摘要
根据GenBank中流产布氏杆菌BPE123基因序列(登录号:NC_006933.1),设计引物,以布氏杆菌病疫苗菌A19全基因组DNA为模板扩增BPE123基因,将目的基因克隆入pEASY-T3载体,测序正确后,双酶切pEASY-T3-BPE123,目的片段BPE123连入pET-32a(+)表达载体,构建表达质粒pET-32a(+)-BPE123,转化大肠杆菌BL21(DE3),IPTG诱导表达;对BPE123基因进行生物信息学分析。结果显示,成功构建了重组菌pET-32a(+)-BPE123-BL21(DE3),诱导表达了融合蛋白BPE123。生物信息学分析显示,BPE123基因N端25个氨基酸是其信号肽,与其相互作用的蛋白大都与菌毛相关,它们是flgF、fliI、motA、BruAb2_0155、BruAb2_0121和BruAb2_0125。布氏杆菌属内BPE123基因序列的同源性高达99%。本研究获得了BPE123蛋白并预测了BPE123基因的相关生物学功能,为进一步探索该蛋白的免疫原性和在布氏杆菌胞内寄生过程中的作用奠定了基础。
According to Brucella abortus BPE123 gene sequence in GenBank,BPE123 gene was amplified by polymerase chain reaction,and then cloned into the pEASY-T3 vector.After sequenced for identification,the gene was cloned into the expression vector pET-32a(+)to construct a recombinant plasmid pET-32a(+)-BPE123.The recombinant plasmid pET-32a(+)-BPE123 was transformed into Escherichia coli BL21(DE3)and induced with IPTG to express the recombinant BPE123 protein.In addition,the BPE123 gene was analyzed by bioinformatics.Results showed that the recombinant plasmid pET-32a(+)-BPE123 was successfully constructed and highly purified protein was expressed.Bioinformatics analysis found that the first 25 amino acids in the N-terminus of BPE123 were essential for its secretory.The Universal Protein Database predicted that most proteins interacting with BPE123 were mostly associated with fimbriae,such as flgF,fliI,motA,BruAb2_0155,BruAb2_0121and BruAb2_0125.The homology of gene sequence of Brucella BPE123 is as high as 99%.In conclusion,this study obtained the BPE123 protein and forecasted its related biological functions.This research laid the foundation for further studies on BPE123protein's biofunction in intracellular parasitic and its immunogenicity.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2015年第4期407-413,共7页
Chinese Veterinary Science
基金
国家高技术研究发展计划(863)项目(2012AA101300)
