Rahimi Mohammad Taghi, Sarvi Shahabeddin, Sharif Mahdi, Abediankenari Saeid, Ahmadpour Ehsan, Valadan Reza, Daryani Ahmad
School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran.
Toxoplasmosis Research Center (TRC), Mazandaran University of Medical Sciences, Sari, Iran.
Iran J Parasitol. 2017 Oct-Dec;12(4):498-505.
The objective of this study was to clone, express and characterize the gene encoding rhomboid 4 () proteins, a vital gene in surface adhesion and host cell invasion process of tachyzoite of in an appropriate expression vector and eukaryotic cell for production of recombinant protein.
RNA was isolated from tachyzoites (RH strain) and complementary DNA was synthesized. Oligonucleotide primer pair was designed based on gene sequence with I and RI restriction sites at 5' end of forward and reverse primers, respectively. gene was amplified by PCR, cloned into pTG19-T vector and the recombinant plasmid was sequenced. The gene was subcloned into pcDNA3 plasmid and expressed in CHO cells as eukaryotic cell. SDS-PAGE and western blotting were performed for protein determination and verification.
Cloning of gene in pTG19-T vector was confirmed by colony-PCR and enzymatic digestion. The results of enzymatic digestion and gene sequencing confirmed successful cloning and subcloning procedures. The nucleotide sequence of the cloned gene showed 99% homology compared to the corresponding sequences of original gene. SDS-PAGE and western blotting analyses of the purified protein revealed a single band having expected size of 65 kDa.
This eukaryotic expression system is an appropriate system for high-level recombinant protein production of gene from tachyzoites used as antigenic component for serological assay and vaccine development.
本研究的目的是在合适的表达载体和真核细胞中克隆、表达并鉴定编码菱形蛋白4（）的基因，该基因是速殖子表面黏附及宿主细胞侵袭过程中的一个关键基因，用于生产重组蛋白。
从速殖子（RH株）中提取RNA并合成互补DNA。根据基因序列设计寡核苷酸引物对，正向引物和反向引物的5'端分别带有I和RI酶切位点。通过PCR扩增基因，克隆到pTG19-T载体中并对重组质粒进行测序。将该基因亚克隆到pcDNA3质粒中并在CHO细胞中作为真核细胞进行表达。进行SDS-PAGE和蛋白质印迹分析以进行蛋白质测定和验证。
通过菌落PCR和酶切鉴定了基因在pTG19-T载体中的克隆。酶切结果和基因测序证实了克隆和亚克隆过程成功。克隆的基因的核苷酸序列与原始基因的相应序列相比显示出99%的同源性。对纯化蛋白的SDS-PAGE和蛋白质印迹分析显示出一条预期大小为65 kDa的单条带。
该真核表达系统是用于从速殖子中高水平重组生产基因的合适系统，该基因可作为血清学检测和疫苗开发的抗原成分。
