犬新孢子虫致密颗粒蛋白7在大肠杆菌中的克隆与表达
Cloning and expression of Neospora caninum dense-granule 7 in E. coli.
作者信息
Kefayat Marziye, Hamidinejat Hossein, Seifiabadshapoori Masoud Reza, Namavari Mohammad Mehdi, Shayan Parviz, Gooraninejad Saad
机构信息
Department of Parasitology, Veterinary Medicine Faculty, Shahid Chamran University, Ahwaz, Iran.
Department of Microbiology, Veterinary Medicine Faculty, Shahid Chamran University, Ahwaz, Iran.
出版信息
J Parasit Dis. 2014 Jun;38(2):196-200. doi: 10.1007/s12639-012-0221-1. Epub 2012 Dec 18.
Neospora caninum is an obligate intracellular protozoan which induces abortion, still birth and neuromuscular disorders in cattle and is an important problem in dairy and beef industry worldwide. The dense granule protein 7 (GRA7) of N. caninum is an immune-dominant protein shared by both tachyzoite and bradyzoites. This study was conducted to produce recombinant GRA7 of N. caninum using a plasmid with high level of expression of this protein in E. coli. For this purpose, a segment of N. caninum DNA corresponding to GRA7 was amplified using PCR. After sequencing, this fragment was cloned into expression vector pMAL-c2X under the control of the lac promoter. Expression of this plasmid in E. coli strain TG1 was identified by western blotting. In this study, pMAL-c2X had a strong promoter to produce high level expression of NcGRA7. This result revealed that this recombinant protein with pMAL-c2X vector may be suitable for developing of diagnostic procedures.
犬新孢子虫是一种专性细胞内原生动物，可导致牛流产、死产和神经肌肉疾病，是全球奶牛和肉牛养殖业中的一个重要问题。犬新孢子虫的致密颗粒蛋白7（GRA7）是速殖子和缓殖子共有的一种免疫显性蛋白。本研究旨在利用一种能在大肠杆菌中高水平表达该蛋白的质粒来生产犬新孢子虫的重组GRA7。为此，使用聚合酶链反应（PCR）扩增了一段与GRA7相对应的犬新孢子虫DNA片段。测序后，将该片段克隆到乳糖启动子控制下的表达载体pMAL-c2X中。通过蛋白质免疫印迹法鉴定该质粒在大肠杆菌TG1菌株中的表达情况。在本研究中，pMAL-c2X具有一个强启动子，可实现NcGRA7的高水平表达。该结果表明，这种带有pMAL-c2X载体的重组蛋白可能适用于诊断方法的开发。
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