Nabavinia Maryam Sadat, Nasab Mahboobeh Naderi, Meshkat Zahra, Derakhshan Mohammad, Khaje-Karamadini Mehrangiz
Microbiology and Virology Research Center, Avicenna Research Institute & Department of Medical Bacteriology & Virology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Avicenna J Med Biotechnol. 2011 Oct;3(4):207-10.
Expressions of recombinant proteins for different applications are important objectives in molecular biotechnology; however, expression of some recombinant proteins is difficult. Several methods have been designed for expression of these proteins. The aim of this study was to construct a vector containing Mtb32C fragment of Mycobacterium tuberculosis (M.tuberculosis) as a fusion partner in order to improve the expression of fused recombinant proteins. Mtb32C was amplified by polymerase chain reaction (PCR). The amplified fragment was ligated into pET21b+ vector. Colony-PCR, enzyme digestion and DNA sequencing methods were used to confirm the recombinant vector. Colony-PCR showed a 420 bp fragment in size corresponding to the correct size of our fragment. In addition the recombinant plasmids sequencing showed the accuracy of the cloned fragment. For confirming the expression, reverse transcriptase (RT)-PCR analysis was performed showing a 420 bp fragment in agarose gel electrophoresis using specific primers. The construction of a vector containing Mtb32C fragment is promising as a fusion partner for future studies as it affected the expression of the fused proteins and increased immune responses against the partner.
为不同应用表达重组蛋白是分子生物技术中的重要目标；然而，一些重组蛋白的表达却很困难。已经设计了几种方法来表达这些蛋白。本研究的目的是构建一个含有结核分枝杆菌（M.tuberculosis）Mtb32C片段作为融合伴侣的载体，以提高融合重组蛋白的表达。通过聚合酶链反应（PCR）扩增Mtb32C。将扩增的片段连接到pET21b+载体中。采用菌落PCR、酶切和DNA测序方法来确认重组载体。菌落PCR显示大小为420 bp的片段，与我们片段的正确大小相符。此外，重组质粒测序显示克隆片段的准确性。为了确认表达，进行了逆转录酶（RT）-PCR分析，使用特异性引物在琼脂糖凝胶电泳中显示出420 bp的片段。构建含有Mtb32C片段的载体作为融合伴侣在未来研究中很有前景，因为它影响了融合蛋白的表达并增强了针对该伴侣的免疫反应。
