Thankappan Sabarinath, Rana Rajneesh, Remesh Arun Thachappully, Rekha Valsala, Nagaleekar Viswas Konasagara, Puvvala Bhavani
Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India.
Vet World. 2017 Sep;10(9):1108-1113. doi: 10.14202/vetworld.2017.1108-1113. Epub 2017 Sep 21.
The present study was undertaken to clone, express and study the immunogenicity of P67 protein of .
P67 gene was amplified from genomic DNA of . The polymerase chain reaction (PCR) product was inserted in pRham N-His SUMO Kan vector and was used to transform competent 10G cells. Recombinant protein expression was done by inducing cells with 0.2% Rhamnose. Purification was done using nickel nitrilotriacetic acid affinity chromatography. Western blot and dot blot analysis were performed to assess the immunoreactivity of P67 protein.
PCR amplicon size of P67 gene was found to be 1500 base pair. The size of the fusion protein with SUMO tag was 79 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The recombinant P67 fusion protein expressed in pRham N-His SUMO Kan vector was found to be immunogenic in both western blot and dot blot analysis.
Western blot and dot blot analysis of P67 protein of revealed that the protein is immunogenic. Further work is needed to evaluate the role of P67 antigen of as an immunodiagnostic agent.
进行本研究以克隆、表达并研究[具体物种]P67蛋白的免疫原性。
从[具体物种]的基因组DNA中扩增P67基因。将聚合酶链反应（PCR）产物插入pRham N-His SUMO Kan载体中，并用于转化感受态10G细胞。通过用0.2%鼠李糖诱导细胞来进行重组蛋白表达。使用镍三乙酸亲和层析进行纯化。进行蛋白质免疫印迹和斑点印迹分析以评估P67蛋白的免疫反应性。
发现P67基因的PCR扩增子大小为1500碱基对。在十二烷基硫酸钠聚丙烯酰胺凝胶电泳分析中，带有SUMO标签的融合蛋白大小为79 kDa。发现在pRham N-His SUMO Kan载体中表达的重组P67融合蛋白在蛋白质免疫印迹和斑点印迹分析中均具有免疫原性。
对[具体物种]P67蛋白的蛋白质免疫印迹和斑点印迹分析表明该蛋白具有免疫原性。需要进一步开展工作以评估[具体物种]的P67抗原作为免疫诊断试剂的作用。
