Identification And Purification Of Hsp70 And Her-2 Protein Complex In The SKBR-3 Breast Cancer Cells

Breast cancer is a kind of disease threatening women's health seriously, whose death rate is the first in female cancers. In recent years, numerous studies have shown that proto-oncogene Her-2 is overexpressed in 20%~ 30%of breast cancer patients, which prompts a high degree of tumor malignancy and could be used as an independent indicator of prognosis. In addition, many studies also have shown that many kinds of heat shock proteins are expressed at different levels in the tissues or cells derived from tumors, in which heat shock protein 70 (Hsp70) is closely related to the occurrence and development of many tumors. With the understanding of the structure and function of Her-2 and Hsp70, the research for cancer therapy has entered a new era. In this study, we try to establish a stable, efficient technology platform for separating and purifying Hsp70 and Her-2 protein complex by purify the protein complex in SKBR-3 cell line. Our work will lay an experimental and theoretical foundation for exploring new individualized cancer immunotherapy.Firstly, the expression of Her-2 in SKBR-3 cell line was detected by flow cytometry. The result showed that proto-oncogene HER-2 was highly expressed in the SKBR-3 cell line. Next, the existing form of Hsp70 and Her-2 protein in the cell line was examined by immunoprecipitation technology. The result indicated that Hsp70 and Her-2 existed in the form of the Hsp70 and Her-2 protein complex in SKBR-3 cells. Then, the SKBR-3 cells were heat-shocked at 42℃for 12 hours. Hsp70 and Her-2 protein complex was purified by ConA Sepharose and ADP agarose affinity chromatograpHy after the heat-shocked cells were lysed in the lysis buffer. The purified products were detected by SDS-PAGE and Western dot-blot methods. The results showed that we got the partially purified Hsp70 and Her-2 protein complex. Lastly, the purified products were concentrated with PEG20000 and quantified by BCA kit. The concentration of the total protein in the purified products was 258μg/mL. The completion of the work provides a basis for research Hsp70/Her-2 protein complex-based immunotherapy of breast cancer further.