Objective To study the role of protective antigen(PA)and N-terminal segment of lethal factor (LFn)in the entrance of EGFP(enhanced green fluorescent protein)into HeLa cells. Methods The DNA fragments encoding LFn and EGFP were amplified,respectively,and cloned into the plasmid pET-21 a(+)one after another to construct a recombinant plasmid pET-LFn-EGFP. The plasmid was txansformed into BL21 cells to express LFn-EGFP protein under the induction of IPTG. The protein was purified by Ni chelating chromatography. After incubation with LFn-EGFP in the presence of PA or not, the HeLa cells were analyzed by flow cytometry or laser confocal microscopy. Results The fusion protein LFn-EGFP was purified by over 90% homogeneity and retained the ability of LF to bind with PA when incubated with J774A.1 macrophage cells,and could get into HeLa cells. Conclusion The LFn-EGFP could enter the HeLa cells in a PA independent pathway. But PA could help more LFn-EGFP molecules enter into HeLa cells.

Recombinant Fl-V (rFl-V) fusion protein is the main ingredient of the current candidate vaccine against Yersinia pestis infection, which has been under investigation in clinical trial in USA. We investigated the soluble expression conditions of rF1-V in Escherichia coli BL21 (DE3) that we constructed before. After scale-up and optimization of fermentation processes, we got the optimized fermentation process parameters: the culture was induced at the middle exponential phase with 50 µmol/L of IPTG at 25 °C for 5 h. Soluble rFl-V protein was isolated to 99% purity by ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography and gel filter chromatography. The protein recovery was above 20%. Protein identity and primary structure were verified by mass spectrometry and Edman sequencing. Results of purity, quality and western blotting analysis indicated that the target protein is a consistent and properly folded product. Furthermore, the immunogenicity of various antigens formulated with aluminum hydroxide adjuvant was evaluated in mice. Serum antibody titers of 4 groups including 20 µg rFl, rV and rFl-V and 10 µg rFl+10 µg rV, were assayed by ELISA after 2 doses. The antibody titers of anti-Fl with 20 µg rFl-V were obviously higher than titers with other groups; meanwhile there were no significant difference of anti-V antibody titers among them. These findings confirm that rFl-V would be the active pharmaceutical ingredient of the plague subunit vaccine.
Adjuvants, Immunologic ; Animals ; Antibodies, Bacterial ; blood ; Antibody Formation ; Antigens, Bacterial ; immunology ; Blotting, Western ; Chromatography, Ion Exchange ; Enzyme-Linked Immunosorbent Assay ; Mice ; Plague ; prevention & control ; Plague Vaccine ; immunology ; Recombinant Fusion Proteins ; immunology ; Vaccines, Subunit ; immunology ; Yersinia pestis

Objective:To understand the current status of screening, diagnosis, and treatment and analyze the factors influencing micro-elimination strategy, so as to achieve hepatitis C elimination in hospital.Methods:Anti-HCV and HCV RNA test results of patients from October 2017 to September 2020 were retrospectively analyzed. Anti-HCV positive rates and factors influencing different genders, ages, places of residence and departments were analyzed. After comparing anti-HCV-positive patients with HCV RNA-positive patients with duplicate entries in "Name" and "Date of birth", the data were divided into three categories: anti-HCV positive without HCV RNA test, HCV RNA positive in single test, and HCV RNA positive many times in multiple tests. The above three types of patients were followed-up by telephone. According to the hospital follow-up results, current status of diagnosis and treatment and the factors influencing the micro-elimination strategy of hepatitis C were studied and analyzed. The comparison of data between groups were performed using χ2 or χ2 continuity-correction test. Results:Anti-HCV positive detection rate was 1.34% (899/66 866). The positive rate of male patients aged 40 and over residing in cities was significantly higher than female patients under 40 years old residing in rural areas, and the difference was statistically significant ( χ2 = 55.178, 264.11, 36, 351, P < 0.05). There were 90 (10.02%) and 809 cases (89.98%) in outpatient and inpatient departments, respectively, with no statistically significant difference between the two ( χ2 = 0.002, P > 0.05). The total number of anti-HCV positive cases were 196 in Gastroenterology (22.0%), 75 in Respiratory and Critical Care Medicine (8.3%), 74 in Neurology (8.2%), 63 in Orthopedics (7.0%) and 55 in Endocrinology departments (6.1%), and the difference in the positive rate among different departments were also statistically significant ( χ2 = 271.585, P < 0.05). Among the 480 cases who were followed-up, 215 (44.79%) were lost to follow-up, 84 cases (39.07%) were unregistered, 77 cases (16.04%) were untreated, 15 cases (19.48%) were unaware of their state of illness, 46 cases (59.74%) were diagnosed without concern, 16 cases (20.78%) were diagnosed but did not take medicine, 60 cases were under treatment, and 29 cases were mostly on counterfeit drugs (48.33%). Conclusion:Comprehensive diagnosis and treatment education to non-specialist clinicians and timely manner regular follow-up of patients is a key factor and an important link to formulate a simple, easy and sustainable model to improve the efficiency of screening, diagnosis, and treatment of hepatitis C micro-elimination strategy in hospital. In addition, it will also play an important role in achieving the strategic goal of "eliminating hepatitis C as a public health threat by 2030".