The continuing spread of the newly emerged H7N9 virus among poultry in China, as well as the possibility of human-to-human transmission, has attracted numerous efforts to develop an effective vaccine against H7N9. The use of nanoparticles in vaccinology is inspired by the fact that most pathogens have a dimension within the nano-size range and therefore can be processed efficiently by the immune system, which leads to a potent immune response. Herein, we report a facile approach to increase antigen size to achieve not only fast but also effective responses against the recombinant HA/H7N9 protein via a simple conjugation of the protein onto the surface of nanodiamond particles. In this study, trimeric Haemagglutinin (H7) that is transiently expressed in N. benthamiana was purified using affinity chromatography, and its trimeric state was revealed successfully by the cross-linking reaction. The trimeric H7 solution was subsequently mixed with a nanodiamond suspension in different ratios. The successful conjugation of the trimeric H7 onto the surface of nanodiamond particles was demonstrated by the changes in size and Zeta-potential of the particles before and after protein coating, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Western-blot analysis. Next, biofunction of the protein-nanodiamond conjugates was screened using a haemagglutination assay. A mixture containing 5 µg of trimeric H7 and 60 µg of nanodiamond corresponds to a ratio of 1:12 (w/w) of agglutinated chicken red blood cells at HA titer of 1024, which is 512-fold higher than the HA titer of free trimeric H7. After the 2nd and 3rd immunization in mice, ELISA and Western blot analyses demonstrated that the physical mixture of trimeric H7 protein and nanodiamond (1:12, w/w) elicited statistically significant stronger H7-specific-IgG response demonstrated by higher amounts of H7N9-specific IgG (over 15.4-fold with P < 0.05 after the second immunization). These results indicated a potential effect inherent to nanodiamond towards modulating immune systems, which should be further evaluated and broadly applied in nanovaccine development.

Endophytic fungi are promising sources for the production of podophyllotoxin-an important anticancer compound, replacing depleted medical plants. In this study, the endophytes associated with Dysosma difformis-an ethnomedicinal plant species were isolated to explore novel sources of podophyllotoxin. Fifty-three endophytic fungi were isolated and identified by morphological observation and ITS-based rDNA sequencing, assigning them to 27 genera in 3 divisions. Fusarium was found the most prevalent genus with a colonization frequency of 11.11%, followed by Trametes (9.26%) and Penicillium (7.41%). Phylogenetic trees were constructed for the endophytic fungi community in two collection sites, Ha Giang and Lai Chau, revealing the adaptation of the species to the specific tissues and habitats. Cytotoxic activity of endophytic fungal extracts was investigated on cancer cell lines such as SK-LU-1, HL-60, and HepG2, demonstrating strong anti-cancer activity of six isolates belonging to Penicillium, Trametes, Purpureocillium, Aspergillus, and Ganoderma with IC₅₀ value of lower than 10 µg/mL. The presence of podophyllotoxin was indicated in Penicillium, Trametes, Aspergillus and for the first time in Purpureocillium and Ganoderma via high-performance liquid chromatography, which implied them as a potential source of this anti-cancer compound.

Plant endophytes are an interesting group of microorganisms that colonize the internal tissues of living plants and do not cause any disease symptoms in the host plant. It exists in different parts of plants, such as roots, leaves, and stems, and significantly affects the formation of metabolic products in plants by promoting the accumulation of important secondary metabolites. The present study focused on analyzing the diversity and distribution of endophytic fungi related to different tissues from samples of the medicinal plant Dysosma difformis (Hemsl & E.H. Wilson) T.H. Wang collected in Ha Giang and Lai Chau, in which the isolates from roots were 27 strains (50.94%), 12 strains from stems (22.64%), and 14 strains from leaves (26.42%). Based on the isolates, we analyzed the fungal diversity through seven different diversity indices. The results showed that isolates’ diversity was similar to the endophytic fungal population in tissues of D. difformis distributed in different vegetation in Vietnam. Specifically, Shannon's index showed the highest diversity in roots (H′ = 2.673), followed by stems (H′ = 2.162) and leaves (H′ = 2,054). Similarly, species richness was highest in roots (Dmg = 4,551; Dmn = 3,079) and stem (Dmg = 4,024; Dmn = 3,175) and lowest in leaves (Dmg = 3.41; Dmn = 2,405). However, the Simpson diversity index showed that the endophytic fungal community was most abundance in leaves (1-D = 0.911), followed by stems (1-D = 0.897) and roots (1-D = 0.867). In addition, the Sorensen index of 0.615 shows the average similarity in species composition between the two sites, Ha Giang and Lai Chau. This is the first report on the diversity of endophytic fungi isolated from D. difformis, paving a potential way for screening endogenous fungal strains capable of producing important secondary compounds.

Virus tả lợn Châu Phi (ASFV) là nguyên nhân gây ra tả lợn Châu Phi - một trong những bệnh truyền nhiễm nguy hiểm nhất ở lợn với tỷ lệ tử vong 100%. Tuy nhiên, hiện nay vẫn chưa có vắc xin được cấp phép cho phòng, chống ASFV. CD2v, một glycoprotein ở lớp vỏ ngoài của ASFV, đóng vai trò quan trọng trong quá trình lây nhiễm virus và giảm miễn dịch ở vật chủ, là kháng nguyên tiềm năng để phát triển vắc xin tiểu đơn vị phòng ASFV. Trong nghiên cứu này, trình tự CD2v của chủng VNUAHY-ASFV/Vietnam2019 được tối ưu mã cho biểu hiện trên hệ thống baclovirus - tế bào côn trùng và đặt tổng hợp nhân tạo trong vector pUC57. Toàn bộ cấu trúc CD2vFull-Foldon-His được cắt nối vào vector pLIB để tạo vector tái tổ hợp pLIB-CD2vFull-Foldon-His. Vector pLIB-CD2vFull-Foldon-His được dùng làm khuôn để thiết kế vector pLIB-CD2vED-Foldon-His mang trình tự biểu hiện vùng ngoại bào của CD2v dạng trimer bằng kit gây đột biến định hướng Q5 dựa trên phản ứng PCR với enzyme Polymerase ADN Q5 High-Fidelity. Vector pLIB-CD2vED-Foldon-His được dùng để thiết kế vector pLIB-CD2vED-His cho biểu hiện CD2vED dạng monomer bằng bộ kit Q5. Hai vector pLIB-CD2vED-Foldon-His và pLIB-CD2vED-His được biến nạp vào E. coli DH10Bac để tạo bacmid tái tổ hợp cho biểu hiện kháng nguyên CD2v trên hệ thống baculovirus - tế bào côn trùng, định hướng sản xuất vắc xin phòng ASF.

The baculovirus expression vector system (BEVS) is one of the strongest tools for producing recombinant proteins such as pharmaceuticals and vaccines. In this study, the multivalent CD2v extracellular domain (CD2v ED) of the African swine fever virus (ASFV) was expressed in Sf9 insect cells using BEVs. Bacmid carrying the gene encoding CD2v ED-Foldon-His was transfected into Sf9 insect cells using the lipid transfer complex Cellfectin II and Opti-MEM I Reduced Serum Medium. The transfection process was observed under microscopy and protein expression would be assessed by western blot. The results indicated the transparent differences in infected cells, including the swelling of cells, the increase of nuclei, granular and vesicular appearance, cessation of cell growth, detachment from the flask bottom, and cell lysis in comparison with normal cells that are round, small, and regular size. The western blot results illustrated the expression of CD2v ED-Foldon-His in trimeric and oligomeric forms with bands of 100 kDa and more than 250 kDa, respectively. Most importantly, the expression of CD2v ED-Foldon-His in adherent culture insect cells was optimized with two main factors, namely multiplicity of infection (MOI) and infection time. The results demonstrated the highest yield of CD2v ED-Foldon-His protein was 4.4 mg/L at a MOI of 5 pfu/cell and 5 days post-infection, which was confirmed by captured pictures throughout the infection time. This is the first study on the expression of the oligomeric CD2v extracellular domain on a baculovirus-insect cell system, aiming to produce subunit vaccines against ASFV. Besides, the study also presented a practical procedure for enhancing the expression of recombinant proteins in this system.

Expression of a taste-modifying protein, miraculin, in by-2 tobacco cell line (Nicotiana tabacum L. Cv Bright Yellow 2)