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羊种布鲁氏菌Rev.1株VjbR基因的克隆、原核表达及生物信息学分析

8243 2019-12-16 11:12:12 杭天宇,宋前进,金 铭,等 中国动物检疫
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本研究旨在构建表达载体pET-30a-VjbR,进而表达布鲁氏菌VjbR蛋白;利用生物软件分析该蛋白生物信息学信息,为进一步研究该蛋白奠定基础。以布鲁氏菌Rev.1株基因组为模板,扩增VjbR基因序列,将其克隆至原核表达载体pET-30a(+)中,获得重组质粒pET-30a-VjbR,并进行原核表达、Western-blot检测及该基因的生物信息学分析。结果显示:本试验成功克隆并表达了VjbR基因;经对表达产物进行SDS-PAGE分析纯化,发现本试验得到较纯蛋白;经Western-blot检测,发现该基因表达蛋白可与布鲁氏菌羊阳性血清发生特异性反应,具有良好的反应原性;通过生物信息学系列软件统计分析,发现VjbR蛋白无跨膜区,无信号肽,且该蛋白共有15个抗原决定簇,在二级结构中,α-螺旋的氨基酸有131个,占比为49.81%。布鲁氏菌VjbR基因的成功表达与纯化,为进一步制备该蛋白的单克隆抗体及iELISA诊断试剂盒的研制奠定了基础。

Cloning,Prokaryotic Expression and Bioinformatics Analysis of VjbR Gene of Brucella melitensis Rev.1 Strain

In order to construct the expression vector(pET-30a-VjbR)to express the VjbR protein of Brucella,and to analyze the bioinformatics information of the protein by biological software so as to lay a foundation for further relevant study. Taking the genome of Brucella melitensis Rev.1 strain as a template,VjbR gene sequence was amplified and then cloned into the prokaryotic expression vector,pET-30a(+),to obtain the recombinant plasmid,pET-30a-VjbR,then the prokaryotic expression,western-blot and bioinformatics analysis were respectively carried out. The results showed that VjbR gene was successfully cloned and expressed;the purer protein was received after SDS-PAGE analysis and purification for the expressed products;it was detected that the expressed protein could specially react with the positive sheep serum through western-blot,showing good reactivity;according to the analysis by series of bioinformatics software,it was found that VjbR protein was with no any transmembrane domain or signal peptide,it had 15 antigenic determinants. In the secondary structure,the number of α-spiral amino acids was 13,accounting for 49.81%. In conclusion,the successful expression and purification of VjbR gene of Brucella would lay a foundation for further preparation of monoclonal antibodies and development of iELISA kit.

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