PilQ的分子特征与功能分析：一种新型分泌素结构域
Molecular characterization and Functional Analysis of the PilQ: a Novel Secretin Domain in .
作者信息
Faezi Sobhan, Nikokar Iraj, Elmi Ali, Ghasemi Yusuf, Farahbakhsh Mojtaba, Salimi Chirani Alireza, Mahdavi Mehdi
机构信息
Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran.
Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran.
出版信息
Avicenna J Med Biotechnol. 2018 Jan-Mar;10(1):34-40.
BACKGROUND
Type 4 pili (T4P) is an important virulence factor of . T4P pass the outer membrane through a large oligomeric channel made of a single PilQ protein that is most highly conserved at their C-termini. To develop a functional vaccine that can be used in clinical application, the secretin domain of the PilQ (PilQ) was produced as a recombinant protein.
METHODS
A 981 fragment of C-terminal of the secretin () from was designed into the prokaryotic expression vector pET28a. The presence of the gene in the recombinant construct (pET28a/) was assessed by double digestion and PCR. After transformation, expression of the recombinant PilQ was induced by addition of IPTG. The expressed recombinant protein was purified by a modified method using a HisTrap affinity column and finally confirmed by SDS-PAGE. The functional activities of the produced PilQ confirmed by Western blot analysis and twitching inhibition assay.
RESULTS
The PCR and enzymatic digestion results showed the presence of the gene in the construct. The protein electrophoresis showed that the molecular weight of the recombinant PilQ is approximately 37 . The Western blot analysis confirmed the specificity of specific IgG against the PilQ protein. The PilQ protein showed high biological activity in all of these standard assays.
CONCLUSION
Since, the PilQ protein plays an important role in the biogenesis of pili; and thus, the primary establishment of ; it seems that it can be used as a candidate vaccine or an adjuvant in the future studies.
背景
4型菌毛（T4P）是……的一种重要毒力因子。T4P通过由单个PilQ蛋白构成的大型寡聚通道穿过外膜，该蛋白在其C末端最为保守。为开发一种可用于临床应用的功能性疫苗，制备了PilQ的分泌素结构域（PilQ）作为重组蛋白。
方法
将来自……的分泌素（）C末端的981个片段设计到原核表达载体pET28a中。通过双酶切和PCR评估重组构建体（pET28a/）中……基因的存在情况。转化后，通过添加IPTG诱导重组PilQ的表达。使用HisTrap亲和柱通过改良方法纯化表达的重组蛋白，最后通过SDS-PAGE进行确认。通过蛋白质印迹分析和抽动抑制试验确认所产生的PilQ的功能活性。
结果
PCR和酶切结果表明构建体中存在……基因。蛋白质电泳显示重组PilQ的分子量约为37……。蛋白质印迹分析证实了针对PilQ蛋白的特异性IgG的特异性。PilQ蛋白在所有这些标准试验中均表现出高生物活性。
结论
由于PilQ蛋白在菌毛生物合成中起重要作用；因此，……的初步建立；在未来的研究中，它似乎可以用作候选疫苗或佐剂。
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