title: [Targeted detecting HER2 expression with recombinant anti HER2 ScFv-GFP fusion antibody].
abstract: To verify the reliability of targeted detecting HER2 positive cancer cells and clinical pathological tissue specimens with a recombinant anti HER2 single chain antibody in single chain Fv fragment (scFv) format, we have constructed the fusion variable regions of the ScFv specific for HER2/neu. labeled a green-fluorescent protein(GFP). The humanized recombinant Anti HER2 ScFv-GFP gene was inserted into pFast Bac HT A, and expressed in insect cells sf9. Then the recombinant fusion protein Anti HER2 ScFv-GFP was properly purified with Ni2+-NTA affinity chromatography from the infected sf9 cells used to test the specificity of the fusion antibody for HER2 positive cancer cells. Firstly, the purified antibody incubated with HER2 positive breast cancer cells SKBR3, BT474 and HER2 negative breast cancer cells MCF7 for 12 h/24 h/48 h at 37 degrees C, in order to confirm targeted detecting HER2 positive breast cancer cells by Laser Confocal Microscopy. Furthermore, the same clinical pathological tissue samples were assessed by immunohistochemistry (IHC) and the fusion antibody Anti HER2 ScFv-GFP in the meanwhile. The data obtained indicated that the recombinant eukaryotic expression plasmid pFast Bac HT A/Anti HER2 ScFv-GFP was constructed successfully In addition, obvious green fluorescent was observed in insect cells sf9. When the purified fusion antibody was incubated with different cancer cells, much more green fluorescent was observed on the surface of the HER2 positive cancer cells SKBR3 and BT474. In contrast, no green fluorescent on the surface of the HER2 negative cancer cells MCF7 was detected. The concentration of the purified fusion antibody was 115.5 microg/mL, of which protein relative molecular weight was 60 kDa. The analysis showed the purity was about 97% and the titer was about 1:64. The detection results of IHC and fusion antibody testing indicated the conformity. In summary, the study showed that the new fusion antibody Anti HER2 ScFv-GFP can test HER2 positive cancer cells, indicating a potential candidate method for clinical HER2 positive specimens detection.
authors.affiliation: Zhejiang Provincial Key Laboratory of Medical Genetics, School of Life Sciences, Wenzhou Medical College, Wenzhou 325035, Zhejiang, China.
authors.forename: Guohui
authors.initials: G
authors.lastname: Gao
authors.forename: Chong
authors.initials: C
authors.lastname: Chen
authors.forename: Yanmei
authors.initials: Y
authors.lastname: Yang
authors.forename: Han
authors.initials: H
authors.lastname: Yang
authors.forename: Jindan
authors.initials: J
authors.lastname: Wang
authors.forename: Yi
authors.initials: Y
authors.lastname: Zheng
authors.forename: Qidi
authors.initials: Q
authors.lastname: Huang
authors.forename: Xiaoqu
authors.initials: X
authors.lastname: Hu
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000473:pathology; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D061986:MCF-7 Cells; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D061987:Sf9 Cells; D057127:Single-Chain Antibodies / Q000235:genetics
chemical_list: D049452:Green Fluorescent Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C508053:ERBB2 protein, human