[Toxicity reduction of human islet amyloid polypeptide by Trim-Away technique in insulinoma cells].

Human islet amyloid polypeptide (hIAPP, also known as amylin) is a co-secreting protein of insulin in human pancreatic ß-cells. It is encapsulated in vesicles and secreted out of the cells with insulin. hIAPP can promote insulin secretion and regulate blood glucose homeostasis in the body under the normal physiological conditions. However, hIAPP misfolding or excessive accumulation can cause toxic effects on the ß cells, which in turn affect cell function, resulting in type 2 diabetes mellitus (T2DM) for the affected individuals. In order to eliminate the excessive accumulation of hIAPP in the cell and to maintain its normal synthetic function, we have adopted a new protein degradation technology called Trim-Away, which can degrade the target protein in a short time without affecting the mRNA transcription and translation synthesis function of the target protein. First, we overexpressed hIAPP in the rat insulinoma cells (INS1) to simulate its excessive accumulation and analyzed its effect in INS1 cells by measuring the release of LDH (lactate dehydrogenase), CCK8 activity and PI-Annexin V positive ratio. Results showed that excessive accumulation of hIAPP caused ß cell apoptosis. Second, real-time quantitative PCR analysis and ELISA detection showed that the synthesis and secretion of insulin were hindered. We used Trim-Way technology to specifically eliminate the excessive accumulation of hIAPP protein in hIAPP overexpressing INS1 cells. Cell activity experiments confirmed that clearance of hIAPP reduced the cell death phenotype. Further ELISA experiments confirmed that INS1 cells restored insulin secretion ability. This study examined the toxic effect of hIAPP excessive accumulation in INS1 cells and demonstrated the cytotoxicity clearance effect of Trim-Way technology in pancreatic ß-cells. Our research has provided a new strategy for using Trim-Away technology for treatment of diabetes.

[Preparation of monoclonal antibodies against human Apo B100 and establishment of a sandwich ELISA system].

AIM: To prepare the monoclonal antibodies (mAbs) against human Apo B100 and establish a double-antibody sandwich ELISA system for human Apo B100 detection. METHODS: The BALB/c mice were immunized with human Apo B100 antigen. After cell fusion and screening, the hybridoma cells were cultured in serum-free medium and the supernatant was purified to obtain mAbs using protein A. The affinity, subtype, specificity and epitope of the mAbs were characterized and the sandwich ELISA system was established. RESULTS: Four hybridoma cell lines 4-1-2, 4-2-2, 4-3-2 and 4-6-3 were obtained. The affinity of the anti-Apo B100 mAbs was up to 1×109 L/mol and nearly had no cross reaction with other relevant proteins. Linear detection of the sandwich ELISA system using 4-3-2 and 4-6-3 covered a range from (1.3-80) ng/mL, and its sensitivity was 1.24 ng/mL. The intra-assay coefficient of variation (CV) and inter-assay CV were less than 10% and 15%, respectively, and the recovery rate was above 90%. CONCLUSION: The mAbs against human Apo B100 have been prepared and a sandwich ELISA system for human Apo B100 detection has been established successfully, which provide a basis for human Apo B100 detection and disease diagnosis.

[The application of two-dimensional liquid phase chromatography to separation of total protein of Leymus chinensis shoot].

The total proteins of Leymus chinensis were extracted from eight-week-old seedlings grown in the greenhouse by TCA-acetone precipitation method. After the lysis buffer was replaced by the start buffer, proteins were fractionated along the first dimension using chromatofocusing (CF). Subsequently the fractions with pI values between 8.5 and 4.0 collected after the first dimension separation were further fractionated by nonporous silica reverse-phase high-performance liquid chromatography (NPS-RP-HPLC, HPRP). With ProteoVue software the pI/UV map was generated to show the protein expression profile of Leymus chinensis. Some experiments were tested to optimize the fraction procedure. Three different elution gradients were employed to get the optimal chromatogram. Comparison of the protein expression pattern between 2D-PAGE and 2D-LC indicated that 2D-LC was a powerful tool in protein fraction. Reproducibility and veracity of the protein patterns were confirmed in different injections of the same sample. A method to fractionate the total protein of Leymus chinensis shoot with two-dimensional liquid chromatography (2D-LC) was founded.