Preparation a Recombinant Form of Pneumolysin Protein from Streptococcus pneumoniae.

A recombinant form of pneumolysin from Streptococcus pneumoniae was obtained. By using Vector NTI Advance 11.0 bioinformatic analysis software, specific primers were designed in order to amplify the genome fragment of strain No. 3358 S. pneumoniae serotype 19F containing the nucleotide sequence encoding the full-length pneumolysin protein. A PCR product with a molecular weight corresponding to the nucleotide sequence of the S. pneumoniae genome fragment encoding the full-length pneumolysin was obtained. An expression system for recombinant pneumolysin in E. coli was constructed. Sequencing confirmed the identity of the inserted nucleotide sequence encoding the full-length recombinant pneumolysin synthesized in E. coli M15 strain. Purification of the recombinant protein was performed by affinity chromatography using Ni-Sepharose in 8 M urea buffer solution. Confirmation of the recombinant protein was performed by immunoblotting with monoclonal antibodies to pneumolysin.

Preparation of recombinant atoxic form of exotoxin A from Pseudomonas aeruginosa.

Nucleotide sequences encoding full-length protein of Pseudomonas aeruginosa exotoxin A and its atoxic form were cloned and expressed in Escherichia coli cells. Purified recombinant exotoxin and immune rabbit sera protected mice from exotoxin A.

[Production of hybrid protein OprF-OprL of Pseudomonas aeruginosa].

AIM: Production of preparation consisting of amino acid sequences of 2 proteins of outer membrane--OprF and OprI--of P. aeruginosa and study of its protective properties from experimental P. aeruginosa infection. MATERIALS AND METHODS: Nucleotide sequences coding OprF protein (1 kb) as well as its C-terminal region (0.6 kb) and OprI protein (0.25 kb) were integrated into pQE-30 plasmid (QIAGEN). And oprF gene (C-terminal region of oprF in variant 2) and oprI gene were combined and cloned sequentially into a single vector. E. coli M15 strain cells (QIAGEN) were used for the production of producent strains of recombinant proteins. Protein products were analyzed by electrophoresis in polyacrylamide gel by Lammle. Purification of recombinant proteins was performed by affinity chromatography in Ni-sepharose columns. Live virulent culture P. aeruginosa PA-170015 strain was used for the analysis of protective properties of recombinant proteins. RESULTS: 2 hybrid recombinant proteins were produced including amino acid sequences of F and I proteins of outer membrane (OprF and OprI) of P. aeruginosa. Recombinant protein 1 included whole size sequences of OprF and OprI and protein 2--C-terminal region (including amino acid residues 192-342) of OprF and whole size sequence of OprI. These recombinant proteins after 2 immunizations protected mice from the experimental intraperitoneal infection with P. aeruginosa. Hybrid protein consisting of whole size sequences had the best protective effect. CONCLUSION: The results obtained open a perspective for further immunobiological testing of hybrid recombinant protein OprF-OprI with the aim of creating immunopreparations for prophylaxis of P. aeruginosa infection.

[Mass spectrometry detection of monomeric renalase in human urine].

Renalase is a recently discovered secretory protein, which is suggested to play a role (which still remains elusive) in regulation of blood pressure. Earlier it was purified from urine of healthy volunteers by means of ammonium sulfate fractionation and subsequent affinity chromatography (Xu et al. (2005) J. Clin. Invest., 115, 1275). The resultant purified preparation of renalase contained 2 proteins with molecular masses of 35 and 67-75 kDa. The authors believed that the latter represents a dimerization (aggregation) product of the 35 kDa protein. In this study we have detected relanase in urinary samples of 2 of 6 volunteers only after immunoaffinity enrichment of urinary samples subjected to ammonium sulfate precipitation. Electrophoresis of the purified preparation also demonstrated the presence of 2 proteins with molecular masses of 35 and 66 kDa, respectively. Mass spectrometry analysis of these proteins identified 35 and 66 kDa proteins as renalase and serum albumin, respectively. Thus, our results do not support suggestion on formation of renalase dimers and they indicate that urinary renalase excretion significantly varies in humans.

[Immunobiological properties of recombinant atoxic forms of the Pseudomonas aeruginosa exotoxin A].

AIM: Evaluation of immunobiological properties of recombinant atoxic forms of the Pseudomonas aeruginosa exotoxin. MATERIALS AND METHODS. 3 recombinant atoxic forms of the P. aeruginosa exotoxin A were produced and studied: aTox1, consisting only of exotoxin A domain 1; aTox1,2, consisting of domain 1 and 2; and aTox1,2,delta3, consisting of both domain 1 and 2, and part of domain 3. RESULTS: aToxl,2 and aTox1,2,delta3 had distinctive antigenic properties. Formulations based on these recombinant proteins were immunogenic and protected animals from exotoxin A in experimental conditions. CONCLUSION: These results maybe used to construct direct-action immunobiological formulations.

[A novel vector for construction of a cDNA library].

A new original vector pEM-(dT)40(f+) has been prepared. It can be used for cDNA library construction from polyadenylated mRNA, isolated from various sources. The pGEM-(dT)40f(+) is initially transformed into single stranded and then into a linear form and its (dT)40 tail at 3'-end is used as the vector-primer for synthesis of the first strand cDNA. The use of a synthetic oligonucleotide complementary to the vector and recombinant DNA results in vector cyclization and synthesis of the second strand cDNA. This approach significantly simplifies cDNA library construction, it does not require PCR reaction (which can induce artifact mutations in cDNA sequences) and restrictase treatment.

[Study of protective properties of recombinant atoxic form of exotoxin A and recombinant outer membrane protein F of Pseudomonas aeruginosa].

AIM: To obtain recombinant atoxic form of Pseudomonas aeruginosa exotoxin A and assess its protective properties during simultaneous administration with recombinant protein F in experiment. MATERIALS AND METHODS: Genetic methods were employed for construction of deletion variant of P. aeruginosa exotoxin A gene, whereas Escherichia coli cells were used for transformation. Purification of proteins was performed by common method. White outbred mice were used for immunization and experimental infection was produced by intraperitoneal administration of live virulent culture of P. aeruginosa strain PA103. RESULTS: The gene coding defect form of P. aeruginosa exotoxin A with lacked 106 C-terminal aminoacid residues was cloned. Synthesized protein was nontoxic and immunogenic. Recombinant variants of anatoxin A and outer membrane protein F of P. aeruginosa protected animals from experimental infection caused by toxigenic strain PA103 of P. aeruginosa if were administered separately or concomitantly. Nonetheless, the highest protective effect was observed after immunization with both proteins. CONCLUSION: Studied recombinant toxoid and OprF could be the candidates for inclusion in vaccines for prevention of pseudomonas infection.

[Isolation and study of perspective probiotic strain of spore-forming bacteria from Bacillus genus].

AIM: To study physiologic, biochemical as well as antagonistic characteristics of isolated strain of Bacillus pumilus in comparison with known spore-forming bacteria from B. subtilis species. MATERIALS AND METHODS: Four strains of Bacillus spp. were used in the study: B. subtilis 3H, B. subtilis 534, B. subtilis 1719, B. pumilus isolated from the environment. The following strains from the collection of Tarasevich State Institute of Standardization and Control for Immunobiologicals were used for determining of antagonistic activity: Staphylococcus xylosus 25, Proteus mirabilis 24a, S. aureus "Nikiforov", S. aureus "Filippov", P. vulgaris 177, Shigella flexneri 337, Escherichia coil O111:H55, S. sonnei 170, Pseudomonas aeruginosa 9022, Candida albicans 690; clinical test-strains of yeast fungi: C. albicans (3 strains), C. haemuloni, C. tropicalis, Rhodotorula spp., Debaryomyces hansenii. RESULTS: Identification of isolated strain on combination of morphologic, tinctorial, cultural and biochemical characteristics showed that studied strain belonged to B. pumilus species. It did not have hemolytic and lecitinase activities, was resistant to several groups of antibiotics and had index of adhesion 1.76 +/- 2.5. In experiments in vivo the strain was non-toxic, non-toxigenic and avirulent. In preparations of isolated DNA, plasmids were not found. B. pumilus had high antagonistic effect against opportunistic and pathogenic bacteria as well as yeast fungi. Comparative assessment of tested strains from Bacillus genus showed that the isolated strain was not inferior than other representatives of spore-forming bacteria on spectrum and intensity of aforementioned characteristics. CONCLUSION: Low-adhesive, safe, plasmid-less strain of B. pumilus, which have intensive antagonistic properties, could be considered as a candidate for the development of new probiotic drugs for medical or veterinary use.

[Immunobiological properties of recombinant L peptide from the Pseudomonas aeruginosa outer membrane].

The gene encoding plasma membrane protein L (OprL) was cloned and expressed in E. coli. It was shown that the recombinant protein had immunogenic properties and protected laboratory animals from experimental P. aeruginosa infection. Rabbit hyperimmune sera raised against recombinant L protein possessed antimicrobial activity and prevented infection with P. aeruginosa.

[Study of immunogenic properties of recombinant outer membrane proteins F and L from Pseudomonas aeruginosa].

Recombinant outer membrane proteins OprF, OprL, and C-terminal part of OprF protein (OprF(192-342)) was synthesized in Escherichia coil M15 cells. It was shown that in rabbits immunized with chromatographically purified recombinant proteins mounted specific antibody response. In experiments in vitro obtained hyperimmune to OprF, OprL, and OprF(192-342) rabbit sera inhibited growth of P. aeruginosa culture and in experiments in vivo protected mice from infection caused by this microorganism.

[Influence of antisense RNA and sequences of viral transactivators traps on RNA synthesis of HTLV-1 virus].

Significant number of scientific publications devoted to inhibition of viral replication by antisense RNA (asRNA) genes shows that this approach is useful for gene therapy of viral infections. To investigate the possibility of suppression of HTLV-1 virus reproduction by asRNA we constructed recombinant plasmids containing asRNA genes against U3 long terminal repeats region and X gene under the control of promoter of myeloproliferative sarcoma virus (MPSV) or without such promoter. Using stable calcium-phosphate transfection method with subsequent selection in the presence of G-418, RaHOS line-based cell clones carrying both asRNA genes and sequences able to bind HTLV-1 transactivator proteins (i.e. "traps" of viral transactivators, TVT) were obtained. Data from dot-hybridization analysis of viral RNA extracted from RaHOS cell clones showed that TVT sequences are able to suppress the viral RNA synthesis on 90% and asRNA against X gene synthesis--on 50%.

[Obtaining of outer membrane protein I of Pseudomonas aeruginosa and assessment of its antigenic properties].

Gene of outer membrane protein I (OprI) of Pseudomonas aeruginosa was cloned in Escherichia coli cells. Synthesized protein OprI contained additional sequence of 6 histidines on the N-terminus, which allowed its chromatographic purification in Ni-agarose. Obtained recombinant ptotein specifically reacted with hyperimmune rabbit serum against whole-cell P. aeruginosa and stimulated synthesis of specific antibodies in immunized mice and rabbits. Obtained hyperimmune rabbit sera against recombinant protein OprI had directive antimicrobial activity against P. aeruginosa.

[Evaluation of properties of Pseudomonas aeruginosa recombinant outer membrane protein F (OprF)].

Study showed that synthesis of specific IgG occurs in rabbits immunized with recombinant outer membrane protein F (OprF) of Pseudomonas aeruginosa and that these antibodies inhibit grow of P. aeruginosa in vitro. In vivo studies on mice showed that rabbit hyperimmune sera and recombinant OprF are both able to protect animals from intraperitoneal challenge with P. aeruginosa.

[Preparation of Pseudomonas aeruginosa recombinant outer-membrane protein F and the study of its antigenic properties].

In Escherichia coli M15, the gene of P. aeruginosa recombinant outer-membrane protein F (OprF) was cloned. OprF, chromatographically purified on Ni-agarose and containing an additional sequence of 6 histidines on the N-end, was obtained. The purified OprF specifically reacted with rabbit serum, hyperimmune to P. aeruginosa, and in the mice injected with this protein specific IgG antibodies were synthesized. The optimum concentrations of P. aeruginosa OprF were selected for further tests of its protective properties from infection induced by P. aeruginosa.

[Effective method of DNA purification from contaminated matters and optimization of amplification]. / Effektivnyi metod ochistki DNK iz zagriaznennykh ob''ektov i optimizatsiia amplifikatsii.

A new modification of DNA purification has been developed. It includes: 1) standard treatment of biological material with proteinase K followed by phenol-chlorophorm extraction; 2) subsequent sample purification using micro-columns packed with Dowex-50 and Sephadex G-50. Oligonucleotide primers often used for DNA typing in man by means of polymerase chain reaction have also been modified. These are VNTR (variable number of tandem repeats) loci of apoB and D17S5. The increase of stability and specificity of amplification of VNTR loci of apoB and D17S5 was achieved by increase of primer length and amplification cycle. The sensitivity of this mode of amplification is 2-4 ng DNA-template. Employment of the nested amplification for apoB locus increased sensitivity of this method up to a few copies of DNA.