Screening and Identification of the Peptides Specifically Binding to Human Non-small Cell Lung Cancer NCI-H1299 Cells / 中国肺癌杂志

BACKGROUND@#Non-small cell lung cancer (NSCLC) is the most common histological type of lung cancer, and one of the malignant tumor with the highest mortality. As the main part of the optical molecular imaging probe, peptide can realize the early screening and diagnosis of tumor and improve the survival rate of patients. The aim of this study was to screen the small-molecule peptide that highly binds to NSCLC NCI-H1299 cells using in vivo phage display technology and to identify their binding specificity by in vitro experiment.@*METHODS@#To prepare a tumor-bearing nude mouse model of NCI-H1299 cells, after 3 rounds of in vivo screening with Ph.D.-C7CTM Peptide Library, phage clones were randomly picked, using immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) to identify the affinity of phage clones to NCI-H1299 cells. The positive monoclonal phages DNA was extracted and sequenced to obtain the amino acid sequence of the peptides. The peptides with the highest repetition rate was chemically synthesized and labeled with fluorescein (FITC) to prepare optical molecular probe. We preliminary identified the specificity of the probe binding to lung cancer cells by in vitro experiment.@*RESULTS@#After three rounds of in vivo screening, the phages enrichment rate was 341.3 times compared with the first round. Immunohistochemical staining showed that with the increase of screening times, the phages binding to tumor tissues continued to increase, and the binding amount was significantly higher than normal tissues; ELISA results showed that 20 clones among the 30 randomly selected phage clones were positive. After sequencing, the peptide with the highest repetition rate was synthesized and named NSP1; Methyl thiazolyl tetrazolium assay (MTT) and would healing assay showed that NSP1 will not affect cell proliferation and migration. Flow cytometry and immunofluorescence showed specific binding of NSP1 to NCI-H1299 cells.@*CONCLUSIONS@#We successfully obtained the peptide NSP1 that specifically binds to lung cancer NCI-H1299 cells by in vivo phage display, which provide a theoretical basis for NSCLC early diagnosis and targeted therapy.

The inhibition of accessory gene regulator C specific binding peptides on biofilm formation of Staphylococcus epidermidis on the surface of polyvinyl chloride in vitro / 中国修复重建外科杂志

Objective: To investigate the effect of accessory gene regulator C (agr C) specific binding peptides (named N1) on the biofilm formation of Staphylococcus epidermidis on the surface of polyvinyl chloride (PVC) materials in vitro. Methods: Firstly, the two strains (ATCC35984, ATCC12228) were cultured with N1 at concentrations of 100, 200, 400, 800, and 1 600 μg/mL, respectively. The control group was cultured with agrC specific binding unrelated peptides (named N0) at the same concentrations and the absorbance ( A) value was measured after 24 hours to determine the optimal bacteriostatic concentration of N1. The two strains were cultured with N1 and N0 of the optimal concentration, respectively. The A values were measured at 6, 12, 18, 24, 30, and 48 hours to observe the effect of N1 on the biofilm formation ability of Staphylococcus epidermidis. On this basis, the surface structure of the biofilm on the surface of PVC material was observed by scanning electron microscopy after 6, 12, 18, 24, and 30 hours of incubation with PVC material sheet. The thickness of the biofilm was observed by laser confocal microscopy after 6, 12, 18, and 24 hours of incubation with ATCC35984 strain. Results: The optimal bacteriostatic concentration of N1 was 800 μg/mL. ATCC 12228 strain did not form obvious biofilm after being cultured with N1 and N0. When ATCC35984 strain was cultured with N1 and N0 for 12 hours, the difference in biofilm formation ability between groups N1 and N0 was statistically significant ( P0.05). Scanning electron microscopy examination showed that mature biofilm structure was observed in ATCC35984 strain and was not observed in ATCC12228 strain. Laser confocal microscopy observation showed that the number of bacteria in the group N1 was significantly lower than that in the group N0 at 12 hours, and the most of bacteria were dead bacteria. There was no significant difference in the number of bacteria at 6, 18, and 24 hours, and the most of them were live bacteria. The biofilm thickness of group N1 was significantly lower than that of group N0 at 12 and 18 hours ( P<0.05). Conclusion: The intensity of N1 inhibiting the formation of Staphylococcus epidermidis biofilm is dose-dependent. During the aggregation period, N1 can inhibit the biofilm formation by hindering the bacterial growth and aggregation. The inhibition effect on mature biofilm is not obvious.

Screening and identification of the polypeptides specifically binding to the adhesion protein of Myco-plasma genitalium / 中华微生物学和免疫学杂志

Objective To screen and identify the polypeptides specifically binding to the adhesion protein of Mycoplasma genitalium(MgPa) by using the Ph. D.-12TM phage display peptide library for further understanding the biological function and the possible pathogenic mechanism of the MgPa. Methods The Ph. D.-12TM phage display peptide library was used for 3 rounds of biopanning with the purified recombinant MgPa ( rMgPa) as the given target. The phages were collected for amplification after biopanning. The single strand DNA of phage clones were extracted and purified by using the sodium iodide method for further se-quencing. ELISA, competitive binding assay and dot immunobinding assay were performed to analyze the specific binding of positive phages to rMgPa. Results A significant enrichment of phages was achieved after 3 rounds of biopanning. Eleven different phage exogenous sequences (P1-P11) were detected among the 38 phages randomly selected from the agar. Two core sequences were deduced according to the repeating times of amino acids among the 11 polypeptide sequences, which were V-H-W-D-F-R-Q-W-W-Q-P-S and D-W-S-S-W-V-H/Y-R-D-P-Q-T/S. Ten out of the 11 representative phages ( P1-P10 ) specifically combined with the rMgPa. Conclusion Two polypeptides specifically binding to rMgPa were successfully screened out, which provided the tool for further investigation on the biological function of MgPa and the pathogenic mecha-nism of Mycoplasma genitalium.

Selection of peptide specifically binding to bladder carcinoma by using phage display in vivo / 中国免疫学杂志

Objective:To screen the peptide binding to human bladder carcinoma cells specifically by using phage display technology in vivo.Methods: Nude mice were inoculated with bladder carcinoma cells BIU87 for establishing tumor-bearing mice model.The Ph.D.-C7CTM Peptide Library was injected intravenously via tail vein.Then we screened Phage containing exogenous peptides binding to bladder transitional carcinoma cells specifically.The phage peptide homed to the tumor tissues was obtained after 3 rounds screening in vivo.The phage clones affinity to BIU87 were identified by immunohistochemistry and ELISA.The positive peptide was synthetized by chemical methods after sequencing the positive monoclonal phage DNA.The tumor cell specificity of target peptide was identified by confocal laser scanning microscope and flow cytometry.Results:After 3 rounds screening in vivo,enrichment rate of phage was 4.334×102 times.Immunohistochemistry results showed that the dyeing of the tumor tissue had a rising trend following each round of phage screening,while liver had a lot of non-specific binding phage because the phages were metabolized through liver and kid-ney.The 30 phage clones were identified by ELISA and 10 clones had a strong affinity on BIU87 among 24 positive clones.Three amino acid sequences of positive phage clones were obtained.The highest rate of repeat sequences CSSPIGRHC(8/10) named NYZL1 and the FITC-C6-NYZL1 peptide was synthesized.Our results showed that it could bind to bladder carcinoma cells BIU87 specifically.Conclusion:We obtained the small molecular peptide NYZL1 binding to human bladder carcinoma specifically by means of phage display in vivo,which provide a theoretical basis for bladder carcinoma early diagnosis and targeted therapy.

Research progress in receptor binding assay by mass spectrometry / 中国药学杂志

OBJECTIVE: To explain that receptor-ligand binding assay is an important drug screening method, which can quickly and inexpensively study the interactions between the targeted receptor and the potential ligands in vitro and provide information about the relative binding affinity of ligand-receptor. METHODS: The correlative literature and abroad were collected and analyzed. RESULTS AND CONCLUSION: The concept of MS binding assay based on mass spectrometric quantification of nonlabeled markers represents a promising alternative to conventional radioligands binding without inherent drawbacks. The technique will play an important role in the design and screening of receptor-targeting pharmaceuticals.

Preparation of Anti-HER2 Monoclonal Antibody-paclitaxel Immunoconjugate and Its Biological Evaluation / 华中科技大学学报（医学）（英德文版）

Anti-HER2 monoclonal antibody (Sc7301)-paclitaxel (TAX) immunoconjugate was prepared and its specific binding to tumor cells was investigated in this study.Sc7301 was conjugated to　TAX by the active ester method and then the TAX-Sc7301 immunoconjugate was obtained.After purification and labeling by Cyano-fluorescein isothiocyanate (FITC),the specific binding of TAX-Sc7301to HER2-positive tumor cells (SKOV3) and HER2-negative tumor cells (HepG2) was evaluated respectively.TAX-Sc7301 (20 nmol/L) showed distinct specific binding to SKOV3 cells rather than HepG2cells.And the uptake of the immunoconjugate by SKOV3 cells was increased with the TAX-Sc7301concentration (3-48 nmol/L) and the incubation time (P＜0.05).It was concluded that the TAX-Sc7301immunoconjugate is potentially applicable as a targeted agent against HER2-positive tumor cells.

Development of an Immuno-PCR Protocol for Detection of a Small Amount of Antigen / 대한진단검사의학회지

BACKGROUND: Immuno-PCR has been known as a highly sensitive and specific method, yet no standardized protocol is available. We analyzed each step of immuno-PCR to develop a reliable standardized method. METHODS: We made a protocol modified from several methods reported previously, and performed immuno-PCR, but false positive reactions were noted. To reduce the false positivity, we investigated the buffer reagents and biotin-labelled oligo-nucleotide probe. Using a finally determined protocol, we compared the detection-limits of the immuno-PCR and ELISA methods. RESULTS: Streptavidin was identified as a main reagent causing a non-specific binding, thus it was replaced by neutravidin. The employment of CAS block as a dilution buffer for the biotin-labelled oligo-nucleotide probe and Casein block as a buffer for the detection antibodies resulted in a dramatic reduction in the false positive reactions. The standardized immuno-PCR detected angiogenin antigen at a concentration as low as 5 fg/mL, while an ELISA method detected 5 pg/mL. CONCLUSIONS: The immuno-PCR procedure newly described in this study was ultra-sensitive with no false positivity. This method can be utilized as an epochal tool for detection of a small amount of antigen which would not be discovered by ELISA method.

Binding experiments of 911-FITC with Immunocytes / 中国海洋药物

Lymphocytes from thymus, spleen, macrophages and endothelial cells w.ere incubated with the fluorescent probe of 911-FITC and the colouring method with flow cytometry and fluorescent microscope was reviewed in this paper. The results indicated that 911-FITC could specifically bind to lymphocytes, macrophages and endothelial cells. These findings suggested that 911 could play a immunomodulatory effect by direct combination with immu-nocytes.

Specific binding of folate conjugated PGA to FR-positive tumor cells / 中国药理学通报

Aim To investigate the specific binding of folate conjugated PGA to FR-positive tumor cells.Method Folate-PGA and PGA were radiolabeled with 125I by the Iodogen method to examine the binding of PGA to FR positive HeLa cells and SKOV3 cells, or FR negative A549 cells. Results 125I-folate-PGA showed specific bound to HeLa cells and SKOV3 cells; Scatchard analysis of the data estimated the Kd of binding to be 0.11 nmol?L-1 and 0.25 nmol?L-1 respectively. 125I-folate-PGA showed virtually little specific binding to A549 cells which lack folate receptors. Conclusions folate-PGA displayed high affinity and good targeting activities for FR-positive tumor cells and the data warranted further studies for enzyme prodrug therapy.