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Objective: To screen the recombinant hemagglutinin (HA) protein with high expression level and hemagglutinin inhibitory activity for B/Yamagata influenza virus. Methods: Based on the HA of B/Singapore/INFTT-16-0610/2016, the peptide sequences of GCN4pLL trimerization motif, GCN4pLL-cysteine, and GCN4pLL-the sequences from C-terminus of lamprey variable lymphocyte receptor (VLR)-B antibodies were fused to C-terminus of HA ectodomain, to obtain HA mutant genes BY-T, BY-LLc, and BY-PLc. They were inserted into pFastBac1 separately and expressed using baculovirus expression vector system. After expression identification, Strep-Tactin affinity chromatography purification was performed to purify the mutant proteins followed by identification of the degree of oligomerization and hemagglutination activity of the purified mutant proteins. Anti-HA Ig G titer and hemagglutination inhibition (HAI) antibody titer in sera of mice immunized with HA mutants were detected. Results: All HA mutants were effectively expressed. The BY-T mutant had the highest expression level, was easy to purify, had good purity, and existed in a polymeric form. The expression levels of BY-LLc and BY-PLc are secondary and exist in a highly polymerization form. The hemagglutination activities of the three HA mutant proteins were comparable, and all of them could effectively induce anti-HA Ig G and HAI antibodies. Conclusion: The HA protein ectodomain mutant fused with oligomeric motif GCN4pLL at the C-terminus is easy to purify and has hemagglutination activity, which can induce anti-HA Ig G and HAI antibodies in mice. The results provide a reference for the development strategy of recombinant protein vaccines against influenza virus B/Yamagata lineage. [ABSTRACT FROM AUTHOR]

Objective: To screen the recombinant hemagglutinin (HA) protein with high expression and good hemagglutination activity for H3N2 influenza virus. Methods: Based on the HA ectodomain gene of A/MINNESOTA/41/2019 (H3N2) strain, GP67 signal sequences and trimerization motifs were respectively fused to the N-and C-terminals of the full-length HA ectodomain gene and the near transmembrane domain two residues truncated gene. The recombinant genes were expressed in baculovirus system. At the same time, the corresponding recombinant HA proteins without containing trimerization motifs were generated. The expressions of these recombinant HA proteins were identified by Western blot. The recombinant proteins were purified by affinity chromatography. Then, the hemagglutination titer and stability of the recombinant proteins were analyzed. Results: Four kinds of HA recombinant proteins were effectively expressed, among which the expression level of HA ectodomain full-length fusion trimerization motif (HA-T) was the highest, and the yield of it was up to 30 mg per liter suspension medium purified by Strep tag affinity chromatography. The cross-linking analysis showed that HA-T was in the form of stable trimer of HA. In addition, the hemagglutination activity analysis showed that HA-T had the highest activity, and its hemagglutination titer can reach 29. Dynamic light scattering showed that it was stable at 4 ℃ for 3 months. Conclusions: HA-T has the advantages of high expression, good stability, high hemagglutination activity and easy purification. The research provides a useful reference for the development strategy of HA recombinant protein vaccines. [ABSTRACT FROM AUTHOR]

BACKGROUND: The aperture of bone tissue engineering scaffold is one of the important indexes to evaluate its biological performance, and seeking the best combination of aperture and mechanical properties has always been an urgent problem to be solved in bone tissue engineering. OBJECTIVE: To prepare three-dimensional scaffolds with different aperture gradients and to study their effects on the biological behavior of MC3T3-E1 cells. METHODS: The three-dimensional nano-hydroxyapatite scaffolds with uniform pore size were prepared by mixing wax spheres of specific diameter (300, 500, 800 μm) with hydroxyapatite as pore-forming agent using the modified particle leaching method, which were recorded as the D300, D500, and D800 scaffolds, respectively. The wax spheres of three diameters were piled up in order from small to large. The pore gradient nano-hydroxyapatite three-dimensional scaffold was prepared by the modified particle leaching method, which was recorded as the D-Gradient scaffold. The porosity of scaffolds of the four groups was measured. MC3T3-E1 cells were inoculated on scaffolds of the four groups. The cells cultured separately were used as the blank control group. Cell proliferation activity, alkaline phosphatase activity, cell adhesion, cytoskeleton rearrangement and the transcription level of osteogenic differentiation marker factors were detected. RESULTS AND CONCLUSION: (1) The porosity of scaffolds of the four groups was 60%-80%. (2) After 24 hours of inoculation, the scaffolds in each group could promote the proliferation of MC3T3-E1 cells. The proliferation index was: D-Gradient group > D500 group > D800 group > D300 group > blank control group. The scaffolds in the D300 group, D500 group, and D-Gradient group promoted the secretion of alkaline phosphatase in MC3T3-E1 cells. The amount of alkaline phosphatase secretion was: D300 group > D500 group > D-Gradient group > D800 group ≈ blank control group. The scaffolds in the D300 group, D500 group, and D-Gradient group promoted the expression of osteopontin and osteocalcin mRNA in MC3T3-E1 cells. The expression levels of the two genes from high to low were: D300 group > D500 group > D-Gradient group > D800 group ≈ blank control group. (3) Immunofluorescence staining after 48 hours of inoculation showed that compared with the blank control group, MC3T3-E1 cells on the four scaffolds showed better adhesion and locomotor activity. The fluorescence intensity of focal adhesion actin from high to low was: D-Gradient group > D500 group > D800C group > D300 group > blank control group. (4) It is concluded that the aperture gradient scaffolds promote the growth, proliferation, adhesion, and osteogenic differentiation of MC3T3-E1 cells. [ABSTRACT FROM AUTHOR]

In order to identify the key factors affecting the quality development of the Yellow River basin and effectively solve the existing problems, based on the particularity and new development trend of the Yellow River basin and problem-oriented, 13 factors influencing the high-quality development of the Yellow River basin were selected from three aspects of economy, ecology and society. The AHP-DEMATEL method was used to quantitatively explore the relationship among various influencing factors and identify key influencing factors. The results show that innovation ability, human resources, human destruction, educational resources, income level and its gap are the key factors affecting the realization of collaborative high-quality development in the Yellow River basin. It suggests to strengthening personnel training, enhancing the innovation ability of enterprises, rationally allocating educational resources and inclining to the Yellow River basin appropriately, improving the transportation infrastructure of the basin and solving the problems of low income level and large gap among residents. [ABSTRACT FROM AUTHOR]

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