Guangzhou Baiyunshan Bai Di Bio-technology Co., Ltd.

Objective: To construct a dual-siRNA expression vector targeting Survivin and Livin and identify its activity, and provide evidence for down-regulation of both genes simultaneously.Methods: The published siRNA sequences targeting Survivin and Livin, resp., were chosen to construct the dual-siRNA-expressed vector pSilencer2.1-Sur-Liv.This vector was then transfected into HepG2 cells and the mRNA levels of these two genes were detected.This vector was also transfected into Hela reporter cells, which expressed DsRed2-SURVIVIN and GFP-LIVIN, for examining the florescence.Results: The recombinant vector pSilencer2.1-Sur-Liv was successfully constructed.After transfected with pSilencer2.1-Sur-Liv, the mRNA levels of Survivin and Livin in HepG2 cells were obviously decreased and the florescence in Hela reporter cells was reduced.Conclusion: The dual-siRNA-expressed vector targeting Survivin and Livin had been constructed successfully and may effectively suppress the expressions of Survivin and Livin.

The quality control method of quant. testing of endotoxin in rhGM-CSF was established.Kinetic chromogenic method was applied and quant. method of testing endotoxin in rhGM-CSF was established via reliability experiment of standard curve and interference experimentIn the reliability experiment of standard curve, R² values of the curve were all above 0.98, which indicated the validity of the standard curve, in the interference experiment, the sample was redissolved and diluted five times before 0.5 EU/mL of standard endotoxin was added into the sample as recovered solutionThe recovery rates of endotoxin were all close to 100%.Under the dilution ratio, the interference of sample concentration was minor to the endotoxin experiment, in addition, the contents of endotoxin in two batches of samples were all lower than the limited value through routine testing, and the test results were consistent with that of gel method.The kinetic chromogenic method is applicable to the quant. testing of endotoxin in rhGM-CSF.

Objective: To establish the quant. detection for the bacterial endotoxin in recombinant human granulocyte/macrophage colony-stimulating factor for injection (rhGM-CSF).Methods: The endpoint chromogenic assay, stipulated in was used in Chinese Pharmacopoeia (2010 Edition) was adopted for the determination of rhGM-CSF.Results: No obvious interference action was found in the 2-fold dilutions of rhGM-CSF.Recovery was 50%-200%.The results were consistent with those of the gel method.Conclusion: Quant. detection of bacterial endotoxin in rhGM-CSF by endpoint chromogenic assay is entirely feasible in practice.