Objective To observe and compare the atomic force microscopy (AFM) images of Yersinia pestis EV76 and the changes in the morphology of the bacteria treated with normal serum and F1 antibody from rabbit,and to explore the immunoassay method to detect Yersinia pestis by AFM. Methods The Yersinia pestis were treated with normal serum and F1 antibody from rabbit and control buffer. All the prepared samples were observed and analyzed by AFM. The changes in the cell surface structures were probed and characterized through sectional analysis,especially the changes of Ra and Rq value. Results The normal morphology of Yersinia pestis was oval in shape with a relatively smooth surface, the size dimension of which was about 1.1-1.3 μm in length with a section profile of 0.8-1.0 μm in width and 0.04-0.06 μm in step height. The step height of the bacteria treated with the normal serum and F1 antibody was obviously enlarged. The shape of the bacteria treated with F1 antibody changed irregularly. Furthermore, the surface of the bacteria was more roughened. Conclusion The morphological characters of Yersinia pestis has been acquired through its AFM images. The morphology of Yersinia pestis treated with F1 antibody has changed greatly, and the index of roughness can be regarded as the distinguished index to detect Yersinia pestis by AFM.

Objective: To design and prepare a novel bi-specific chimeric antigen receptor (CAR)-T cell targeting both CD20 and CD19 antigen on B lymphocyte surface, and to detect its killing effect on B lymphocyte tumors as well as its treatment efficacy on immunodeficiency B-NSG mouse with leukemia. Methods: Bi-specific CAR molecule of CD20 (human originated)/CD19 (murine originated) scFv was constructed and packaged into lentiviral vector in 293 cells, and then transfected into T lymphocytes from healthy donors to prepare BiCAR-T cells. K562-CD19-GFP cells (with positive CD19 expression), K562-CD20-GFP cells (with positive CD20 expression) and Nalm6-Luc-GFP cells expressing luciferase were constructed as target cells. After being co-incubated with above mentioned targets cells, the cytotoxic effects of BiCAR-T cells on target cells were evaluated via LDH release assay, and the secretion of IFN-γ by BiCAR-T cells was evaluated by ELISA. Nalm6-Luc-GFP cells were used to construct the mouse model of leukemia and BiCAR-T cells were transfused via tail veins; the treatment efficacy of BiCAR-T cells on tumor bearing mice was evaluated with small animal imaging method. Results: After 7 days’incubation, the BiCAR-T cells originated from healthy donors amplified about 20-50 times with a positive rate of 10%~92%, indicating successful preparation of BiCAR-T cells. Under an effector∶target ratio of 10∶1, the killing rates of BiCAR-T cells against Nalm-6, K562-CD19-GFP and K562-CD20-GFP cells were significantly higher than that of control cells [(76.7±7.4)% vs (8.7±2.4)%, (93.3±5.2)% vs (46.7±6.2)%, (51.0±0.8) vs (30.7±0.5)%, all P<0.01]. Compared with control group, BiCAR-T cells co-incubated with Nalm-6 cells also secreted significantly more IFN- γ [(872.7±7.7) vs (101.0±5.3) pg/ml, P<0.01). Animal experiment showed that BiCAR-T cells had significant efficacy on B-NSG mice with leukemia; NSG leukemia mice treated with BiCAR-T cells all lived up to 70 days (till they were mercy killed) and leukemia cells disappeared at about 50 days, while the mice in PBS and T lymphocytes group all died at (19±3) d and (20±1) d, respectively. Conclusion: Bi-specific CAR molecules expressing CD19 and CD20 were successfully designed and BiCAR-T cells were successfully prepared. The BiCAR-T cells can effectively kill CD19 and/or CD20 tumor cells and secret large amounts of IFN-γ after co-incubation with target cells, exerting significant treatment efficacy on B-NSG immunodeficiency mouse with leukemia.