Designing a recombinant chimeric construct contain MUC1 and HER2 extracellular domain for prediagnostic breast cancer

Abstract

Breast cancer is the most common cancer among women in the world. One of the approaches for diagnosis of breast cancer is detection of its tumor-associated markers. Mucin 1 (MUC1), a tumor-associated antigen, is a transmembrane glycoprotein expressed by normal epithelial cells and overexpressed by carcinomas of epithelial origin. Also, human epidermal growth factor receptor-2 (HER2/erbB-2) belongs to the one of four members of tyrosin kinase type 1 family in which overexpression of HER2 is associated with malignancy in breast cancer. This study was aimed to bioinformatics analysis and designing a recombinant chimeric protein containing MUC1 and HER2 antigens to express in prokaryotic host (Escherichia coli) as breast cancer diagnosis tools. The immunogenic sequences of MUC1 and HER2 were extracted and fused together by a linker. The chimeric construct was analyzed by bioinformatics softwares. The optimization and purification, evaluation of the expression of chimeric protein was performed using Western blotting, ELISA, and immunohistochemistry. The results showed that the chimeric construct was stable and immunogenic domains were exposed. The pET-28a vector containing chimeric gene had high level of protein expression. The recombinant chimeric protein was confirmed using Western blotting, and it was investigated using ELISA and IHC. Then, the MUC1 and HER2 combined peptides can be used as coating antigens in ELISA for detection of antibodies against MUC1 or HER2 in human serum.

Genetically modified proteins: functional improvement and chimeragenesis

“…92,93 The bioinformatics analysis of immunogenic sequences of the tumor-associated antigens MUC1 and HER2 allowed combining it in the hybrid protein for using as the breast cancer antigens in ELISA for detection of antibodies against MUC1 or HER2 in human serum (Table 1). 92 The optimized chimeric gene composed of the structural subunits of colonization factor antigens (CFA), labile toxin subunit B and the binding subunit of heat-labile and heatstable toxoid, was designed to provide broad-spectrum protection against seven enterotoxigenic E. coli (ETEC) strains, the most common cause of bacterial diarrhea, inducing anti-CFA and antitoxin immunity (Table 1). 93 The above will show that alterations in protein's thermoactivity, stability, pH-dependence and foldability are generally special cases for the rational improvement of the valuable protein function.…”

Recombinant Expression and Purification of Human Cystatin C Biomarker in Escherichia coli for Prediagnostic Renal Disorders

“…The mice serum was collected after checking the antibody against CysC protein by indirect ELISA and stored at -70°C until use. 26 Enzyme-Linked Immunosorbent Assay Analysis Enzyme-linked immunosorbent assay analysis (ELISA) was used to determine the antibody specific responses. Microplates were coated with 5 µg/mL of recombinant antigen and used standard kit Cystatin C (NSO, M1-A146) for evaluation of recombinant antigen.…”

Phase 1 clinical trial demonstrated that MUC1 positive metastatic seminal vesicle cancer can be effectively eradicated by modified Anti-MUC1 chimeric antigen receptor transduced T cells

“…Another anti-MUC1 scFv, of HMFG2, was identified, differing from SM3 by five amino acids and exhibiting a 7.4-fold-higher affinity for unglycosylated MUC1, which was sufficient to enable T-cells grafted with this CAR to proliferate robustly when plated on anchored glycan-free MUC1. Accordingly, HMFG2-CD28-OX40-CD3z CAR was tested in a preclinical model, which resulted in a significant delay of tumor growth (Wilkie et al, 2008;Maher and Wilkie, 2009;Gheybi et al, 2014).…”