The recombinant fusion protein CFP10–ESAT6–dIFN has protective effect against tuberculosis in guinea pigs

Development of effective vaccine candidates against tuberculosis (TB) is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein CFP10–ESAT6–dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute TB. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response. Double intradermal immunization of guinea pigs with the tested fusion protein (2 × 0.5 µg) induces a protective effect against subsequent Mtb infection. The immunized guinea pigs do not develop the symptoms of acute TB and their body weight gain was five times more as compared with the non-immunized infected guinea pigs. The animal group immunized with this dose of antigen displays the minimum morphological changes in the internal organs and insignificant inflammatory lesions in the liver tissue, which complies with a decrease in the bacterial load in the spleen and average Mtb counts in macrophages

Modification of Natural Clays for Use in the Processes of Sewage Treatment

The paper deals with the modification of clay sorbents in order to improve the efficiency of of copper ions extraction from wastewater. The study examined the elemental, mineralogical and phase composition of kaolin and montmorillonite clays. The authors analyzed the sorption capacity of natural sorbents under static conditions before and after modification with hydrochloric acid, sodium chloride and sodium hydroxide. The work also determines the change in the physical and chemical properties of the sorbent after modification. The most optimal modification conditions for the purification of wastewater from copper ions are distinguished

Three Parts of the Plant Genome: On the Way to Success in the Production of Recombinant Proteins

Recombinant proteins are the most important product of current industrial biotechnology. They are indispensable in medicine (for diagnostics and treatment), food and chemical industries, and research. Plant cells combine advantages of the eukaryotic protein production system with simplicity and efficacy of the bacterial one. The use of plants for the production of recombinant proteins is an economically important and promising area that has emerged as an alternative to traditional approaches. This review discusses advantages of plant systems for the expression of recombinant proteins using nuclear, plastid, and mitochondrial genomes. Possibilities, problems, and prospects of modifications of the three parts of the genome in light of obtaining producer plants are examined. Examples of successful use of the nuclear expression platform for production of various biopharmaceuticals, veterinary drugs, and technologically important proteins are described, as are examples of a high yield of recombinant proteins upon modification of the chloroplast genome. Potential utility of plant mitochondria as an expression system for the production of recombinant proteins and its advantages over the nucleus and chloroplasts are substantiated. Although these opportunities have not yet been exploited, potential utility of plant mitochondria as an expression system for the production of recombinant proteins and its advantages over the nucleus and chloroplasts are substantiated

CRISPR/Cas9-Mediated Targeted DNA Integration: Rearrangements at the Junction of Plant and Plasmid DNA

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We studied the presence and extent of DNA rearrangements at the junction of plant and transgenic DNA in five lines of Arabidopsis thaliana suspension cells carrying a site-specific integration of target genes. Two types of templates were used to obtain knock-ins, differing in the presence or absence of flanking DNA homologous to the target site in the genome. For the targeted insertion, we selected the region of the histone H3.3 gene with a very high constitutive level of expression. Our studies showed that all five obtained knock-in cell lines have rearrangements at the borders of the integrated sequence. Significant rearrangements, about 100 or more bp from the side of the right flank, were found in all five plant lines. Reorganizations from the left flank at more than 17 bp were found in three out of five lines. The fact that rearrangements were detected for both variants of the knock-in template (with and without flanks) indicates that the presence of flanks does not affect the occurrence of mutations

Oral Immunogenicity of Plant-Made Mycobacterium tuberculosis ESAT6 and CFP10

Two lines of transgenic carrot plants producing Mycobacterium tuberculosis proteins (ESAT6 and CFP10) have been constructed. The target proteins are present in carrot storage roots at a level not less than 0.056% of the total storage protein (TSP) for ESAT6 and 0.002% of TSP for CFP10. As has been shown, oral immunization of mice induces both the cell-mediated and humoral immunities. These data suggest that the proteins in question are appropriate as a candidate edible vaccine against tuberculosis

The Role of Carbonic Anhydrase αCA4 in Photosynthetic Reactions in <i>Arabidopsis thaliana</i> Studied, Using the Cas9 and T-DNA Induced Mutations in Its Gene

An homozygous mutant line of Arabidopsis thaliana with a knocked out At4g20990 gene encoding thylakoid carbonic anhydrase αCA4 was created using a CRISPR/Cas9 genome editing system. The effects of the mutation were compared with those in two mutant lines obtained by the T-DNA insertion method. In αCA4 knockouts of all three lines, non-photochemical quenching of chlorophyll a fluorescence was lower than in the wild type (WT) plants due to a decrease in its energy-dependent component. The αCA4 knockout also affected the level of expression of the genes encoding all proteins of the PSII light harvesting antennae, the genes encoding cytoplasmic and thylakoid CAs and the genes induced by plant immune signals. The production level of starch synthesis during the light period, as well as the level of its utilization during the darkness, were significantly higher in these mutants than in WT plants. These data confirm that the previously observed differences between insertional mutants and WT plants were not the result of the negative effects of T-DNA insertion transgenesis but the results of αCA4 gene knockout. Overall, the data indicate the involvement of αCA4 in the photosynthetic reactions in the thylakoid membrane, in particular in processes associated with the protection of higher plants’ photosynthetic apparatus from photoinhibition