亚基融合蛋白PDHA及PDHc E1β亚基蛋白PDHB。纯化表达产物并分别免疫新西兰兔(Oryctolagus cuniculus)制备多克隆抗体,进而应用Western blot及酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)对M.b PDHc E1α亚基和PDHc E1β亚基在细胞内的分布进行了初步研究。结果表明,经IPTG的诱导,pdhα与pdhβ基因在大肠杆菌BL21(DE3)中均成功表达,重组蛋白r PDHA及r PDHB的分子量分别约为40和37 k D,且均有良好的免疫原性,而Western blot及ELISA结果证实E1α和E1β在牛支原体细胞膜上和胞浆中均有分布且分布量相当。本研究结果为进一步研究M.b生物学功能提供了基础资料。更多还原

【Abstract】 Mycoplasma bovis(M.b) is one of the important pathogenic mycoplasmas. It can cause a variety of diseases if cow(Bos taurus) infected with the M.b. Pyruvate dehydrogenase complex E1(PDHc E1) is a speed limit of enzymes that involve in biological sugar metabolic process, and widely exists in microbe, mammals and higher plants. It directly impact on pyruvate to acetyl co A. According to the gene sequences of M.b PG45 strain in Gen Bank, two pairs of primers were designed. PDHc E1 component subunit alpha(pdhα)(Gen Bank No. KU355295) and PDHc E1 component subunit beta(pdhβ)(Gen Bank No. KU3552956) genes of M.b Wuwei strain were amplified by PCR. After finished sequencing and point mutation, the genes were clonedinto prokaryotic expression plasmid p ET-28a(+), respectively. Then the recombinant plasmids p ET-pdhα and p ET- pdhβ were transformed into Escherichia coli BL21(DE3), respectively. After induced expression by isopropyl β- D- 1- thiogalactopyranoside(IPTG), the fusion protein recombinant PDHA(r PDHA) and recombinant PDHB(r PDHB) were purified and anti- serum against r PDHA or r PDHB was prepared respectively through immunizing New Zealand rabbit(Oryctolagus cuniculus). Subsequently, subcellular location of E1α and E1β in M.b was performed using Western blot and enzyme linked immunosorbent assay(ELISA). The results showed that the recombinant proteins successfully expressed in Escherichia coli BL21(DE3) and their molecular weights were approximately 40 and 37 k D, respectively. Western blot and ELISA analysis indicated that the E1α and E1β of M.b equally distributed in both membrane and the cytoplasm. This study provides foundational data for further investigation of biological functions of M.b.更多还原