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*

Objective:To investigate the effects of iron metabolism and oxidative stress level on blood glucose control during pregnancy in patients with gestational diabetes mellitus (GDM).Methods:A total of 139 pregnant women who received prenatal examination between January 2020 and June 2021 in Wenzhou Central Hospital were included in this study. They were divided into GDM group ( n = 68) and control group ( n = 71) according to oral glucose tolerance test results at 24-48 weeks of gestation. Clinical data were collected. Iron metabolism, oxidative stress and blood glucose levels were measured. The relationships between iron metabolism and oxidative stress levels and blood glucose control in GDM were analyzed. Results:There was no significant difference in age between the GDM and control groups ( P > 0.05). Body mass index, fasting blood glucose, fasting insulin, glycosylated hemoglobin, nuclear factor-κB (NF-κB), malondialdehyde (MDA), ferritin (SF), serum iron, transferrin (TRF) and insulin resistance index (IRI) in the GDM group were (24.11 ± 3.05) kg/m 2, (4.92 ± 0.67) mmol/L, (10.56 ± 2.21) pmol/mL, (6.15 ± 0.62)%, (20.50 ± 1.72) μg/L, (20.34 ± 2.92) μmol/L, (70.77 ± 7.01) μg/L, (30.18 ± 4.25) μmol/L, (3.93 ± 0.69) g/L and (2.50 ± 1.03), respectively, which were significantly higher than those in the control group [(21.41 ± 2.86) kg/m 2, (4.69 ± 0.62) mmol/L, (5.76 ± 2.09) pmol/mL, (5.37 ± 0.58)%, (15.43 ± 1.55) μg/L, (12.93 ± 2.17) μmol/L, (42.53 ± 8.86) μg/L, (18.81 ± 3.85) μmol/L, (2.89 ± 0.53) g/L and (1.74 ± 0.89)] ( t = 5.39, 2.10, 13.16, 7.66, 18.27, 17.03, 20.78, 16.54, 9.99, 4.66, all P < 0.05). Superoxide dismutase (SOD), total antioxidant capacity (TAOC) and insulin sensitivity index in the GDM group were (21.49 ± 3.52) U/L, (10.87 ± 1.34) kU/L and (3.28 ± 0.46), respectively, which were significantly lower than those in the control group [(26.28 ± 3.95) U/L, (13.28 ± 1.52) kU/L, (3.86 ± 0.53), t = 7.54, 9.90, 6.88, all P < 0.05]. Multivariate logistic regression analysis revealed that SOD, TAOC, NF-κB, MDA, SF and TRF were independent influential factors of GDM occurrence [ OR (95% CI) = 1.57 (1.09-2.26), 3.15 (1.71-5.80), 2.18 (1.32-3.61), 3.27 (1.58-6.76), 2.12 (1.29-3.50), 1.23 (0.99-1.53), 3.65 (1.89-7.04), all P < 0.05]. SOD and TAOC levels were negatively correlated with IRI ( r = -0.75, -0.84, both P < 0.05), while NF-κB, MDA, SF, serum iron and TRF were positively correlated with IRI ( r = 0.93, 0.96, 0.98, 0.07, 0.92, all P < 0.05). Conclusion:Increased levels of iron metabolism and oxidative stress are risk factors for the occurrence of GDM, and they are closely related to the degree of insulin resistance. GDM screening should be carried out in advance in pregnant women with increased levels of iron metabolism and oxidative stress indicators, which plays a positive role in clinical diagnosis and treatment of GDM.

Animals ; Antibodies, Neutralizing ; Antibodies, Viral ; Foot-and-Mouth Disease Virus

Objective To investigate the clinical efficacy of purging fu-organs traditional Chinese medicine (TCM)therapy for treatment of patients with severe pneumonia and sthenia-heat. Methods According to random number table method,71 patients with sthenia-heat of severe pneumonia were divided into a treatment group (35 cases)and a control group(36 cases). Conventional basic treatment was given to both groups,and additionally, small chengqi decoction was applied nasogastrically for the therapy in treatment group for 2 weeks. The clinical pulmonary infection score(CPIS),Marshall score,integration score of TCM syndromes and the mortalities in 28 days and 60 days were used to compare the clinical efficacy of the two groups. Results With the prolongation of treatment,the CPIS,Marshall score and integration score of syndromes in the two groups were gradually decreased. In treatment group,CPIS and Marshall scores were lower than those of control group on the 4th day ,and there were statistically significant differences(CPIS score:5.8±1.7 vs. 6.8±1.9,Marshall score:5.3±2.3 vs. 6.6±2.7,both P<0.05);the above 2 scores in treatment group were also lower than those of control group on the 7th and 14th day after treatment(7th day CPIS score:5.3±1.5 vs. 5.6±1.4,Marshall score:5.1±1.9 vs. 5.7±1.8;14th day CPIS score:3.9±1.7 vs. 4.4±2.3,Marshall score:4.2±1.9 vs. 4.9±2.5),but there were no statistically significant differences(all P>0.05). In addition,the integration scores of syndromes were significantly decreased on the 4th, 7th and 14th day in the treatment group significantly lower than those in the control group(4th day:7.6±2.3 vs. 10.6±2.7,7th day:7.4±2.5 vs. 9.2±2.1,14th day:6.1±1.9 vs. 8.3±2.4,all P<0.05). However,there were no statistically significant differences in mortality rates in 28 days and 60 days respectively between control group and treatment group(28 days:16.7% vs. 11.4%,60 days:25.0% vs. 20.3%,both P>0.05). Conclusion Purging fu-organs therapy not only can decrease the CPIS and Marshall scores of patients with sthenia-heat of severe pneumonia,but also can improve their syndromes.