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Jul/Aug/Sep 2013; 2(3)

Re-Anchored GPC3-hAFP542-550 Fusion Protein Localizes to Hepatocellular Carcinoma Cell Membrane and Induces Cytokine Secretion by Lymphocytes

Dongye Yang, Jingbo Yang, Caihong Li, Zhen Zhou, Meixue Ma, Yangqing Zhang, Laiqiang Huang

+Author Affiliations

Dongye Yang

Division of Gastroenterology & Hepatology, the Second XiangYa Hospital of Central South University, Changsha 410011, China

Center of Bioscience and Biomedicine & Shenzhen Key Laboratory of Gene and Antibody therapy, Tsinghua Campus in University Town, Shenzhen 518055, China

Jingbo Yang

Division of Gastroenterology & Hepatology, the Second XiangYa Hospital of Central South University, Changsha 410011, China

Caihong Li

Division of Gastroenterology & Hepatology, the Second XiangYa Hospital of Central South University, Changsha 410011, China

Zhen Zhou

Division of Gastroenterology & Hepatology, the Second XiangYa Hospital of Central South University, Changsha 410011, China

Meixue Ma

Division of Gastroenterology & Hepatology, the Second XiangYa Hospital of Central South University, Changsha 410011, China

Yangqing Zhang

Center of Bioscience and Biomedicine & Shenzhen Key Laboratory of Gene and Antibody therapy, Tsinghua Campus in University Town, Shenzhen 518055, China

Laiqiang Huang

Center of Bioscience and Biomedicine & Shenzhen Key Laboratory of Gene and Antibody therapy, Tsinghua Campus in University Town, Shenzhen 518055, China

Corresponding Author:

Dongye Yang, dongyeyang2013@live.com

Abstract

α-Fetoprotein (AFP) is expressed in most human hepatocellular carcinomas (HCC ). Peptide fragments of AFP such as hAFP542–550 could serve as a potential immunotherapy target (recognized by T cells). Glypican-3 (GPC3), a new marker of HCC , attaches at its C-terminus to the exocytoplasmic surface of tumor cells by a glycosylphosphatidylinositol anchor. In this study, we sought to create fusion proteins containing chimeric genes of hAFP542-550 and GPC3 as an initial step in the development of a vector-based, membraneanchored DNA vaccine against HCC . Recombinants containing chimeric genes of hAFP542-550 and GPC3, with or without enhanced green fluorescent protein (EGFP) (termed pGPC3+afp-EGFP and pGPC3+afp, respectively) were constructed successfully. Expression of pGPC3+afp-EGFP was detected by RT-PCR and Western blot; membrane localization of fusion proteins was confirmed by fluorescence and confocal microscopy, as well as by Western blot detection of fusion proteins only in the membranous protein fraction but not in the supernatant or soluble protein fractions extracted from transfected HE K 293 cells. Preliminary antitumor function of the 2 fusion proteins was tested; the enzyme-linked immunosorbent spot (ELISPOT) assay identified secretion of tumor necrosis factor–α and interferon-γ/interleukin 2 by peripheral blood lymphocytes. Recombinants of pGPC3+afp-EGFP and pGPC3+afp were confirmed as membrane–re-anchored fusion proteins that induced secretion of multiple antitumor cytokines.

Received January 30, 2013.

Revised April 13, 2013.

Accepted April 23, 2013.

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Published September 20, 2013;

DOI:10.7178/ig.38

Immunogastroenterology

2013;2(3): 177-184.

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