Objective To obtain DNA oligonucleotide aptamers which can specifically bind to MPT64 protein from Mycobacterium tuberculosis by SELEX(systematic evolution of ligands by exponential enrichment)technology. Methods A random ssDNA library with in vitro synthesized 78 nucleotides in length was subiected to 12 rounds of selection by SELEX method against MPT64 protein. The binding ability of the aptamers to the protein was examined by biotin-streptavidin-horseradish peroxidase system. Results The selective system used was as follows:in PCR amplification,annealing temperature was 65℃ and the concentration of Mg2+ was 1.5 mmol/L in optimizing library, and when preparation of ssDNA with asymmetrical PCR amplification, 0.75 mmol/L of Mg2+ was used. When using the plate for ELISA as the substrate for the selection, the pattern of electrophoretic band of PCR product after the tenth round selection became unitary and denser than that of the first round. The binding assay demonstrated that A value at 450 nm of the tenth round increased by 9.18 times as compared with that of the first round. The results showed that the affinities of the aptamers were different. The highest A value at 450 nm was 1.606, and the lowest 0.572. Conclusion A set of aptamers with considerable binding affinity to MPT64 protein are successfully picked out from the initial random DNA library.

Objective To construct the recombinant plasmid of protein CFP10-MPT48-TB8.4 of Mycobacterium tuberculosis and to investigate the diagnosis potential of this fusion protein in tuberculosis serodiagnosis.Methods The recombinant fusion protein CFP10-MPT48-TB8.4 was expressed, and identified by Western blot.The ELSIA based on the purified fusion protein was done,and used for screening in 230 cases of clinical serum samples including pulmonary tuberculosis patients ( n =150 ),pulmonary disease patients other than tuberculosis (n =70) and health controls (n =103 ).The test result was analyzed by Medcale11.5 software.Results The fusion protein CFP10-MPT48-TB8.4 was successfully expressed with a purity over 95％.Specific immunogenicity of the recombinant protein was confirmed by Western blot.The overall sensitivity and specificity obtained of ELISA were 56.7％ (85/150) and 90.8％ ( 157/173 ),respectively.The specificity was 85.7 ％ (60/70) in non-tuberculosis group and 94.2％ (97/103 ) in healthy group,respectively.Conclusion The recombinant protein of CFP10-MPT48-TB8.4 has a high sensitivity and specificity and may be a potential candidate antigen in tuberculosis serodiagnosis.

Objective To clone and express of Rv0091 encoding protein in Mycobacterium tuberculosis,identify and characterize of the enzyme activities.Methods Construct the Rv0091 prokaryotic expression plasmid,the vector was transformed into E.coli strain BL21trxB.After induced by IPTG,recombinant protein was purified by Ni2+-NTA chromatography and analyzed for purity by SDS-PAGE gels stained with Coomassie Blue.Immunological activity was identified by Western blot.The recombinant protein molecular weight was identified by Mass spectrometry.The enzyme-coupled assay detectes enzyme activity.Results The expression plasmid pET32a-Rv0091 was constructed and expressed in E.coli.BL21trxB,and the optimum expression system was conformed.The purity of the recombinant protein was more than 95％.Western blot analysis confirmed that recombinant protein was one of Mycobacterium tuberculosis proteins.Mass spectrometry identified the relative molecular weight and theoretical molecular weight was basically the same.Enzyme assay showed the recombinant protein able to catalyze the substrate MTA.Enzymatic properties showed that the optimal buffer for the phosphate and Hepes buffer,the poor thermal stability of the enzyme,the optimal temperature of 37℃,optimal pH10-12,when the pH ≤7,the protein denaturation and loss of some vitality.Conclusion The recombinant protein methylthioadenosine nucleosidase(MTAN) was obtained and enzyme activity was detected and plays a key role in the metabolism of Mycobacterium tuberculosis.

Objective To study the relationship of two variants( -871A/G and -336A/G) polymorphisms of the DC-SIGN gene with the susceptibility to pulmonary tuberculosis in Chinese population.Methods Two hundred and thirty-seven tuberculosis cases and 244 controls were genotyped by pyrosequencing in this case-control study. The analysis of the relationship of the -871A/G and -336A/G polymorphisms with their susceptibility of pulmonary tuberculosis(PTB) and the relationship of the two variants with their clinical correlation of tuberculosis was performed by chi-square test. Results The genotypic frequencies of A/G + G/G and A/A of - 871, 37.6%, 62.4% respectively in cases, and 43.4%, 56. 6%respectively in controls, had no significant difference in statistics. And the genotypic frequencies of A/G + G/G and A/A of -336, 12. 2% ,87.8% respectively in cases, and 14.3% ,85.7% respectively in controls, had also no statistical difference between two groups. Interestingly, a significant association is disclosed between the promoter variant - 336G allele and fever in patients ( P = 0. 037, OR = 0. 191, 95 % CI:0. 040-0. 907 ). Conclusion The single nucleotide polymorphism of -871A/G and -336A/G in DCSIGN gene promoter might not be associated with the susceptibility to tuberculosis in Chinese. Tuberculosis patients with -336G allele are significantly protected fever.

The aim of this study is to construct a bifunctional fusion protein, which can conjugate both human red blood cells and antibodies against classical swine fever virus (CSFV). We respectively amplified 2E8ScFv and mE2 genes from different recombinant vectors, in which 2E8ScFv gene is the single chain Fv gene against H antigen of human red blood cells, whereas mE2 gene is the main antigen coding region gene of CSFV E2 protein. We used overlap extension PCR to obtain an artificial fusion gene segment 2E8mE2 containing genes of Both 2E8ScFv and mE2, then ligated into the expression vector pET-DsbA and expressed in Escherichia coli BL21(DE3) PlysS host cells, after induced with IPTG the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. We purified the fusion protein and renatured it from inclusion bodies to obtain a native state of well biological activity. The Erythrocyte agglutination test results indicated that the fusion protein can conjugate both human red blood cells and antibodies of CSFV.
Erythrocytes ; immunology ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Single-Chain Antibodies ; biosynthesis ; genetics ; Viral Envelope Proteins ; biosynthesis ; genetics

OBJECTIVETo obtain the C7C peptide ligands of Mycobacterium tuberculosis by affinity screening based on the phage-displayed random C7C peptide library, and preliminarily identify the binding capacity of the peptide to Mycobacterium.

METHODSInactive Mycobacterium tuberculosis reference strain H37Rv was used as the target molecule to screen the Ph. D.-C7C peptide library, and Mycobacterium bovis, BCG was used for reverse screening. After 4 rounds of affinity screening, single phages eluted by H37Rv and BCG were selected for DNA sequencing. ELISA was used to detect the binding affinities of different single phage clones. The cyclic peptides displayed by the phage clones showing the highest appetency were synthesized in vitro with fluorescent markers. Fluorescence microscopy and flow cytometer was used to detect the binding affinities of synthesized cyclic peptides, comparing with linear binding peptides obtained before.

RESULTSAfter 4 rounds of biopanning, phages that could bind with target molecules were remarkable enriched. 16 common sequences were obtained by sequencing analysis of single phages. With ELISA, phage SB1, SB5, SB8 and SB26 all showed higher affinity with H37Rv and BCG, the ratio to negative control of which were ≥ 2.1, but could not bind to the 3 nonmycobacteria, which were identified as the positive clones. Based on the results of flow cytometer detection, the affinities to H37Rv of 4 cyclic peptides SB1, SB5, SB24, SB26 were (73.2 ± 6.3)%, (63.2 ± 5.3)%, (32.9 ± 3.1)%, (89.4 ± 7.0)%, and to BCG were (65.6 ± 6.1)%, (48.6 ± 4.5)%, (10.3 ± 1.8)%, (86.6 ± 7.9)%, separately, which were all higher than H8 ((4.0 ± 1.0)%, (5.5 ± 1.2)%) . From the results of fluorescence microscopy observation, all of the fluorescent labeled cyclic peptides SB1, SB5, SB24, SB26 could bind to H37Rv and showed higher fluorescence intensities, which also had certain affinities to other 18 mycobacteria, but the fluorescence intensities were lower than H37Rv, and didn't bind to 3 non-mycobacteria.

CONCLUSIONBased on the replacement of linear 7 peptide library with C7C peptide library, new ligands of Mycobacterium tuberculosis were achieved successfully, which showed significantly higher binding affinities to mycobacteria.

Base Sequence ; Enzyme-Linked Immunosorbent Assay ; Ligands ; Mycobacterium tuberculosis ; Peptide Library ; Peptides

OBJECTIVETo induce Mycobacterium tuberculosis (MTB) resistance with ofloxacin (Ofx) of stepwise increasing concentration in vitro, investigate stability to fluoroquinolone (FQs) antibiotic of MTB, and analyze the molecular mechanism and mutation specialty of drug resistance preliminarily.

METHODSMTB Standard strain H37RV and 24 clinical isolates susceptible to Ofx were selected and experimentally serially subcultured in liquid culture medium containing increasing concentration of Ofx and induced the drug resistance to Ofx. Variety of Minimal Inhibitory Concentrations (MICs) to FQs drugs were detected by microwell-MIC-test method. Mutations of quinolone resistance determining region (QRDR) of gyrA gene were sequenced and identified. Relationship of different mutation sites and drug resistant degree were analyzed. A total of 6 MTB clinical isolates resistant to Ofx and induced drug resistant isolates in vitro were serially subcultured in liquid culture medium without drug. Variety of drug resistant stability, including MIC and mutation of gyrA gene were detected.

RESULTSMIC values of 21 Ofx susceptible isolates after induction were eight times higher than before, which were induced to drug resistant strains successfully and also resistant to Lfx and Mfx. Hot mutations of QRDR of gyrA gene were detected by sequencing, except one strain. Mutation of codon 94 occurred in 60% (12/20) of the strains with mutations and corresponding value of 50% Minimal Inhibitory Concentrations(MIC50) was ≥ 8 µg/ml. In all, 4 of 6 MTB clinical isolates resistant to Ofx harbored mutation of codon 90 (67%) , but the corresponding value of MIC50 was 2 µg/ml. After 21 serially subcultured in liquid culture medium without drug, MIC values of 6 clinical isolates resistant to Ofx were not changed obviously and mutations were also not changed. After 11 times serially subcultured in culture medium without drug, MIC values of induced drug resistant strains were also not changed obviously, but new mutations were detected in QRDR of 3 isolates.

CONCLUSIONMTB strains resistant to three kinds of FQs antibiotic were obtained by induction in vitro with Ofx. Codons 88, 94 mutations of QRDR of gyrA gene were related to the high level FQs drug resistance of MTB. Drug resistant stability of MTB to FQs was strong, and it is difficult for MTB to resume susceptibility.

Antitubercular Agents ; pharmacology ; DNA Gyrase ; genetics ; Drug Resistance, Bacterial ; genetics ; Microbial Sensitivity Tests ; Mycobacterium tuberculosis ; drug effects ; genetics ; isolation & purification ; Ofloxacin ; pharmacology

Cell culture is an important tool for biological research.To better understand the pathogenesis and therapeutic methods of the tumors,the three-dimensional cell culture is applied by more and more researchers to create a culture environment that closes to the tumor microoenvironment.Thanks to the advances in the tissue engineering technology,many kinds of models of the three-dimensional cell culture achieve wide accessibility.Compared with the traditional two-dimensional cell culture,the three-dimensional cell culture is better in simulating physiological features of the human histology and cells,including cell proliferation and differentiation,the interaction of cell to cell and cell to matrix.This paper reviews the progress of multi-cellular tumor spheroids (MCTS) culture technique of the three-dimensional cell culture for treatment of bladder cancer.

The optimum harvest time of Tulipa edulis was explored based on biomass accumulation and medicinal quality evaluation. Samples were taken from bud stage (Feb 13th) to dormancy stage (May 14th) and the growth indexes, organs biomasses, drying rate, contents of water-soluble extract and polysaccharides were determined. The results showed that biomass distribution of T. edulis varied with growth center and the bulb gained maximum biomass allocation in the whole growth period. The total biomass accumulation and bulb biomass accumulation　increased in the whole growth period and peaked in fructescence stage. No differences were observed in bulb biomass among fructescence stage, withering stage and dormancy stage. The correlation between bulb biomass allocation and other morphological indexes varied with the harvest time. Bulb dry weight biomass had negative correlation with some morphological indexes of aerial part of T. edulis at bud stage, flower stage and fructescence and had significant positive (P<0.05) or extremely significant positive correlation（P<0.01）with other morphological indexes except for root at bearing fruits stage. The drying rate of bulb of T. edulis increased with the extension of harvest time and peaked in dormancy stage. The water-soluble extract of T. edulis bulb was the highest in pre-growing-stage. The tendency of polysaccharides contents showed a W-shape variation during the harvesting period. The polysaccharides content was the lowest in fructescence stage and was the highest in dormancy stage. Considering the yield and medicinal quality of T. edulis bulb, the optimum harvest time of T. edulis is in the withering stage or early stage of dormancy.

ObjectiveTo establish a rapid method for evaluating the heterozygosity of Murraya paniculata germplasm materials and provide as a foundation for developing germplasm breeding and innovation measures for M. paniculata. MethodSingle nucleotide polymorphisms （SNPs） were screened from the genome resequencing data of 65 plants of M. paniculata. A self-written script was used to transform 20 SNPs into restriction fragment length polymorphism （RFLP） markers. Polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） was employed to detect the 20 RFLP markers in 12 M. paniculata germplasm accessions， and the heterozygosity of M. paniculata germplasm accessions was calculated based on the number of enzyme-cutting bands at the 20 RFLP marker sites. Plink was used to calculate the whole genome heterozygosity of 12 M. paniculata germplasm accessions， and the results obtained with different methods were compared. ResultThere was no significant difference in the heterozygosity calculated by the PCR-RFLP method and the genome resequencing method. The PCR-RFLP and genome resequencing methods identified 8 and 9 germplasm accessions， respectively， with a heterozygosity level less than 30%. Seven germplasm accessions with heterozygosity less than 30.00% were calculated by both methods. ConclusionThe PCR-RFLP method established in this study for evaluating the heterozygosity of M. paniculata germplasm demonstrates the precision of 87.5% and the accuracy of 77.8%. This method serves as a reference for developing heterozygosity evaluation methods in other medicinal plant germplasm resources.