Application of doggerel in classroom teaching of medical microbiology / 中华医学教育探索杂志

Medical microbiology is an important basic subject in medical colleges.In view of the multifarious and abstract nature of the specialized course and the dull atmosphere in classroom teaching,we have adopted a doggerel teaching method in order to enliven the classroom atmosphere and improve the class teaching quality.After long-term teaching practice,it's been proved that the proper use of doggerel in classroom teaching can not only refresh boring theory and enhance students' learning interesting,but also simplify the complicated context and improve their memorizing ability and learning efficiency,posing a significant impact on the teaching effects of medical microbiology.

Transcription factor p53 inhibits dengue virus infection through typeⅠinterferon signaling pathway / 中华微生物学和免疫学杂志

Objective To investigate the role of a transcription factor p 53 in dengue virus infec-tion.Methods A plasmid expressing siRNA specific for p 53 gene was constructed and then used to prepare HepG2 cell line with a suppressed expression of p 53 protein.The expression of p53 protein was detected by Western blot assay .A wild type control group and a siRNA group were set up by infecting wildtype HepG 2 cells and p53 low expressing HepG2 cells with type 2 dengue viruses,respectively.The virus titers in two dif-ferent cells were determined by plaque forming assay using Vero cells .Indirect immunofluorescence assay was performed to detect virus multiplication .The apoptosis of virus infected cells were analyzed by flow cytome-try.ELISA was performed to analyze the levels of IFN-βsecreted by infected cells from two groups .Results Compared with wildtype control group ,the cells in siRNA group showed a suppressed expression of p 53 pro-tein,suggesting that the HepG2 cell line with low p53 protein expression was successfully established .The vi-rus titer in supernatants of the cells from siRNA group was about 100-fold higher than that of wildtype control group at 24 hours after viral infection .Fluorescence activated cell sorting analysis showed that the numbers of green fluorescence labeled cells were remarkably increased in siRNA group .We speculated that p53 protein might play a role in the inhibition of dengue virus infection as indicated by the observed results .The numbers of apoptotic cells showed no significant difference between two groups .However,the level of IFN-βsecreted by wildtype HepG2 cells was six times higher than that of the cells in siRNA group .Conclusion p53 pro-tein might inhibit dengue virus infection through the activation of type Ⅰ interferon signaling pathway rather than enhance cell apoptosis .

Practice and thinking on innovation education of medical microbiology / 中华医学教育探索杂志

Innovation education was introduced in medical microbiology teaching practice,including updating the innovative education concept,reforming teaching methods and means,constructing the teaching content system and practice platform adapting to innovation education.

Exploration of bioinformatics courses for eight-year medical students / 中华医学教育探索杂志

The need of eight-year clinical students for bioinformatics undergraduate courses is described.In addition,the measures and experiences on textbooks choosing,teaching content assignment,teaching methods designing and test means innovation are also discussed.All these provide a reference implementation for the development of eight-year clinical bioinformatics courses.

The construction strategy of gene multimers and their expression modes / 医学研究生学报

The great attempts to clone a gene in the form of multimers were motivated either by the need to produce copious amounts of the particular DNA fragments or by the desire to obtain a large supply of the gene product of interest.The arrangement of the gene multimeric copies is in identical tandem orientation,this head-to-tail arrangement of gene multimers could be constructed by the strategies of tandem repeats,PCR amplification,chemical concatenation and isocaudarners.The expression mode of the gene multimers may be different based on variable construction strategy.

Identification and characterization of the integrated site of pseudomonas aeruginosa phage PaP3 / 医学研究生学报

Objective: Identification of the attachment site of phage PaP3 within the genome of Pseudo-monas aeruginosa PAS. Methods:The full genome of lysogenic bacteria was cleaved by Pst Ⅰ and produce a large fragment of more than 45 000 bp, which was subsequently digested by EcoR Ⅰ. Then the fragment containing DNA sequence of phage and bacteria was cloned into pFastBacTMHT A vector, and the result of sequencing indicated the right hybrid site attR. AttL was isolated by PCR on the base of integration mechanism. And then attP and attB were indentified according to the nucleotide sequences of attR and attB. Results:A sequence of 21 bp(5'-GGTCGTAGGTTCGAATCCTAC-3') was defined to be the core site of integration, which was located at t-RNAPro gene in the genome of phage PaP3 and t-RNALys gene in the genome of Pseudomonas aeruginosa PA3. The attP and attB flanked with a set of inverted repeat and direct repeat. Conclusion:The integrated site of PaP3 within the genome of PA3 was identified and characteriged, which could be of value in investigating the mechanism of integration and gene flow between different species in the natural world.

Application of carrier molecule PaP3.30 in fusion expression of small bioactive peptides / 医学研究生学报

Objective:To screen and clone a carrier molecule for the expression of small bioactive peptides at high levels. Methods: A carrier molecule, PaP3.30, was screened out from the genome of Pseudomonas aeruginosa phage PaP3 and its gene was cloned by PCR method and inserted into pQE 32 expression plasmid, this recombinant plasmid was named pQE PaP30. The peptide antibiotics hPAB ? gene was then inserted into pQE PaP30 and induced to express the fusion protein in Escherichia coli . The ability of PaP3.30 to express other bioactive peptides was evaluated by fusing 6 different origins, varies in sizes and isoelectric points selected peptides to it. Results: After fused to PaP3.30, the peptide antibiotics hPAB ? could express as fusion protein above 30% of total bacterial proteins. Six selected peptides were also expressed by the level of 35%～44% total bacterial proteins when fused to carrier molecule, PaP3.30. Conclusion: The new carrier molecular, PaP3.30, is versatile in the expression of small bioactive peptides.

Molecular modeling of peptide antibiotics hPAB-?and its artificial mutants construction / 医学研究生学报

Objectives: To design the mutants of peptide antibiotics hPAB ? based on its molecular structure. Methods: The three dimension structure of hPAB ? was constructed by protein homology modeling method. The mutant molecules were designed and generated by PCR and inserted into pQE CP4 expression plasmid. The recombinant plasmids were identified by PCR and DNA sequencing and then transformed into Escherichia coli JM109 to express target fusion proteins. Results:Peptide hPAB ? shows one ? helical and three ? sheet in its structure. Its ? helical regions seem play a key role in the formation of active oligomer. Aside from positioning Thr 7 and Lys 10 into contact positions, the orientation of the ? helix is conserved about the oligome core, forming a ridge around it. Additionally, the dipoles of the helices would overlap to create a positively charged region near the core. These dipoles may be offset, however, by the presence of Asp 4 at the base of the helix. Two mutant molecules, hPAB ? 38 and hPAB ? 34, were designed by deleting N or/and C terminal 2～5 amino acid residues based on hPAB ? structure. The recombinant plasmids containing the mutants gene can express interest fusion proteins in E. coli JM109 successfully. Conclusions: Design, cloning and expression of the mutants of peptide antibiotics hPAB ? lay down the foundation for screening of the mutant of shorter peptide chain and having high or same antimicrobial activity.

Reconstruction of engineered bacteria in preparing recombinant human peptide antibiotic ? / 第三军医大学学报

Objective To reconstruct the new engineered bacteria expressing hPAB-? triploids so as to improve the outputs of recombinant human peptide antibiotic ?. Methods The recombinant plasmid pQE31-hPAB-?(3) was transformed into E. coli. M15 to screen the new engineered bacteria expressing hPAB-? triploids. The stabilities of phPAB-?(3)/M15 were observed in continuous cultures. The expression levels of the fusion peptides of interest and the bacterial yields of the new engineered bacteria phPAB-?(3)/M15 were compared with that of phPAB-?(3)/JM109 in different fermentation scales. Results Genetic stability of the recombinant plasmid and phPAB-?(3)/M15 was 100 after 10 passages. Take bacterial yields into account, the new engineered bacteria phPAB-?(3)/M15 was better than phPAB-?(3)/JM109 at the similar expression levels of the target proteins by “t” test analysis (P

Screening of recombinant bacterium for expression of human peptide antibiotic hPAB-? multimers and evaluation of its fermentation / 医学研究生学报

Objective: To screen the best genetic engineering bacterium for the production of peptide antibiotic hPAB-? and evaluate its fermentation level in bottle. Methods:After analysis of the interest fusion protein expression levels of 8 recombinant bacteria containing 1-8 copies of human peptide antibiotic hPAB-? expressing plasmid respectively,2-5 copies expressing bacteria were chosen for the further study of their bacteria yield,expression forms of the target protein, dissolution of the inclusion bodies and the efficiency of fusion protein purification by affinity chromatography, then the best engineering bacterium with the certain copies of interest peptide expressing plasmid was screened out and its optimal fermentation parameters in bottle were also studied. Results:The recombinant bacterium transformed by 3 copies of interest peptide expressing plasmid was the best candidate for its bacteria yield (3.153 g/L) and fusion protein expression level (27.7%) were the highest among 1-8 copies candidates. The inclusion bodies of 3 copies target fusion protein could be easily dissolved by 8 mol/L urea and captured by Ni-NTA column. The elution of the fusion protein could be directly cleaved to monomer by adding 2 mol/L hydroxylamine, adjusting pH to 9.0 and incubating at 45℃ for 2 h. The optimal fermentation conditions of the selected recombinant bacteria were: culture the organisms with modified M9-CAA media at 37℃ and 160 r/min to (A 600 )≈2.5, then add IPTG to the final concentration 100 ?mol/L to induce the expression of target fusion protein for 5 h. Conclusion:The engineering bacterium containing 3 copies interest peptide recombinant expressing plasmid is the best candidate for the production of peptide antibiotic hPAB-?,and its fermentation parameters are confirmed.

Achievements and prospect of research work on important pathogenic microorganisms / 解放军医学杂志

Objective To review the current situation and developments of researches into important pathogenic microorganisms domestically and abroad,and to suggest the orientation of research work and development in pathogenic microbiology in PLA.Methods The achievements and advances of research work achieved domestically and abroad in the past five years regarding important viruses(such as hepatitis viruses,human immunodeficiency virus,influenza virus,encephalitis viruses and hantaanvirus)and bacteria (such as Mycobacterium tuberculosis,Streptococcus suis serotype 2,Yersinia pestis,Bacillus anthracis and Helicobacterp ylori)were retrieved and reviewed using intelligence research methods.Results Infectious diseases caused by pathogenic microorganisms were the most severe hazards to health and life of human beings.Especially in the past thirty years,newly emerging infectious diseases and recurrence of previonsly controlled infectious diseases had received wide attention.Infectious diseases control had been greatly improved owing to the increasing discoveries in the knowledge about pathogenic microorganisms.Conclusions During the period of "Twelfth Five-Years Plan" ,a big team of science and technology personnel with strong innovative ability in the domain of medical microbiology should be brought up in PLA;and a number of advanced and consummate research bases and technology platforms should be built up;to apply for and realize a batch of major research projects,strive to make a number of scientific achievements with innovation and important application prospects,improve the transformation efficiency of scientific and technological achievements and contribution of scientific and technological progress,and strive to achieve important progresses and breakthrough in mainstream research.

Cloning of LVA Ca 2＋ channel and its primary structure features / 细胞与分子免疫学杂志

Aim To clone LVA calcium channel (also known as T-type calcium channel) from INS-1 cell line derived from rat pancreatic β cells. Methods RT-PCR, 3′ -RACE and 5′ -RACE were used to clone coding sequence of the whole gene. DNA sequencing and genomic DNA walking were used to identify the primary structure features and exons. Results The cloned gene, named as α 1 G-INS, was composed of 6 864 bp, encoding 2 288 amino acids, which shares 96.3％ identity to α 1G, the neuronal T-type calcium channel. Compared to α 1G, the amino acids in membrane-spanning regions of four transmembrane domains and intracellular loop LI-II of α 1G-INS were highly conserved, but there are three distinct regions. This discrepancy was due to alternative mRNA splicing. Scattering on other regions of the molecule there were 10 single amino acid substitutions, which could be explained as site-mutations of the gene. Conclusion A new isoform,α 1G-INS, of T-type calcium channel is successfully cloned from rat insulin-secreting cell line INS-1, which is significant to further understanding many Ca2＋ -involved biologically essential processes.

Experimental Study on Target Cells of Pseudomonas aeroginosa Exotoxin A / 第三军医大学学报

Pseudomonas aeroginosa exotoxin A(PEA) was purified from the strain PA103 and labelled with Ⅰ~(125).Then autoradiographic tracing,histopathological observations with light and electron microscopes,and antiserum protestion test were carried out on inbred mice BALB/C.The results indicate that the liver is the target organ of PEA,and both the hepatocytes and kupffer cells the target cells,on which the severe damage may be responsible for the acute death of the toxicated animals.

Experimental identification of endonuclease activity of the putative gene tls from phage PaP3 / 解放军医学杂志

Objective To experimentally verify the putative function of gene tls from Pseudomonas aeruginosa phage PaP3. Methods The gene tls was amplified from the genome of phage PaP3 by PCR and subcloned into pMD18-T vector. Then the gene tls cut down from the vector was inserted into the plasmid pQE31, which could give the 6-His tag at the N' side of the expressed protein. The recombinant vector pQE-tls was transformed to E. coli JM109. After induction with IPTG, the expressed bacteria was resuspended and sonicated, then the inclusion body was obtained after centrifugalization. The inclusion body was then dissolved with lysis buffer. The inclusion body solution, which has the target protein, was then purified by affinity chromatography and renatured by dialysis. Finally the nuclease activity of the fusion protein H6-TLS was tested on the plasmid pMD-cos which contains the cutting site of terminase large subunit. Results The expression plasmid pQE31-tls was successfully constructed, and the target protein yield was up to 30% of the total bacterial proteins. Meanwhile the substrate vector pMD-cos was also successfully constructed. After purification and renaturation, the fusion protein H6-TLS could partially cut the substrate vector pMD-cos. Conclusion The fusion protein H6-TLS was successfully expressed, purified and renatured, and it was shown to possess nuclease function. The experiment lays the foundation for further research of the gene tls.