Expression, purification and identification of an immunogenic fragment in the ectodomain of prostate-specific membrane antigen.

The present study aimed to identify, express and purify an immunogenic fragment in the ectodomain of prostate-specific membrane antigen (PSMA) within a fusion protein. The PSMA amino acid sequence published in National Center for Biotechnology Information GenBank was used to determine sequence homology and immunogenic index analyses, additionally using BLASTN, Protean and ExPASy software to predict the polypeptide sequences of immunogenic epitopes. The gene sequence encoding the ectodomain of the polypeptide immunogenic fragments, containing the identified immunogenic epitopes, was generated using whole-gene synthesis. Prokaryotic expression vector pET-32a-r-ectodomain-PSMA was constructed and the recombinant plasmids were transformed into competent BL21 (DE3) Escherichia coli, which was followed by induction of recombinant protein expression using isopropyl-ß-D-thiogalactopyranoside. Fusion proteins were isolated and purified using affinity chromatography and their immune activity was subsequently investigated using western blot analysis. Purified protein was used to immunize BALB/c mice in order to generate polyclonal antibodies, and the binding of polyclonal antibodies to prostate cancer cell lines in vitro was evaluated using flow cytometry. A total of 3 polypeptide fragments with high specificity were identified following analysis using numerous software packages, and the gene sequences encoding regions containing the 2 most immunogenic fragments were synthesized and successfully inserted into the prokaryotic expression vector pET-32a-r-ectodomain-PSMA. The recombinant PSMA protein fragment had a molecular weight of ~50 kDa and 95% purity. Western blot analysis revealed that the r-ectodomain-PSMA fusion protein specifically bound to the anti-PSMA ectodomain monoclonal antibody. Flow cytometry demonstrated that polyclonal antibodies raised against these recombinant proteins could specifically bind to PSMA-positive LNCaP cells, but not to PSMA-negative PC-3 cells. An immunogenic fragment in the ectodomain of PSMA was successfully expressed and purified. The present study, therefore, provides a basis for the preparation of an anti-PSMA small humanized monoclonal antibody.

Comparison of tumor markers using different detection devices.

BACKGROUND: With the development of proteomics, tumor markers have attracted increasing attention for the early diagnosis and treatment of lung cancer. As biochip technology and nanotechnology continues to grow, rapid and highly sensitive joint detection of multi-tumor markers has become possible. METHODS: Eighty-six patients with lung cancer and 42 healthy controls were recruited for this study. Based on analysis of the detection results, we plotted four standard tumor marker graphs, and compared the results of the highly sensitive nanogold probe and protein chip detection with the results of electrochemiluminescence immunoassay and Dickkopf-1 (DKK1) detection used in the clinic. We then analyzed the relationship between the detection results and our clinical data. RESULTS: Four plotted standard protein graphs all had stages with sound linear relationships. It was found in a correlation analysis of the detection results that overall the two methods showed consistency. CONCLUSION: We developed a detection method for ultra-trace protein that can detect four tumor markers, namely carcinoembryonic antigen, cytokeratin-19 fragments, neuron-specific enolase, and DKK1 in a highly sensitive way within 1.5 hours by magnifying the signal of nanogold deposition based on protein chips and nanogold probes. By comparing the results from the different detection devices, we have developed an experimental basis for detection of tumor markers in the clinic.

Differentiation of human adipose-derived stem cells induced by recombinantly expressed fibroblast growth factor 10 in vitro and in vivo.

The adipogenesis effect of fibroblast growth factor 10 (FGF10) has been demonstrated in many studies. The aim of this study is to render a novel method which can continuously induce hypodermal adipose-derived stem cell (ADSC) differentiation and maturation in vivo and in vitro using FGF10. We constructed a recombinant pcDNA3.0-FGF10-MSC which can continuously express FGF10 by transfected FGF10 into a human mesenchymal stem cell (MSC) clone, and we cultured ADSCs from human subcutaneous resected adipose tissue. An in vitro and in vivo coculture system of pcDNA3.0-FGF10-MSC and ADSCs was then established. We observed the characteristics of ADSCs, monitored the adipogenesis-related transcription factor CAAT/enhancer binding protein-ß, peroxisome proliferator-activated receptor-γ, and measured the adipose tissue layer of carrier animals. The results showed that FGF10 secreted from pcDNA3.0-FGF10-MSC could induce ADSC differentiation into mature adipocytes consistently. The study demonstrated that FGF10 can promote the adipogenesis effect in situ, and the autotransplantation of a carrier continuously secreting FGF10 may be utilized for increasing local subcutaneous adipose tissue in cosmetology.