牛结核分枝杆菌早期标识性分泌性蛋白MPB70、MPB83基因的串联表达及其抗原性鉴定
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摘要
为获得一种具有抗原性的牛结核分枝杆菌(MB)感染早期标识物MPB70、MPB83分泌性蛋白的串联抗原蛋白,本研究利用DNAStar生物信息软件对MB感染早期标识性分泌性蛋白MPB70、MPB83抗原结构区域进行预测后设计合成相应引物,并在两个片段之间引入16位柔性多肽,通过重叠延伸PCR技术获得MBP70与MBP83基因的抗原优势区核苷酸串联片段,将其连接至原核表达载体pGEX-6p-1中经诱导、表达,结果显示构建的重组表达载体获得高效表达,目的抗原蛋白的分子质量约58 ku,纯化后重组蛋白的纯度>90%,经免疫BALB/c小鼠4次后,抗体效价达到1∶51 200。本实验获得了具有明显抗原活性的MB感染早期分泌性蛋白串联抗原MPB70+MPB83,为后续进一步研发MB早期感染标识物快速检测试剂盒奠定了基础。
To acquire a tandemly expressed protein of MPB70 and MPB83, the early infection markers of Mycobacterium bovis, the antigenic regions of MPB70 and MPB83 were predicted by DNAStar software. The tandem nucleotide fragments of MPB70 and MPB83 genes were amplified by overlapping extension PCR. In order to ensure the correct expression of the two genes, nucleotide sequence coding for sixteen flexible peptides were introduced between the two fragments. The fusion gene was cloned into prokaryotic expression vector pGEX-6 p-1. After transforming of E.coli BL21(DE3), the recombinant Escherichia coli expressed 58 ku target protein after 1 mmol/L IPTG induction. The fusion protein(purity>90%) was purified by affinity chromatography. Four BALB/c mice were subcutaneously immunized with the mixture of the recombinant protein and Freund's adjuvant four times at the interval of fourteen days. The titer of antibody against the recombinant protein reached up to 1 ∶51,200 after immunization, which indicated that the fusion protein had good antigenicity. This study laid a foundation for the development of rapid detection kit for early infection markers of bovine mycobacterium tuberculosis.
To acquire a tandemly expressed protein of MPB70 and MPB83, the early infection markers of Mycobacterium bovis, the antigenic regions of MPB70 and MPB83 were predicted by DNAStar software. The tandem nucleotide fragments of MPB70 and MPB83 genes were amplified by overlapping extension PCR. In order to ensure the correct expression of the two genes, nucleotide sequence coding for sixteen flexible peptides were introduced between the two fragments. The fusion gene was cloned into prokaryotic expression vector pGEX-6 p-1. After transforming of E.coli BL21(DE3), the recombinant Escherichia coli expressed 58 ku target protein after 1 mmol/L IPTG induction. The fusion protein(purity>90%) was purified by affinity chromatography. Four BALB/c mice were subcutaneously immunized with the mixture of the recombinant protein and Freund's adjuvant four times at the interval of fourteen days. The titer of antibody against the recombinant protein reached up to 1 ∶51,200 after immunization, which indicated that the fusion protein had good antigenicity. This study laid a foundation for the development of rapid detection kit for early infection markers of bovine mycobacterium tuberculosis.
引文
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[2]师清博,赵明明,王春凤,等.结核分枝杆菌感染与巨噬细胞凋亡的免疫机制研究进展[J].中国兽医学报,2017,37(09):1811-1816.
[3]Silva M R.Risk factors for human Mycobacterium bovis infections in an urban area of Brazil[J].Mem Inst Oswaldo Cruz,2018,113(8):e170445.
[4]顾训平.奶牛结核病的诊断要点与预防措施分析[J].当代畜牧,2016,(20):50.
[5]郭爱珍,陈焕春.牛结核病流行特点及防控措施[J].中国奶牛,2010,(11):38-45.
[6]孙建文,达剑森.两种奶牛结核病检测方法的比较研究[J].中国动物保健,2016,18(05):70-73.
[7]李金岭.排除牛结核变态反应中禽结核干扰的研究[J].吉林农业大学学报,2008,(03):352-355.
[8]Wiker H G.MPB70 and MPB83--major antigens of Mycobacterium bovis[J].Scand J Immunol,2009,69(6):492-499.
[9]Sabio Y,Garcia J V.Expression of MPB83 from Mycobacterium bovis in Brucella abortus S19 induces specific cellular immune response against the recombinant antigen in BALB/c mice[J].Microbes Infect,2010,12(14-15):1236-1243.
[10]张喜悦,路广计,雪瑞,等.我国北方地区的牛结核分枝杆菌病流行病学调查[J].中国预防兽医学报,2018,(40)4:350-352.
[11]Tashakkori M M,Tebianian M,Tabatabaei M,et al.Cloning,expression,and purification of recombinant protein MPT-64from a virulent strain of Mycobacterium bovis in a prokaryotic system[J].Int J Mycobacteriol,2016,Suppl 1(5):S249.
[12]平晓坤,植广林,陈绚姣,等.牛分枝杆菌PtpA基因的原核表达及其多克隆抗体的制备[J].中国兽医科学,2018,48(04):464-471.
[13]王晓龙,卢天成,王秀然,等.鹿结核分枝杆菌MPB70蛋白的表达与免疫原性鉴定[J].中国畜牧兽医,2016,43(12):3135-3140.
[14]陈爽.牛分枝杆菌新型鸡尾酒抗原诊断方法的建立及其初步应用[D].长春:吉林农业大学,2017.
[15]布日额,王金良,吴金花,等.牛乳腺炎无乳链球菌PI-2a菌毛岛辅助蛋白AP1、BP主要抗原域串联表达及其免疫活性鉴定[J].中国病原生物学杂志,2017,12(07):609-613.
[16]吴金花,布日额,王金良,等.奶牛乳腺炎无乳链球菌sip、pgk及FbsA基因主要抗原区域的融合表达及抗原性鉴定[J].中国兽医学报,2017,37(07):1292-1299.