滑液支原体亚甲基四氢叶酸脱氢酶(MTHFD)的原核表达及亚细胞定位  

Prokaryotic Expression and Subcellular Localization of Methylenetetrahydrofolate Dehydrogenase(MTHFD)in Mycoplasma synoviae

作　　者：张宏燕 岳亚辉 邢小勇[1] 龙翠琴 武小椿 温峰琴[1] 包世俊[1] ZHANG Hong-Yan;YUE Ya-Hui;XING Xiao-Yong;LONG Cui-Qin;WU Xiao-Chun;WEN Feng-Qin;BAO Shi-Jun(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China)

机构地区：[1]甘肃农业大学动物医学院,兰州730070

出　　处：《农业生物技术学报》2021年第12期2407-2415,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(32072863)。

摘　　要：滑液支原体(Mycoplasma synoviae,MS)是一种重要的禽类致病性支原体,感染鸡(Gallus domesticus)和火鸡(Meleagris gallopavo)后引起渗出性滑膜炎、关节炎、跗跖部肿胀和呼吸道炎,造成养鸡业的重大经济损失。亚甲基四氢叶酸脱氢酶(methylenetetrahydrofolate dehydrogenase,MTHFD)催化5,10-亚甲基四氢叶酸生成5,10-甲炔基四氢叶酸,该酶是叶酸代谢中的关键酶,参与嘌呤核苷酸的合成。为研究滑液支原体MTHFD蛋白的免疫原性及其在MS中的分布,本研究参照GenBank中MS WVU1853株双功能5,10-亚甲基四氢叶酸脱氢酶/5,10-亚甲基四氢叶酸环化水解酶(bifunctional 5,10-methylenetetrahydrofolate dehydrogenase/5,10-methenyltetrahydrofolate cyclohydrolase,folD)基因序列设计引物,利用PCR扩增MS folD基因并对其序列进行分析和优化。结果表明,MS folD基因全长837 bp,与GenBank中其他MS菌株folD基因的序列相似性达99.6%。folD基因克隆至pET-28a(+)质粒后转化至大肠杆菌(Escherichia coli)BL21(DE3)感受态细胞,诱导后SDS-PAGE检测表达情况。结果显示重组蛋白(recombinant protein,r)MS MTHFD成功表达,且相对分子质量约为36 kD。纯化蛋白免疫新西兰兔(Oryctolagus cuniculus)制备抗血清,并利用Western blot、ELISA与免疫荧光试验对rMS MTHFD的免疫原性和MTHFD在MS中的分布进行分析。结果表明,rMS MTHFD蛋白具有良好的免疫原性,MTHFD在MS的细胞膜和细胞浆中均有分布,但在胞浆中的含量较多。本研究结果为滑液支原体MTHFD生物学功能的深入研究提供了基础资料。Mycoplasma synoviae(MS)is an important pathogenic mycoplasma in poultry,which can cause exudative synovitis,arthritis,tarsometatarsal swelling and respiratory tract inflammation in chickens(Gallus domesticus)and turkeys(Meleagris gallopavo),causing great economic losses in chicken industry.Methylenetetrahydrofolate dehydrogenase(MTHFD)is a key enzyme in folic acid metabolism,catalyzing 5,10-methylenetrahydrofolate to 5,10-methoxytetrahydrofolate and participating in the synthesis of purine nucleotides.To study the immunogenicity of MTHFD protein of MS and its distribution in MS,primers were designed according to the folD(bifunctional 5,10-methylenetetrahydrofolate dehydrogenase/5,10-methenyltetrahydrofolate cyclohydrolase)gene of MS WVU1853 strain in GenBank.On the basis of sequence analysis and gene optimization,the full-length folD gene was successfully obtained through PCR,which the length of CDS of folD is 837 bp and the sequence similarity was as high as 99.6%with other MS strains.The prokaryotic expression vector pET-folD was constructed and transformed into E.coli BL21(DE3).After induction,the recombinant protein(r)MS MTHFD was expressed through SDS-PAGE and molecular weight of rMS MTHFD was approximately 36 kD.The antiserum was prepared by immunizing New Zealand rabbits(Oryctolagus cuniculus),Western blot,ELISA and immunofluorescence test were used to analyze the distribution of MTHFD in MS.The results showed that MTHFD protein was distributed in MS cell membrane and cytoplasm,but more in cytoplasm.The study build up a basis for in-deepth research of biological characteristics of MS MTHFD.

关 键 词：滑液支原体(MS) 亚甲基四氢叶酸脱氢酶(MTHFD) 原核表达 亚细胞定位 

分 类 号：S852.62[农业科学—基础兽医学]