马鹿布氏杆菌25kDa外膜蛋白基因的克隆、序列分析及其表达研究

目的克隆马鹿布氏杆菌ML分离株25kDa外膜蛋白(Omp25)基因,并在大肠杆菌中表达。方法运用RT-PCR技术从马鹿布氏杆菌分离株中扩增出Omp25基因,将其克隆入pMD18-T中进行核苷酸序列测定和遗传变异分析,并将目的基因亚克隆到大肠杆菌表达载体pET28a中诱导表达。结果该基因全长642bp,编码213个氨基酸;其中前23个氨基酸残基构成信号肽。在推导的氨基酸序列中,存在两个跨膜区,但没有潜在的N-联糖基化位点。与GenBank中已经登录的布氏杆菌其他11个分离株相比,马鹿布氏杆菌分离株Omp25基因变异较小,以散在的点突变为主,Omp25基因核苷酸和推导氨基酸序列的同源性分别为99.1%-99.4%和99.2%-99.5%之间。转化重组质粒pETOMP25的大肠杆菌BL21(DE3)在IPTG的诱导下,可表达出具有反应原性的目的蛋白,表达量占菌体蛋白的18.6%。结论马鹿布氏杆菌分离株与流产型布氏杆菌其它分离株Omp25基因同源性很高,可能是一个毒力较强的野毒株。

Cloning, sequence analysis of 25kDa outer membrane protein gene of wapiti''''s Brucella abortus isolate and its expression

In order to cluone,seauence and express the outer membrane protein 25(Omp25) gene sequence of wapiti's Brucella abortus strain ML.The Omp25 gene of wapiti's Brucella abortus was amplified by RT-PCR and sequenced. Then the interesting gene was subcloned into pET28a for expression.The results showed that the full length of Omp25 gene was 642bp,which encoded 213 amino acids.The initiative 23 amino acids consist of signal peptide.There were two transmembrane regions but no N-glycosylation sites in the deduced amino acid sequence.Compared with 11 other isolates reported in GenBank,there was slightly variation in the nucleotide sequence of Omp25 gene of wapiti's isolate.The identities of nucleotide sequence and deduced amino acid sequences among 11 strains were 99.1%-99.4% could 99.2%-99.5% respectively.The E.coli strain BL21(DE3) transformed by pETOMP25 could express a recombinant protein with reactivity,which amounts to 18.6% in the total protein of the induced bacteria.In conclusion the Omp25 protein gene of wapti's Brucella abortus shares high identities with other isolates of Brucella abortus, which may be a virulent strain.