Regression analysis to select native-like structures from decoys of antigen-antibody docking / 生物工程学报

Given the increasing exploitation of antibodies in different contexts such as molecular diagnostics and therapeutics, it would be beneficial to unravel properties of antigen-antibody interaction with modeling of computational protein-protein docking, especially, in the absence of a cocrystal structure. However, obtaining a native-like antigen-antibody structure remains challenging due in part to failing to reliably discriminate accurate from inaccurate structures among tens of thousands of decoys after computational docking with existing scoring function. We hypothesized that some important physicochemical and energetic features could be used to describe antigen-antibody interfaces and identify native-like antigen-antibody structure. We prepared a dataset, a subset of Protein-Protein Docking Benchmark Version 4.0, comprising 37 nonredundant 3D structures of antigen-antibody complexes, and used it to train and test multivariate logistic regression equation which took several important physicochemical and energetic features of decoys as dependent variables. Our results indicate that the ability to identify native-like structures of our method is superior to ZRANK and ZDOCK score for the subset of antigen-antibody complexes. And then, we use our method in workflow of predicting epitope of anti-Ebola glycoprotein monoclonal antibody-4G7 and identify three accurate residues in its epitope.

Expression, purification and characterization of diphtheria toxin mutant CRM197 in Eschrichia coli / 生物工程学报

CRM197 (cross-reacting material 197), a non-toxic mutant of diphtheria toxin, has wide application potential in biopharmaceuticals. However, it is difficult to express CRM197 in bacteria other than Corynebacterium diphtheriae. Here we proposed a new alternative method to produce soluble CRM197 without label in Escherichia coli. In particular, a synthetic gene coding for CRM197, optimized for E. coli codon usage, was cloned in the pET32a (+) vector. Accordingly, the over-expression of the protein was simply induced with IPTG in E. coli BL21 (DE3). The target protein was soluble and accounted for about 40% of the total protein in the supernatant. Following an ultrasonic cytolysis step, the recombinant protein was purified by anion exchange, affinity and desalting chromatography and the purity of the final preparation reached 95%. Cytotoxicity tests showed that the IC₅₀ value of CRM197 was 2.1×10⁷ times the IC₅₀ value of diphtheria toxin, and 9.6 times the IC50 value of diphtheria toxoid, telling that the target protein is safe and non-toxic. Subsequently, we found that both the high dose (20 μg) and the low dose (2 μg) of CRM197 were equally efficient in inducing an immune response against diphtheria toxiod in mice, and the antibodies titer of mice after three immunizations with low dose could reach 1:409 600. In conclusion, our findings provide a highly efficient strategy for the rapid production and purification of unlabeled and soluble recombinant CRM197 in E. coli, with good immunogenicity and safety.

Screening of full human anthrax lethal factor neutralizing antibody in transgenic mice / 生物工程学报

Anthrax is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis. The major virulence factor of B. anthracis consists of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA binds with LF to form lethal toxin (LT), and PA binds with EF to form edema toxin (ET). Antibiotics is hard to work in advanced anthrax infections, because injuries and deaths of the infected are mainly caused by lethal toxin (LT). Thus, the therapeutic neutralizing antibody is the most effective treatment of anthrax. Currently most of the anthrax toxin antibodies are monoclonal antibodies (MAbs) for PA and US FDA has approved ABTHRAX humanized PA monoclonal antibody for the treatment of inhalational anthrax. Once B. anthracis was artificially reconstructed or PA had mutations within recognized neutralization epitopes, anti-PA MAbs would no longer be effective. Therefore, anti-LF MAbs is an important supplement for anthrax treatment. Most of the anti-LF antibodies are murine or chimeric antibodies. By contrast, fully human MAbs can avoid the high immunogenicity of murine antibodies. First, we used LF to immunize the transgenic mice and used fluorescent cell sorting to get antigen-specific memory B cells from transgenic mice spleen lymphocytes. By single cell PCR method, we quickly found two strains of anti-LF MAbs with binding activity, 1D7 and 2B9. Transiently transfected Expi 293F cells to obtain MAbs protein after purification. Both 1D7 and 2B9 efficiently neutralized LT in vitro, and had good synergistic effect when mixed with anti-PA MAbs. In summary, combining the advantages of transgenic mice, fluorescent cell sorting and single-cell PCR methods, this study shows new ideas and methods for the rapid screening of fully human monoclonal antibodies.

Pilot-scale purification of rF1-V fusion protein of Yersinia pestis and characterization of its immunogenicity / 生物工程学报

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.

Single B cell monoclonal antibody technologies and applications / 生物工程学报

Monoclonal antibodies (mAbs) contribute a lot to the development of numerous fields in life science as a pivotal tool in modern biological research. Development of the PCR methods and maturation of antibody production have made it possible to generate mAbs from single human B cells by single cell RT-PCR with successional cloning and expression in vitro. Compared to traditional monoclonal antibody technologies, single B cell technologies require relatively fewer cells, which are highly efficient in obtaining specific mAbs in a rapid way with preservation of the natural heavy and light chain pairing. With so many advantages, single B cell technologies have been proved to be an attractive approach for retrieval of naive and antigen-experienced antibody repertoires generated in vivo, design of rationale structure-based vaccine, evaluation and development of basic B cell biology concepts in health and autoimmunity, and prevention of infectious diseases by passive immunization and therapy for disorders. Accordingly, this review introduced recent progresses in the single B cell technologies for generating monoclonal antibodies and applications.

Production and immunogenicity analysis of conformation-stable fragment-C mutant of tetanus toxin / 生物工程学报

Tetanus is caused by tetanus toxin synthesized by Clostridium tetani. Fragment C (Hc), the 50 kDa carboxy-terminal portion of tetanus toxin, is nontoxic but has receptor protein binding activities, which has been evaluated as a potential new recombinant subunit vaccine to replace the traditional formaldehyde inactivated toxoid vaccine. It is easy for wild Hc (HcW) to form inter- and intra-molecular disulfide bonds and the different conformations changes unstably, which brings difficulties for vaccine production technology. In our study, the Cys 869 of HcW was mutated to A1a and the conformation-stable fragment-C mutant of tetanus toxin (HcM) was constructed. The HcM was expressed, fermented and purified and its stability, receptor binding and immunogenicity were evaluated. The result showed that the HcM got high-level expression and was purified to > 95% of purity. The purified HcM was conformation-stable at different temperature for different time and kept the binding activities with one of its receptor GT1b. Mice given three vaccinations by HcM developed a protective immune response and were 100% protected against an intraperitoneal administration of 1 x 10(3) 50% lethal doses (LD50s) of tetanus neurotoxin. All the results showed that the conformation-stable HcM had potent immunogenicity as a recombinant tetanus vaccine candidate with simple production process and similar immunogenicity with HcW. Whether for routine tetanus therapy or for countries to respond to unexpected events (war, earthquake or other disaster), it is of great significance.

Rapid genetic characterization of a novel Enterobacteria phage and determination of its host recognizing genes / 生物工程学报

We isolated a novel Enterobacteria phage IME08 from hospital sewage, then confirmed it was a double-stranded DNA phage by digesting its genetic material with DNase I, RNase A and several restriction endonucleases respectively. BLAST results of random fragments generated by a random PCR cloning method revealed that it belonged to T4-like virus. We subsequently determined the host recognizing genes (g37 and g38) sequence with a PCR-based "genome jumping" protocol based on highly conserved region at 5' terminus of g37 from four other T4-like Bacteriophages (T4, JS98, T2 and K3). These molecular biological methods enabled us to readily characterize the bacteriophage and efficiently determine the sequence of the genes of interest based on very limited conserved sequence information.

The relationship between carotid atherosclerosis and cerebral infarction recurrence / 中华神经科杂志

Objective To study the clinical significance of carotid atherosclerosis and the relationship between carotid atherosclerosis and cerebral infarction recurrence.Methods Carotid atherosclerosis was assessed using doppler ultrasongraphy in 312 patients with cerebral infarction, the duration of following up was 12 to 18 months,and the characteristics of carotid atherosclersis was compared to patients with or without recurrence cerebral infarction.Results Of the 312 patients with cerebral infarction, 61 patients suffered from new cerebral infarction during following up period, the recurrence rate in patients with atherothrombotic brain infarction was higher than those with lacunar infarction, and the cerebral infarction recurrence usually occurred in the same side of initial stroke of the 57 patients with severe carotid atherosclerosis, 26 patients had a sufference of new infarction, of the 48 patients with high grade stenosis, 25 had a sufference of new cerebral infarction, of the 42 patients with ulcerated plaque, 23 had sufference of new cerebral infarction,showing a recurrence rate significantly higher than those patients with non carotid atherosclerosis or those with mild carotid atherosclerosis. The logistic regression analysis showed that the levels of carotid atheroslerosis , stenosis and ulcerated plaque were positively related to the cerebral infarction recurrence.Conclusions There is a positively relationship between carotid atherosclerosis and cerebral infarction recuurrence, and the level of carotid atherosclerosis is a risk factor of cerebral infarction recurrence.It serves as a risk marker of cerebral infarction recurrence.

Clinical investigation of low molecular weight heparin on acute cerebral infarction / 中国综合临床

Objective To observe the clinical effect of low molecular weight heparin on the acute cerebral infarction (ACI) and the influence on hemodynamics.Methods 143 cases of ACI within 72 hours of the onset of symptoms were randomly divided into two groups:low molecular weight heparin (LMWH) and control groups.The LMWH group was added 0.4 ml LMWH subcutaneously twice a day for 10 days,in addition to the routine treatment.The indexes of hemodynamics were measured before and after treatment.Neurological scoring was used to evaluate the clinical effect.Results The effect in the treatment group was significantly superior to that in the control group (P

The Effects of Carotid Arteriosclerosis on Cognitive Functions and Recent Prognosis in Patients with Cerebral Infarction / 中国临床心理学杂志

Objective: To investigate the effects of carotid arteriosclerosis on cognitive functions and current prognosis in patients with cerebral infarction. Methods: Extracranial carotid arteries were assessed using dopple ultrasongraphy in 112 patients with cerebral infarction. Cognitive functions were evaluated with the Mini-Mental state Examination (MMSE) and five neuropsychological tests assessing memory, attention, calculation psychomotor rapidity and visuospatial perception. Deficits in neurological functions were assessed on admission and 3 to 4 weeks. Results:All neuropsychological measures were found to be poorer in patients with carotid arteriosclerosis than those with no carotid arteriosclerosis, especially in cases with severe carotid arteriosclerosis and severe carotid stenosis. There was a positive relationship between severity of carotid arteriosclerosis and change in cognitive functions. The scores of SSS were higher on admission in cerebral infarction patients with carotid arteriosclerosis. Recent prognosis was also poorer in patients with carotid arteriosclerosis.Conclusions: Significant effect of carotid arteriosclerosis was shown on cognitive functions of patients with cerebral infarction. As cerebral ischemic injury is severe, prognosis in cerebral infarction patients with carotid arteriosclerosis is poor.