Khaleghizadeh Maryam, Forghanifard Mohammad Mahdi, Rad Abolfazl, Farshchian Moein, Hejazi Zahra, Gholamin Mehran, Memar Bahram, Abbaszadegan Mohammad Reza
Department of Microbiology, Damghan Branch, Islamic Azad University, Damghan, Iran.
Human Genetic Division, Immunology Research Center, Avicenna Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.
Avicenna J Med Biotechnol. 2018 Apr-Jun;10(2):75-82.
Cancer/Testis Antigens (CTAs) are a sub-group of tumor-associated antigens which are expressed normally in germ line cells and trophoblast, and aberrantly in a variety of malignancies. One of the most important CTAs is Developmental Pluripotency Associated-2(DPPA2) with unknown biological function. Considering the importance of in developmental events and cancer, preparing a suitable platform to analyze roles in the cells seems to be necessary.
In this study, the coding sequence of gene was amplified and cloned into the retroviral expression vector to produce recombinant retrovirus. The viral particles were transducted to Esophageal Squamous Cell Carcinoma (ESCC) cell line (KYSE-30 cells) and the stable transducted cells were confirmed for ectopic expression of gene by real-time PCR.
According to the critical characteristics of retroviral expression system such as stable and long time expression of interested gene and also being safe due to deletion of retroviral pathogenic genes, this system was used to induce expression of gene and a valuable platform to analyze its biological function was prepared. Transduction results clearly showed efficient overexpression of the gene in target cells in protein level due to high level of GFP expression.
Such strategies can be used to produce high levels of desired protein in target cells as a therapeutic target. The produced recombinant cells may present a valuable platform to analyze the effect of ectopic expression in target cells. Moreover, the introduction of its potential capacity into the mouse model to evaluate the tumorigenesis of these cancer cells leads to an understanding of the biological importance of in tumorigenesis. In addition, our purified protein can be used in a mouse model to produce specific antibody developing a reliable detection of existence in any biological fluid through ELISA system.
癌/睾丸抗原（CTAs）是肿瘤相关抗原的一个亚组，正常情况下在生殖系细胞和滋养层细胞中表达，而在多种恶性肿瘤中异常表达。最重要的CTAs之一是发育多能性相关蛋白2（DPPA2），其生物学功能尚不清楚。鉴于其在发育事件和癌症中的重要性，制备一个合适的平台来分析其在细胞中的作用似乎很有必要。
在本研究中，扩增该基因的编码序列并克隆到逆转录病毒表达载体中以产生重组逆转录病毒。将病毒颗粒转导至食管鳞状细胞癌（ESCC）细胞系（KYSE - 30细胞），通过实时PCR确认稳定转导的细胞中该基因的异位表达。
根据逆转录病毒表达系统的关键特性，如目的基因的稳定长期表达以及由于逆转录病毒致病基因缺失而具有安全性，该系统被用于诱导该基因的表达，并制备了一个用于分析其生物学功能的有价值的平台。转导结果清楚地表明，由于高水平的绿色荧光蛋白（GFP）表达，该基因在靶细胞中在蛋白质水平上实现了高效过表达。
此类策略可用于在靶细胞中产生高水平的所需蛋白质作为治疗靶点。所产生的重组细胞可能提供一个有价值的平台来分析该基因在靶细胞中的异位表达效应。此外，将其潜在能力引入小鼠模型以评估这些癌细胞的肿瘤发生情况，有助于了解该基因在肿瘤发生中的生物学重要性。此外，我们纯化的蛋白质可用于小鼠模型中以产生特异性抗体，通过酶联免疫吸附测定（ELISA）系统可靠地检测任何生物体液中该蛋白的存在。
