Immunogenicity of quadrivalent influenza virus subunit vaccine combined with RFH01 adjuvant in mice / 中华微生物学和免疫学杂志

Objective:To evaluate the immunogenicity of a quadrivalent subunit vaccine combined with RFH01 adjuvant in a mouse model.Methods:Identification tests were performed on four monovalent influenza virus subunit vaccine stock solutions according to the methods described in Part 3 of the Chinese Pharmacopoeia 2020 Edition. In the study of the quadrivalent subunit vaccine combined with RFH01 adjuvant, 460 female BALB/c mice (6-8 weeks old) were randomly divided into 46 groups including experimental groups, vaccine control group, negative control group and blank group with 10 mice in each group. In the study of the quadrivalent subunit vaccine in old and young mice, 80 female 10-month-old and 80 female 10-week-old BALB/c mice were randomly divided into 16 groups ( n=10) including monovalent influenza virus vaccine group, quadrivalent subunit vaccine group, quadrivalent subunit vaccine+ RFH01 adjuvant group, chicken embryo quadrivalent split vaccine control group and PBS group. All mice were immunized by intramuscular injection. At 21 d after the primary immunization, a booster immunization was conducted using the same strategy. Blood samples were collected at 21 d and 42 d after the primary immunization for serum separation. Haemagglutination inhibition (HI) test was performed to detect the antibody levels in mouse serum samples. Results:After the booster immunization, the positive conversion rates in all vaccine+ RFH01 adjuvant groups reached 100%, and the geometric mean titers (GMTs) of serum antibodies were significantly higher than those of the vaccine groups without RFH01 adjuvant. There were significant differences in serum antibody titers between the monovalent/quadrivalent subunit vaccine groups with and without RFH01 adjuvant. After the booster immunization, the titers of serum antibodies against H1N1, H3N2, B/Victoria and B/Yamagata in the 10-week-old mice were significantly higher than those in the 10-month-old mice.Conclusions:The monovalent and quadrivalent influenza virus vaccines in combination with RFH01 adjuvant could elicit higher antibody titers in young (6-10 weeks old) and old (10 months old) mice, showing good immunogenicity.

Preparation and immunogenicity evaluation of recombinant influenza hemagglutinin trimer vaccine / 中华微生物学和免疫学杂志

Objective:To prepare a recombinant hemagglutinin trimer (HA-Tri) vaccine against influenza viruses and to study its immunogenicity in a mouse model.Methods:A stable CHO cell line that could express HA-Tri was constructed. Western blot, single radial immunodiffusion, protein particle size detection and N-glycosylation site analysis were performed for qualitative and quantitative analysis of the recombinant protein. According to the different treatment conditions such as dosage and adjuvant, BALB/c mice were divided into 11 groups and subjected to consistent immunization procedures. Serum neutralizing antibody titers were measured on 56 d after the first immunization to evaluate the immunogenicity of HA-Tri.Results:The constructed CHO cells could secret and express HA-Tri proteins. The HA-Tri proteins were biologically active and capable of forming precipitation rings in the single radial immunodiffusion. The particle size of HA-Tri was approximately 18.79 nm and 10 N-glycosylation sites were detected, including high mannose, complex glycoforms and heterozygous glycoforms. After prime-boost immunization, there was no statistically significant difference in the titers of neutralizing antibodies induced in mice by 3.75 μg of HA-Tri in combination with RFH01 adjuvant and 15 μg of monovalent vaccine stock solution ( P=0.431 2, U=36). Serum antibody titers in the HA-Tri+ RFH01 groups were higher than those in the corresponding HA-Tri groups without RFH01 adjuvant, and the highest titer was induced in the 15 μg HA-Tri+ RFH01 group, which was 1 280. Conclusions:The recombinant HA-Tri protein was successfully prepared. HA-Tri in combination with RFH01 adjuvant could induce humoral immune responses against influenza viruses in BALB/c mice, which would provide reference for the development of influenza virus recombinant subunit vaccines.

Effects of poly(A) tails with different lengths on mRNA expression in vitro and stability of transcription template in Escherichia coli / 中华微生物学和免疫学杂志

Objective:To investigate the effects of poly(A) tails with different lengths on mRNA expression in vitro and the passage stability of transcription template with poly (A) tail in Escherichia coli ( E. coli). Methods:Plasmids with poly(A) tails of 38, 60, 103, 125 and 126 (60 nt+ 6 nt spacer+ 60 nt) nt were designed and constructed. Then the plasmids were linearized by single enzyme digestion and used as transcription template for preparing enhanced green fluorescent protein (EGFP)-mRNA. EGFP-mRNA containing poly(A) tails of different lengths were transfected into 293T cells and the expression of EGFP was detected by flow cytometry. As to stability test, the template plasmids with poly (A) tail of 125 and 126 nt were transformed into E. coli TransStbl3 and Top10 competent cells. Seven clones were selected for culture and plasmid extraction, and then the plasmids were digested by restriction enzyme and detected by capillary electrophoresis. For passage stability, three correctly sequenced clones of each group were selected for continuous passage at 37℃, and the plasmids were extracted and digested every two generations for capillary electrophoresis. At the same time, the correctly sequenced clones of 125 nt group were also passaged at 30℃, and the plasmids were also extracted and digested every two generations for capillary electrophoresis. Results:The transcription templates with poly(A) tail of different lengths were successfully constructed. Flow cytometry showed that the fluorescence expression of the template plasmids with poly (A) tail of 103 and 125 nt were significantly higher than that of 38 and 60 nt. The fluorescence expression of the plasmid with poly (A) tail of 126 nt was significantly higher than that of all other groups. The percentages of stable sequences of the template plasmid with poly(A) tail of 125 nt in TransStbl3 and Top10 competent cells were 76% and 91%, respectively. The results of continuous passage showed that poly(A) tail of 125 nt could be stable to the 4th generation at 37℃ in both TransStbl3 and Top10 competent cells, and stable to the 16th and 10th generations at 30℃. The percentages of stable sequences of the template plasmid with poly(A) tail of 126 nt in TransStbl3 and Top10 competent cells were 95% and 48%, respectively. The results of continuous passage showed that poly(A) tail of 126 nt could be stable to the 12th generation at 37℃ in both TransStbl3 and Top10 competent cells.Conclusions:The length and composition of poly(A) tail in mRNA affected the expression of target protein. Adding a spacer with a length of 6 nt to poly(A) tail and low temperature culture were both helpful to improve the stability of the template plasmid, which provided a reference for the design and preparation of in vitro transcription template of mRNA vaccine.

Prokaryotic expression and serological analysis of the head domain of influenza A virus hemagglutinin / 中华微生物学和免疫学杂志

Objective:To express the head domain of influenza A virus hemagglutinin (HA) in a prokaryotic expression system and to evaluate its immunogenicity.Methods:The genes encoding the HA head domains of H1N1 and H3N2 influenza viruses were cloned into pET-22b(+ ) prokaryotic expression plasmid. After the induction with IPTG, the fusion proteins rH1N1-HA and rH3N2-HA containing HA head domain and His-tag were expressed and obtained from E. coli BL21. SDS-PAGE and Western blot was used to verify the expression of the recombinant proteins. Rabbits were immunized with multiple doses of the purified recombinant proteins to obtain polyclonal antibodies against the HA head domains of H1N1 and H3N2. The immunogenicity of the recombinant proteins was evaluated in BALB/c mice. Results:rH1N1-HA and rH3N2-HA induced protective antibodies (geometric mean titer ≥40) in mice and could be used as protective antigens. Polyclonal antibodies against rH1N1-HA and rH3N2-HA could be used as important materials for Western blot, ELISA and other immunological assays.Conclusions:The HA head domains prepared in this study could be used as protective antigens to induce protective antibodies in mice. Polyclonal antibodies against the HA head domains could be used for immunological and serological studies of influenza A viruses.

A three-dimensional finite element model of mesio-occluso-distal cavity zirconium dioxide inlay and its bonding interface:a stress analysis / 中国组织工程研究

BACKGROUND: Zirconia inlays have good mechanical, biocompatible and aesthetic properties in the field of dental prosthodontics, but there is no consensus on the standards of zirconia inlays for clinical cavity design, dental preparation and selection of bonding materials. OBJECTIVE: To investigate the stress distribution and characteristics of the bonding interface, tooth and periodontal tissues of the zirconia inlay model after 3 M RelyX Unicem or vario-link resin adhesive bonding. METHODS: Micro-CT was used to scan the right mandibular third molars. A three-dimensional finite element model of MOD zirconia inlay with different adhesives (3 M RelyX Unicem resin binder, vario-link resin binder) and cavity types (2, 3, 4 mm in the depth of the joint cavity) was constructed by software Mimics, Goemagic Studio and NX 10.0. Using ANSYS Workbench mesh generation, the stress distribution of each model was analyzed after loading 10 N·mm torque, 45° 175 N, 90° 600 N. RESULTS AND CONCLUSION: (1) After loading 10 N·mm torque, the bonding agent equivalent stress and root surface equivalent stress of the model with 3 mm cavity depth were largest, so the stress on the bonding interface, crown and root was largest. (2) After loading 175 N at 45° lingual direction, the bonding interface of the model with 4 mm cavity depth undertook a higher stress. The stress values of the root and periodontal tissue of 3 M RelyX Unicem resin bonding agent model with the same cavity depth were higher. (3) After loading 600 N at 90°, the root force of the model with 3 mm cavity depth was largest. The crown, root and periodontal tissue of the 3 M RelyX Unicem resin bonding agent model with the same cavity depth undertook a higher stress. The bonding interface of the vario-link resin bonding agent model was under greater stress. (4) The regions of stress concentration areas included the bifurcation zone of the root, the inlay edge line, the roof of pulp chamber, the gingival wall, 1/3 mesial part of the buccal surface near the neck, and 1/3 mesial part of the lingual surface near the neck. (5) All these findings indicate that vario-link resin adhesive and 3 M RelyX Unicem resin adhesive are suitable for the bonding of zirconia inlay, but the vario-link adhesive strength is larger and the bonding interface stress is larger. The factors such as cavity design, residual tooth tissue resistance, retention shape, and periodontal support should be considered comprehensively. The optimization of cavity design, tooth preparation, cushion bottom and high inlay should be adopted, in order to improve the long-term repair effect of zirconia inlay. However, further clinical trials are needed to verify the results of three-dimensional finite element model.

Determination of the Contents of Heavy Metals in 8 Chinese Crude Drugs by UV Spectrophotometry / 中国药房

OBJECTIVE:To determine the contents of heavy metals in commercial Chinese crude drugs and to provide reference for the monitoring of heavy metals in the crude drugs.METHODS:UV spectrophotometry was established to determine the contents of heavy metals in 6 vegetable drugs(Panax quinquefolium,Panax ginseng,Lycium barbarum,Radix et Rhizoma Glycyrrhizae,Bulbus Fritillariae Cirrhosae and Salvia miltiorrhiza)and 2 animal-source drugs(Scolopendra subspinipes mutilans and Bombyx Batryticatus).RESULTS:The linear range of heavy metal plumbum(reference substance)was 0.01～0.50 ?g?mL-1(r=0.997 7),and the average recovery rate was 99.49%(RSD=1.41%).The contents of heavy metals in Panax quinquefolium,Panax ginseng,Lycium barbarum,Radix et Rhizoma Glycyrrhizae,Bulbus Fritillariae Cirrhosae,Salvia miltiorrhiza were 26.13,28.96,26.99,29.98,26.63 and 28.06 ?g?g-1 respectively,and the contents of Scolopendra subspinipes mutilans and Bombyx Batryticatus were on the high side(47.62 and 59.24 ?g?g-1 respectively).CONCLUSION:The method is easy,highly accurate,and it can be used for detection of heavy metals in Chinese crude drugs.