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Chinese Journal of Biotechnology ; (12): 1002-1014, 2012.
Article in Chinese | WPRIM | ID: wpr-342421


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.

Animals , Breast Neoplasms , Diagnosis , Pathology , Female , Genetic Vectors , Genetics , Green Fluorescent Proteins , Genetics , Humans , MCF-7 Cells , Receptor, ErbB-2 , Recombinant Fusion Proteins , Genetics , Sf9 Cells , Single-Chain Antibodies , Genetics
Article in Chinese | WPRIM | ID: wpr-422824


Objective To study the clinical characteristics,pathology,and treatment for papillary carcinoma of the breast.Methods The clinical data of 17 patients of papillary carcinoma of the breast admitted in the First Affiliated Hospital of Wen Zhou Medical College were retrospectively analyzed.Results Papillary carcinoma of the breast accounted for 0.64% of all breast cancer cases hospitalized during last 10 years.All cases had palpable lumps in the breast.12 cases received modified radical mastectomy,2 cases received simple mastectomy,2 cases underwent breast conservation therapy,1 case underwent simple mastectomy plus sentinel lymph node biopsy.15 patients received postoperative chemotherapy,among those 5 cases also received radiotherapy.During a 32.5-month median follow-up ( 1 month to 8 years),one case with bone metastases died 2 years postoperatively and another one died of multimetastases 7 years later.Conclusions The prognosis of papillary carcinoma of the breast is closely related with its pathology type.For intraductal papillary carcinoma low-traumatic therapy is applicable,while in case of infiltrating papillary carcinoma or invasive micropapillary carcinoma ( IMPC ),more aggressive therapies like that adopted for infiltrating ductal carcinoma are recommended.