Pseudorabies (PR) is an infectious disease caused by the pseudorabies virus (PRV), affecting various domesticated and wild animals. Since pigs are the only natural hosts of PRV, PR poses a serious threat to the pig farming industry. Currently, PR is primarily prevented through vaccination with inactivated vaccines or genetically modified attenuated live vaccines. Developing safe and effective genetically engineered vaccines would facilitate the eradication and control of PR. In this study, the PRV vaccine strain Bartha-K61 was used as the reference strain. The gB protein was expressed via the baculovirus-insect cell expression system. Non-denaturing gel electrophoresis confirmed that the gB protein could form a trimeric structure. The purified gB protein was used to immunize mice, and the immune effect was evaluated by a challenge test. The results showed that the gB antigen induced a strong immune response in mice, with the serum-neutralizing antibody titer above 1:70. The lymphocyte stimulation index reached more than 1.29, and the level of (interferon gamma, IFN-γ) release was higher than 100 pg/mL. After immunization, mice were challenged with the virus at a dose of 10⁴ TCID₅₀/mL, 200 μL per mouse, and the clinical protection rate was 100%. Immunohistochemistry, histopathological section, and tissue viral load results showed that the pathological damage and viral load in the gB-immunized group were significantly lower than those in the PBS group. In summary, the gB protein obtained in this study induced strong humoral and cellular immune responses in mice, laying a foundation for developing a recombinant gB protein subunit vaccine.
Animals ; Mice ; Baculoviridae/metabolism* ; Viral Envelope Proteins/biosynthesis* ; Herpesvirus 1, Suid/genetics* ; Pseudorabies/immunology* ; Swine ; Pseudorabies Vaccines/genetics* ; Antibodies, Viral/blood* ; Insecta/cytology* ; Mice, Inbred BALB C ; Female ; Viral Vaccines/immunology*

The variable domain of heavy-chain antibody (VHH) has been developed widely in drug therapy, diagnosis, and research. Escherichia coli is the most popular expression system for VHH production, whereas low bioactivity occurs sometimes. Mammalian cells are one of the most ideal hosts for VHH expression at present. To improve the yield of VHH in Expi293F cells, we optimized the signal peptide (SP) and codons of VHH. Firstly, the fusion protein VHH1-Fc was used to screen SPs. The SP IFN-α2 showed the highest secretion as quantified by enzyme-linked immunosorbent assay (ELISA). Subsequently, codon optimization by improving GC3 and GC content doubled the yield of VHH1 and kept its binding activity to Senecavirus A (SVA). Finally, the mean yields of other 5 VHHs that fused with SP IFN-α2 and codon-optimized were over 191.6 mg/L, and these VHHs had high recovery and high purity in the culture supernatant. This study confirms that SP IFN-α2 and codon optimization could produce VHHs in Expi293F cells efficiently, which provides a reference for the large-scale production of VHHs.
Codon/genetics* ; Protein Sorting Signals/genetics* ; Escherichia coli/metabolism* ; Humans ; Recombinant Fusion Proteins/biosynthesis* ; Interferon-alpha/metabolism* ; Immunoglobulin Heavy Chains/immunology* ; Cell Line ; Immunoglobulin Variable Region/immunology*

Transient expression is the major method to express foot-and-mouth disease virus (FMDV) capsid proteins in mammalian cells. To achieve stable expression of FMDV capsid proteins and efficient assembly of virus like particles (VLPs) in cells, the plasmids of piggyBac (PB) transposon-constitutive expression and PB transposon-tetracycline (Tet) inducible expression vectors were constructed. The function of the plasmids was tested by fluorescent proteins. By adding antibiotics, the constitutive cell pools (C-WT, C-L127P) expressing P12A3C (WT/L127P) genes and the inducible cell pools (I-WT, I-L127P) expressing P12A3C (WT/L127P) genes were generated. The genes of green fluorescent protein, 3C protease and reverse tetracycline transactivator (rtTA) were integrated into chromosome, which was confirmed by fluorescence observation and PCR testing. The cell pool I-L127P has a stronger production capacity of capsid proteins and VLPs, which was confirmed by Western blotting and enzyme linked immunosorbent assay (ELISA), respectively. In conclusion, inducing the chromosomal expression of FMDV capsid proteins was firstly reported, which may facilitate the technical process of mammalian production of FMDV VLPs vaccine and the construction of mammalian inducible expression systems for other proteins.
Animals ; Foot-and-Mouth Disease Virus/genetics* ; Capsid Proteins ; Viral Proteins/metabolism* ; Foot-and-Mouth Disease/prevention & control* ; Tetracyclines/metabolism* ; Viral Vaccines ; Antibodies, Viral ; Mammals/metabolism*

Objective To study the effects of duration of migration from sea-level to high-altitude on cerebral hemodynamic parameters and their ethnic differences.Methods This randomized comparative study recruited 5 groups of healthy male subjects which were native Hans at sea-level (NHS),Han migrants from sea-level to high-altitude (HMSH,where HMSH-a for residence duration of 4 days,HMSH-b for 1 year,and HMSH-c for 5 years),and native Tibetans at high-altitude (NTH).Color duplex Doppler sonography (CDDS) was used to measure bilateral vertebral and internal carotid arterial diameters,mean flow velocities.The heart rate,arterial blood pressure,and arterial oxygen saturation were also recorded simultaneously,and in combination,hemoglobin was measured to assess total cerebral blood flow (TCBF),total cerebrovascular resistance (TCVR),and total cerebral oxygen supply (TCOS).Results After migration to high-altitude,Hans' TCVR and TCBF maintained the level of NHS after transient changes during early stage (P<0.05),and the TCBF was markedly higher than that of NTH (P<0.05);while the blood pressure and TCOS increased abruptly (P<0.05) at the beginning and kept for a relatively long time at a constant level which were higher than those of NTH all the time (P<0.05).Conclusions Han migrants can acclimate to high-altitude by adaptive change of cerebral blood flow over a short time and maintain a constant adaptability which is always weaker than that of native Tibetans.CDDS can be used for non-invasive measurement of actual cerebral blood flow,and is of good value in the study on high-altitude-related cerebral hemodynamics.

OBJECTIVETo investigate the frequency of p16 and p15 gene methylation in multiple myeloma (MM), and its relationship with bone marrow cell apoptosis and clinical outcome.

METHODSTwenty-two patients with MM were studied to detect p16 and p15 gene methylation. Methylation-specific polymerase chain reaction (MSP) was used to detect gene methylation, and terminal transferase-mediated dUTP nick end-labeling (TUNEL) was used to detect cell apoptosis.

RESULTSp16 and/or p15 gene methylatoin was detected in 10 of 22 patients (45.4%). There were 3 patients with p16 gene methylation, 9 patients with p15 gene methylation, and 2 patients with both genes methylation. The incidence of methylation of p15 gene was higher than that of p16 gene (P < 0.05). The patients with p16 and/or p15 gene methylation had a delayed cell apoptosis, poor response to chemotherapy, and a short over-all survival (OS).

CONCLUSIONThe methylation of p16 and/or p15 gene plays a key role in MM apoptosis pathogenesis. The patients with both p16 and p15 gene methylation had a poor prognosis.

Apoptosis ; Cell Cycle Proteins ; Cyclin-Dependent Kinase Inhibitor p15 ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; DNA Methylation ; DNA, Neoplasm ; genetics ; Gene Silencing ; Genes, p16 ; Humans ; Multiple Myeloma ; genetics ; pathology ; Prognosis ; Transcription Factors ; genetics ; Tumor Suppressor Proteins

Objective:To discuss the relationship between blood-lipid change and acute cerebrovascular disease (ACVD) and tocompare the blood-lipid levels in the patients with cerebral hemorrhage (CH group) with those in the patients with cerebralinfarction (CI group). Methods:The blood-lipid levels in 300 patients with ACVD (ACVD group) and 100 patients withother system disease (control group) were tested with automatic biochemistry analysor and compared between the twogroups. Results:There were significant differences between ACVD group and control group as well as between CH group andCI group. Conclusion:Although there were blood-lipid metabolic disturbances in both CH and CI patients, their degrees weresignificantly different, which should be closely observed and controlled in clinic.

Objective To investigate the incidence of p16 gene methylation in hematological malignancies, and the relations between p16 gene methylation and the prognosis of hematological malignancies. Methods Fifty-five patients were studied by restriction enzyme polymerase chain reaction (PCR) to detect p16 gene methylation. Results p16 gene methylation was detected in 6 of 55 patients (10.9%), who were 1 patient with M2a, 1 patient with M5a, 2 patients with chronic myelogenous leukemia (CML) in blast crisis, 1 patient with progressing multiple myeloma (MM), 1 with non-Hogkin's lymphoma(NHL) accompanied by B-ALL, respectively. p16 gene methylation correlates with adverse prognostic features. The patients with p16 gene methylation had poor response to therapy, and died shortly after p16 gene methylation was detected. Conclusion The methylation of p16 gene plays an improtant role in the pathogenesis and the development of some hematological malignancies. The patients with p16 gene methylation have poor prognosis. The detection of p16 gene is a useful tool for evaluating prognosis of hematological malignancies.

Cerebral topographic map (CTM) and electroencephalogram (EEG) can evaluate the functional activity of brain, and the purpose of this study was to objectively compare the effect of intravenous anesthetics on brain. Forty-five patients, ASA grade Ⅰ-Ⅱ, undergoing neurosurgery,were randomly divided into three groups after administration of Innovar 2 ml, and then equivalent effective dose of propofol(2～2.5 mg/kg)(Ⅰ),sodium thiopental (4～6mg/kg)(Ⅰ) or gama hydroxybutyrate sodium (70～80mg/kg)(Ⅲ) was intravenously given respectively. All patients were monitored continuously during induction. The high frequent excitative activities of ? rhythm appeared immediately after each anesthestic was injected. During the most effective period: in group Ⅰ, there were no ? rhythm in EEG and obvious power change on CTM; in group Ⅱ irregular ? rhythm took place in EEG occasionally and great increase in top power on CTM occured, which showed evident elevation compared with group Ⅰ (P