摘要
副结核分支杆菌(MAP)由于其胞内寄生的特性,难以在早期发现,待发现后往往已经不再具备治疗价值,因此寻找并制备出能同时在早晚期都有显著标识作用的抗原蛋白,有利于更好地诊断牛副结核病。通过生物信息学软件对MAP的MAP0862和MAP2191c两种蛋白进行一级、二级及三级结构上的理化性质、抗原性分析及相对位置建模预测,判断串联蛋白重组表达的可行性;使用柔性肽短链序列(G4S)2连接两段序列构建了重组表达载体pET30a-MAP6291c,将其导入至BL21(DE3)原核表达载体中进行诱导表达,并进行Western blot分析。结果显示,预测MAP0862-2191c串联蛋白为一个有多个抗原表位的亲水性的蛋白,两种蛋白在三级结构上无相互影响,因此可以进行原核表达;构建合成的融合基因双酶切结果与预期结果相符;经SDS-PCGE凝胶电泳检测成功表达串联蛋白,大小约68.5 ku,与预期蛋白大小相符;分别选择鼠源抗His抗体及MAP阳性牛血清作为一抗进行Western blot验证,均显示MAP0862-2191c重组蛋白有单一的特异性条带,位置及大小与预期结果一致,证明了该串联蛋白制备的正确性,并且具有良好的反应原性,能够作为牛副结核病的诊断蛋白,为后续建立牛副结核病的诊断方法奠定了良好基础。
Mycobacterium avium subsp.paratuberculosis is difficult to detect at an early stage due to its intracellular parasitic characteristics,and often no longer has therapeutic value after being discovered.Therefore,in order to find and prepare an antigenic protein that can have significant identification function at both early and late stages,and to establish a better detection method for bovine parattuberculosis,in this study,by searching literature and using bioinformatics software,the physical and chemical properties,antigenicity and relative location modeling of the two secreted proteins MAP0862 and MAP2191c after MAP infection were analyzed on the primary,secondary and tertiary structures,so as to judge the feasibility of tandem protein recombination expression.The recombinant expression vector pET30a-MAP6291c was constructed by connecting two sequences with flexible peptides(G4S)2,which was introduced into BL21(DE3)prokaryotic expression vector for induced expression,and Western blot analysis was performed.The results showed that the MAP0862-2191c tandem protein was predicted to be a hydrophilic protein with multiple epitopes,and the two proteins had no interaction on the tertiary structure,so prokaryotic expression could be considered.The results of double enzyme digestion of the synthesized fusion gene were consistent with the expected results.The tandem protein was successfully expressed by SDS-PCGE,and the size was about 68.5ku,which was consistent with the expected size of the protein.Mouse anti-HIS antibody and MAP-positive bovine serum were selected as primary antibodies for Western blot verification.Both results showed that the recombinant protein MAP0862-2191c had a single specific band,the location and size were consistent with the expected results,which proved the correctness and antigenicity of the recombinant protein preparation.It laid a good foundation for the subsequent establishment of diagnostic methods for bovine parattuberculosis.
作者
郝彦儒
郭号
谢梦圆
陈坚
杨文洁
徐晓静
HAO Yan-ru;GUO Hao;XIE Meng-yuan;CHEN Jian;YANG Wen-jie;XU Xiao-jing(Veterinary College,Inner Mongolia Agricultural University,Hohhot,Inner Mongolia 010018,China;The Spirit Jinyu Biological Pharmaceutical Co.,Ltd.,Hohhot,Inner Mongolia 010111,China)
出处
《动物医学进展》
北大核心
2025年第4期28-34,共7页
Progress In Veterinary Medicine
基金
内蒙古自治区科技计划项目(2023YFDZ0058)
呼和浩特市科技创新领域人才项目(2022RC-联合体-1)
内蒙古自治区科技重大专项(2020ZD0006)
呼和浩特市科技重大专项(2022-农-重-1-1)。
作者简介
郝彦儒(1998-),男,山西运城人,硕士研究生,主要从事动物组织胚胎发育及疫病防控研究。;通讯作者:徐晓静。
