人胎盘特异性1（PLAC1）在CHO-K1细胞中的表达

Expression of Human Placenta-specific 1 (PLAC1) in CHO-K1 Cells.

作者信息

Mahmoudian Jafar, Nazari Mahboobeh, Ghods Roya, Jeddi-Tehrani Mahmood, Ostad Seyed Nasser, Ghahremani Mohammad Hossein, Vafaei Sedigheh, Amiri Mohammad Mehdi, Zarnani Amir-Hassan

机构信息

Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran.

Monoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran.

出版信息

Avicenna J Med Biotechnol. 2020 Jan-Mar;12(1):24-31.

PMID:32153735

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7035464/

Abstract

BACKGROUND

Placenta-specific 1 (PLAC1), as a new Cancer/Testis Antigen (CTA), is frequently expressed in a variety of cancers and localized to cytoplasm and plasma membrane. Surface expression of cancer target antigens is of great importance that enables antibody-mediated cancer immunotherapy. The aim of the current study was to express the intact human PLAC1 protein on plasma membrane of a eukaryotic cell as a model for future anti-PLAC1-based cancer immunotherapy.

METHODS

In the first approach, entire human PLAC1 gene including its own Signal Peptide (SP) was cloned into pIRES2-EGFP and LeGO-iG2 vectors and expressed in CHO-K1 cells. In the second approach, cytosolic and Signal-Anchor (SA) sequence of Transferrin Receptor Protein 1 (TFR1) were fused to extracellular portion of PLAC1 and expressed as above. Expression of PLAC1 was then assessed using Reverse Transcription Polymerase Chain Reaction (RT-PCR), Western Blot (WB), Immunocytochemistry (ICC), Immunofluorescence (IF) and Flow Cytometry (FC).

RESULTS

The first approach resulted in the expression of PLAC1 in submembranous but not in the surface of transfected CHO-K1 cells. Using the chimeric human PLAC1 construct, the same intracellular expression pattern was observed.

CONCLUSION

These results indicated that there are some yet unknown PLAC1 localization signals employed by cancer cells for surface expression of PLAC1.

摘要

背景

胎盘特异性1（PLAC1）作为一种新的癌/睾丸抗原（CTA），在多种癌症中频繁表达，定位于细胞质和质膜。癌症靶抗原的表面表达对于实现抗体介导的癌症免疫治疗至关重要。本研究的目的是在真核细胞的质膜上表达完整的人PLAC1蛋白，作为未来基于抗PLAC1的癌症免疫治疗的模型。

方法

在第一种方法中，将包含其自身信号肽（SP）的整个人PLAC1基因克隆到pIRES2-EGFP和LeGO-iG2载体中，并在CHO-K1细胞中表达。在第二种方法中，将转铁蛋白受体蛋白1（TFR1）的胞质和信号锚定（SA）序列与PLAC1的细胞外部分融合，并如上所述进行表达。然后使用逆转录聚合酶链反应（RT-PCR）、蛋白质免疫印迹（WB）、免疫细胞化学（ICC）、免疫荧光（IF）和流式细胞术（FC）评估PLAC1的表达。