牛支原体新疆分离株oppD/F基因克隆及分子特征分析

试验旨在分析牛支原体新疆分离株oppD/F基因序列，并探究其序列特征及编码蛋白的结构和功能。根据GenBank中PG45菌株oppD/F基因序列（登录号：AF130119.1）设计1对引物，应用PCR技术扩增获得分离株oppD/F基因，将oppD/F基因片段克隆至pMD19-T载体中进行测序，在采用生物信息学方法分析其核苷酸序列的基础上对其编码蛋白的基本理化性质、疏水性、可溶性、信号肽、跨膜域、亚细胞定位、磷酸化位点、糖基化位点、二级结构、三级结构和功能等进行分析和预测。结果显示，牛支原体新疆分离株oppD/F基因序列全长1 617 bp，与Mb PG45株和Mb JF4278株的同源性均为100%，处于同一分支；该基因编码蛋白是由539个氨基酸残基组成，不存在信号肽及跨膜结构，是一种稳定的亲水性蛋白质；oppD/F蛋白存在1个AAA家族结构域，50个潜在的磷酸化位点和3个糖基化位点，其二级结构是混合型，其中α-螺旋所占比例最高（61.78%），无规则卷曲次之（20.78%）。功能预测结果显示，oppD/F蛋白在信号传导、受体、结构蛋白、离子通道、免疫应答和胁迫应答等方面的几率均较高。本研究结果为进一步分析牛支原体oppD/F基因的功能提供了一定的理论依据。

Cloning and Bioinformatics Analysis of oppD/F Gene of Mycoplasma bovis Isolate Strain in Xinjiang

This study was aimed to clone and analyze the oppD/F gene of Mycoplasma bovis isolated strain in Xinjiang,explore the sequence characteristics of oppD/F gene and the structure and function of its encoded protein.One pair of specific primers was designed based on the sequence of oppD/F gene of PG45 strain (accession No.:AF130119.1) in GenBank,the sequence of oppD/F gene was amplified by PCR,and it was inserted into pMD19-T vector for sequencing.Its nucleotide sequence,amino acids sequence,the basic physicochemical properties,hydrophobicity,soluble,signal peptide,transmembrane,subcellular localization,phosphorylation site,glycosylation site,secondary structure,tertiary structure,function of oppD/F protein were predicted and analyzed by bioinformatics methods.The results showed that the sequence length of Mycoplasma bovis isolated strain oppD/F gene was 1 617 bp,the sequence and phylogenetic tree analysis indicated that it showed 100% homology with that of Mycoplasma bovis strain PG45 and Mycoplasma bovis isolate JF4278,its were closely relative which were in the same branch.The oppD/F protein encoded a protein composed of 539 amino acids,no signal peptide and transmembrane structure,it was a stable soluble hydrophilic protein.The oppD/F protein had a AAA super family domain,50 potential phosphorylation sites and 3 glycosylation sites.The secondary structure was the mixed,and the alpha helix were the most,accounting for 61.78%,followed by the random coil,accounting for 20.78%.Function prediction showed that the high odds predicted from signal transducer receptor,structural protein,ion channel,immune response and stress response in oppD/F protein.The results could provide a theoretical basis for further exploration of its function of oppD/F gene of Mycoplasma bovis isolated strain.