Application On Detecting The Early Index As Diagnosis In Breast Cancer By Producing Stat3 Protein And Stat3 Antibody

Objective: Breast cancer is the most common malign tumor of woman in the world. Great progress made in recent years cannot still satisfy the needs of early diagnosis in clinic practices. As a result, a new diagnostic sensitive and specific index should be needed to detect and treat breast cancer as early as possibly. We will make the use of anti-stat3 antigen as index to detect breast cancer early by the way of producing high-qualified specific anti-stat3 antigen from rabbit by use of stat3 protein and discover the molecular etiology and offer a new index as early diagnosis of breast cancer in clinic practices while finding a new route to treat.Methods: Sf21 cells were infected with recombinant stat3 gene. After 48h of incubation at 28°C, the transfected cells were collected and sonicated on Sonfier sonicator on ice. Filtered cell extract was loaded onto a Reactive Yellow-3 resin column and Toyopearl AF-Heparin-650M column respectively. The character of Stat3 protein in the fractions were identified by SDS-PAGE, Western blotting. Rabbits were immunized with recombinant mouse Stat3 protein to prepare antibody against Stat3. The purity and specificity of the antibody were analyzed by Western blot. The expression of Stat3 protein was detected by immunohistochemical staining. Level of MDA-MB-231 Stat3 mRNA expression in breast cancer cells MCF-7, tissue of breast cancer and normal breast tissue were detected with RT-PCR. RT-PCR products of stat3 were semi-quantitatively analyzed by Gel-Pro Analyzer software. Ratio of stat3 to ?-actin presented level of mRNA expression.Results: (1) Obtained 200mg protein containing Stat3 from Sf21 cells (2.5×109) extract. There was in the elution of Reactive Yellow-3 resin column with MW 92KDa. Then applied Toyopearl AF-Heparin-650M column and obtained pure protein. The results of SDS-PAGE and Western blotting showed the protein band at approximately 92 kDa, corresponding to STAT3. (2) The titer of serum anti- Stat3 antibody was >1:256000 (A450=0.40) 6 weeks after the immunization. The SDS-PAGE and Western blot analysis showed that there was a obvious protein band with Mr 92000, confirming the specificity of obtained anti-Stat3 antibody. It was displayed the positive expression levels in cytoplasmic and negative in nuclear by immunohistochemical staining with anti-Stat3 antibody that in normal breast tissues; The several positive both in cytoplasmic and nuclear in the poor difference breast tissue; It was displayed the positive both in cytoplasmic and nuclear in the high difference breast tissue. (3) Stat3 mRNA expression in breast cancer High expression level of stat3 mRNA were detected in breast cancer cells MCF-7 and MDA-MB231, ratio of stat3 to action in MCF-7 and MDA-MB231 cells were 0.953±0.019 and 0.872±0.026 respectively. Tissues of breast cancer exhibited stronger stat3 mRNA expression in contrast to normal breast, ratio of stat3 to ?-actin were 0.862±0.022 and 0.023±0.022 respectively.Conclusions: (1) The yield of Stat3 is 6.68 mg from 2.5 billion cells and the purity is >95%. The purified Stat3 protein can be applied to study Stat3's affection, produce anti-Stat3 antibody and invent Stat3 diagnosis kit. (2) The rabbit anti-Stat3 antibody with high titer and specificity has been prepared. This was significant correlation between histodegrate and nuclear Stat3 and correlation between histodegrate and cytoplasmic. Meanwhile this was ignificant correlation between cytoplasmic and nuclear. The result may be usful for early clinical diagnosis of breast cancer. (3) mRNA of breast cancer cells MCF-7,MDA-MB-231,tissues of breast cancerand normal breast tissues There is statistically different between tisssues of breast cancer and normal breast tissues. High expression of Stat3 mRNA is related to breast cancer,this is in accord with immunohistochemical.