摘要
目的 在大肠埃希菌(E. coli)中表达结核分枝杆菌(Mycobacterium tuberculosis,MTB)Rv0674蛋白,纯化后制备多克隆抗体,并检测抗体效价。方法 用PCR技术从MTB H37Rv基因组中扩增Rv0674基因,克隆至p EASYBlunt E1表达载体后,转化入E. coli BL21(DE3)中,用优化后的IPTG诱导表达条件表达Rv0674蛋白,His trap TM HP亲和层析柱纯化目的蛋白;将纯化的蛋白腹腔注射BALB/c小鼠制备多克隆抗体,ELISA和Western blot法检测抗体效价及特异性。结果 成功获得了Rv0674序列正确的重组质粒p EASY-E1-Rv0674,挑选出了高表达Rv0674基因的菌株,确定的最适表达条件为1. 0 mmol/L IPTG 28℃诱导表达8 h,获得了相对分子质量约26 500的可溶性蛋白;免疫BALB/c小鼠后,能刺激小鼠产生多克隆抗体,获得的抗血清具有良好的抗原结合特性,且效价大于1∶51 200。结论 成功获得了高纯度的Rv0674蛋白,并首次制备出小鼠抗Rv0674蛋白的多克隆抗体,为进一步研究Rv0674蛋白的功能和临床诊断结核病(tuberculosis,TB)的应用奠定了基础。
Objective To express Mycobacterium tuberculosis(MTB)Rv0674 protein,prepare polyclonal antibodies(PcAbs)and determine their titers.Methods Rv0674 gene was amplified from MTB H37Rv genome by PCR and cloned into expression vector pEASY-Blunt E1.The constructed recombinant plasmid was transformed to E.coli BL21(DE3)and induced with IPTG under the optimized condition.The expressed Rv0674 protein was purified by His trapTM HP affinity chromatography and injected i.p.into BALB/c mice.The prepared PcAbs were tested for titer and specificity by ELISA and Western blot respectively.Results Recombinant plasmid pEASY-E1-Rv0674 with correct Rv0674 gene sequence was constructed,and a strain for high expression of Rv0674 was screened.The condition for expression was optimized as induction with 1.0 mmol/L IPTG at 28℃for 8 h.A soluble Rv0674 protein with a relative molecular mass of about 26500 was obtained,which induced PcAbs in BALB/c mice.The PcAbs showed good antigen-binding properties,of which the titers were more than 1∶51200.Conclusion Highly purified Rv0674 protein was obtained,and PcAbs against Rv0674 was prepared at first,which laid a foundation of further study on function of Rv0674 protein and its application in the clinical diagnosis of tuberculosis(TB).
作者
杨延辉
董利军
梁忠喆
刘通
张炜
杨玉荣
杨志伟
YANG Yan-hui;DONG Li-jun;LIANG Zhong-zhe;LIU Tong;ZHANG Wei;YANG Yu-rong;YANG Zhi-wei(Ningxia Medical University,Yinchuan 750004,Ningxia Hui Autonomous Region,China)
出处
《中国生物制品学杂志》
CAS
CSCD
2019年第12期1370-1376,共7页
Chinese Journal of Biologicals
基金
国家自然科学基金（81560604、81202567和81460311）
宁夏高等学校一流学科建设项目（NXYLXK2017B07）
宁夏医科大学校级课题（XY201425、XY201527和XY201725）
大学生创新项目
关键词
结核分枝杆菌
Rv0674基因
多克隆抗体
Mycobacterium tuberculosis(MTB)
Rv0674 gene
Polyclonal antibodies(PcAbs)
