ABSTRACT
When we try to establish the gene recombinant yeast cell to screen the androgenic endocrine disruptors, the key procedure is the androgen receptor (AR) expression in the yeast cell. For this purpose, we obtained the GPD (glyceraldehyde-3-phosphote dehydrogenase) promoter from the yeast genosome of W303-1A using PCR system and inserting it into Swa I and BamH I sites of pYestrp2. The new constructed vector was named pGPD. The V5 epitope tag DNA with a 5'-BamH I and a 3'-EcoR I sticky end was cloned into the corresponding site of the pGPD vector to yield the vector of pGPDV5. The 2 723 bp full length AR ORF amplified by PCR from pcDNA3.1/AR was fused to V5 epitope tag DNA in pGPDV5 to give the AR yeast expression vector of pGPDV5/AR. This fused vector was transformed into the yeast cell (W303-1A). Western blot was used to detect the V5 fused protein of AR, in the protocol of which the primary monoclonal antibody (IgG(2a)) of mouse anti-V5 and the polyclonal secondary antibody of goat anti-mouse (IgG) linked to horseradish peroxidase (HRP) were used to detect the specific protein in the given sample of the transformed yeast extract. The result showed that the fused protein of AR was expressed successfully in the yeast cell.
Subject(s)
Humans , Base Sequence , Endocrine Disruptors , Epitopes , Genetics , Genetic Vectors , Genetics , Glyceraldehyde-3-Phosphate Dehydrogenases , Genetics , Molecular Sequence Data , Promoter Regions, Genetic , Receptors, Androgen , Genetics , Recombinant Fusion Proteins , Genetics , Yeasts , Genetics , MetabolismABSTRACT
Objective To investigate the relationship between the plasma IL-6,ACE level and DVH parameters radiation pneumonitis,and to evaluate its clinical value on the prediction of radiation pneumonitis.Methods 60 patients with locally advanced non-small cell lung cancer patients with unresectable or unwilling to surgical resection routinely received three-dimensional conformal radiation therapy.Before radiotherapy,radiotherapy beginning 1 week,2 weeks,3 weeks,4 weeks,5 weeks,6 weeks,8 weeks and 12 weeks,enzyme-linked immunosorbent assay (ELISA) was used to detect IL-6 and ACE content.The evaluation of radiation pneumonitis was accordance with the RTOG acute radiation pneumonitis grading standards.The patients with radiation pneumonitis were included in observation group,otherwise as control group.IL-6,ACE and DVH parameters in two groups were analyzed.The DVH parameters includes V20,V20,MLD and NTP.Results Among all the 60 patients,16 cases were diagnosed as radiation pneumonitis,among whom 5 cases occurred in radiotherapy,10 cases occurred within one month after radiotherapy,1 case occurred in two months after radiotherapy,including the 11 cases of grade Ⅱ and the other 5 cases of grade Ⅲ.In the observation group and the control group,expression level of the IL-6 had statistical significance before and after radiotherapy,ACE had significant difference in 2rd,3rd,4th,5th,6th week after radiotherapy (all P ＜ 0.05).V20,V30 and MLD differences were statistically significant between the observation group and control group [(46.2±4.5) ％ vs (30.5±7.5) ％,(37.5±5.6) ％ vs (20.5±5.6) ％,(20.4±2.3) Gy vs (15.5±3.5) Gy,(25.2±8.2) ％ vs (9.9±4.5) ％,all P ＜ 0.05].Conclusion Plasma IL-6,ACE levels,V20,V30,the MLD,and NTP are related to the occurrence of radiation pneumonitis.