|Table of Contents|

Preparation of human phage displayed single-chain antibody against hypoxia-inducible factor 1 alpha of laryngeal carcinoma and its effect on radiotherapy sensitization

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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

Issue:
2017年第16期
Page:
1625-1630
Research Field:
基础医学
Publishing date:

Info

Title:

Preparation of human phage displayed single-chain antibody against hypoxia-inducible factor 1 alpha of laryngeal carcinoma and its effect on radiotherapy sensitization

Author(s):

ZHOU Hanjing LIU Jingshu LONG Shuzi TANG Cuiping ZHANG Tao

Department of Oncology, Chongqing Key Laboratory of Molecular Oncology and Epigenetics, Department of Radiation Medicine and Oncology, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China

Keywords:

phage-displayed single-chain variable fragment antibody laryngeal carcinoma hypoxia-inducible factor 1 alpha radiotherapy sensitization

PACS:
R392-33; R392.11; R739.65
DOI:
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Abstract:

Objective     To construct a phage-displayed library and identify phage-displayed single-chain variable fragment (scFv) antibody against hypoxia-inducible factor 1 alpha (HIF1α) of laryngeal carcinoma. Methods      Total RNA of positive lymph node tissues adjacent to laryngeal carcinoma was extracted. ScFv gene fragments were amplified by RT-PCR and splicing-overlap-extension (SOE) PCR, then linked into the pCANTAB5E phagemid vector. The recombinant plasmids were transformed into E.coli TG1 cells in order to create the primary phage-displayed scFv library. The primary scFv library was performed by biopanning against HEP2 cells and HIF1α antigen. The properties of the product were identified by Western blotting, and its specificity was detected by ELISA and immunocytochemical assay. CCK8 assay was used to detect its effect on cell viability, and cell clone assay was used to detect its effect on survival fraction. Results     A phage-displayed scFv library targeting HIF1α antigen and HEP2 cells was successfully constructed. The prepared scFv antibody had an apparent molecular weight of 34×103 and down-regulated the expression of HIF1α protein in HEP2 cells. ELISA showed that the obtained scFv antibody had a positive recognition rate of 79% to HIF1α antigen. Immunocytochemical assay indicated that it could specifically bind to HEP2 cells. The obtained scFv antibody decreased the viability of HEP2 cells after X-ray radiation when compared with the cells treated by radiation alone (P<0.05). Colony formation test showed that the sensitization enhancement ratio (SER) of the antibody to radiation was 1.89. Conclusion    A human anti-HIF1α scFv antibody of laryngeal carcinoma is successfully prepared, and it can increase the radiation sensitivity of HEP2 cells.

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Last Update: 2017-08-23