A review of research on staphylococcal phenol-soluble modulins / 中华微生物学和免疫学杂志

Phenol-soluble modulins (PSMs) are a novel family of small amphipathic, alpha-heli-cal peptides with strong surfactant-like properties. PSMs have multiple roles in staphylococcal pathogenesis and are considered as important virulence-associated factors. They may cause lysis of many eukaryotic cells, such as human neutrophils,erythrocytes and dendritic cells;induce the expression of pro-inflammatory cyto-kines;facilitate the structuring and detachment of biofilms;influence the expression of other genes. This re-view summarizes the classification,structure,regulatory effect on gene expression and biological function of PSMs. The potential avenues to target PSMs for drug development against staphylococcal infections are also evaluated in this paper.

Development of international genetically engineered machine competition in China / 生物工程学报

Synthetic biology is a fast moving interdisciplinary branch of biology and engineering. To educate the next generation of synthetic biology scientists, the International Genetically Engineered Machine (iGEM) competition was established. In the past eleven years, many Chinese teams have participated in this event, but no thorough review and analysis have been carried out. In this paper, we collected the data and information of the Chinese teams from the iGEM website and analyzed the number, distribution and performance of Chinese teams in iGEM competition. We also described contributions made by the Conference of China iGEMer Community (CCiC) organization. The contributions to China higher education made by the iGEM competition were also summarized. Finally, we proposed several suggestions for the development of the iGEM competition in China. We envision the iGEM competition will continue to promote the innovative education and cultivation of the next-generation synthetic biology scientists in China.

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.

Identification of the nanobacteria in prostatic fluid of CPPS / 中华泌尿外科杂志

Objective To identify the nanobaeteria in prostate fluid of patients with CPPS.Methods Expressed prostatic secretion(EPS)and urine specimens were collected by Meares-Stamey way from CPPS patients(n=100)and normal controls(n=100).The specimens were cultured and nanobacteria was identified by indirect immunofluoreseenee staining with rnonoelonal antibody.The morphological features were observed by using transmission electron microscopy(TEM). Results The positive rate of nanobaeteria in the EPS cdture of CPPS patients and controls were 43% and 5% respectively,with significant statistical difference(X2=39.58,P＜0.01).By TEM,the sizes of NB ranged from 100 to 500 nm and appeared eoccoid-ccccobacillary shape. Conclusion Nanobaeteria infection may exist in EPS of CPPS patients.

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.

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.

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.

Determination of binding ability of PaP3’s terminase large subunit to cos site / 第三军医大学学报

Objective To investigate the binding ability of the terminase large subunit of Pseudomonas aeruginosa bacteriophage PaP3 to the cos site. Methods The gene tls was amplified from the genome of bacteriophage 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 a 6-His tag at the N-terminal of the expressed protein. The recombinant vector pQE-tls was transformed to E.coli. JM109, after induction with IPTG, the expressed bacteria were resuspended and sonicated, then after centrifugation, the inclusion body was obtained. The inclusion body was dissolved with lysis buffer, then the tagged protein was purified by Ni-NTA affinity chromatography and renatured by dialysis. Finally the DNA-binding ability of the fusion protein rTLS was determined by EMSA. Results The expression plasmid pQE31-tls was successfully constructed, and the target protein yield was up to 30% of the total bacterial proteins. After purification and renaturation, the fusion protein rTLS can partially bind the cos fragment. Conclusion The fusion protein rTLS was successfully expressed, purified and renatured. The rTLS has the specific DNA-binding activity. The present work lays the foundation for the further research of the gene tls.

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

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.

Nanobacteria: isolation from prostatic calculus and identification of its 16S rRNA gene / 第三军医大学学报

ObjectiveTo explore the distribution of nanobacteria in prostatic calculus and investigate its role in the formation of prostatic calculus. MethodsThe stones of 40 patients with prostatic calculus was used to isolate and culture the possible bacteria. The genomes of obtained bacteria were extracted, and the 16S rRNA was amplified by PCR followed by sequencing. ResultsThe obtained specific fragment had a 98% resemblance with 16S rRNA of nanobacteria: Score=2 480 bits (1 290), Expect=0.0; Identities=1 387/1 409 (98%), Gaps=4/1 409 (0%); Strand=Plus/Plus. ConclusionNanobacteria is proved existing in the stones of prostatic calculus patients by PCR and sequencing.

Construction of recombinant plasmid harboring human peptide antibiotic gene hPAB-? and its expression in E. coli / 解放军医学杂志

Objective To construct the recombinant plasmid with a human peptide antibiotic hPAB-? gene and to make it expressed in E. coli. Methods To replace the CNBr cleavage site in plasmid pFAST-hPAB-? (CNBr), a pair of primers containing the hydroxylamine cleavage site were designed, and the amplified PCR fragments were cloned into pFAST-HTa plasmid to produce pFAST-hPAB-? (HA), which was then transformed into E. coli DH10B. The constructed plasmid was identified by Ehe Ⅰ/Hind Ⅲ digestion and direct DNA sequencing. An Ehe Ⅰ/Hind Ⅲ digested fragment from pFAST-hPAB-? (HA) was subcloned into pQE32-CP to construct pQE32-CP-hPAB-?, which was transformed into E. coli JM109. The bacteria containing the expression plasmid were induced to express the fusion protein by IPTG. SDS-PAGE was carried out to analyze the molecular weight, expression quantity and expression form of the target fusion protein. After captured by Ni-NTA affinity column, the fusion protein was subjected to hydroxylamine cleavage analysis. Results An expected 230bp fragment was obtained by digesting pFAST-hPAB-? with Ehe Ⅰ/Hind Ⅲ. After this fragment was cloned into pQE32-CP, the recombinant plasmid was confirmed to contain the correct target sequence by DNA sequencing. The recombinant plasmid pQE32-CP-hPAB-? could express a desired protein with a relative molecular weight about 27kD, and its expression level reached 43 percent of the total bacterial proteins. The inclusion bodies were lysed by 8mol/L urea, and the fusion protein could then be captured by Ni-NTA column and cleaved by 2mol/L hydroxylamine at pH9.0. Conclusion The recombinant plasmid pQE32-CP-hPAB-? has been successfully constructed, and it can express the desired hPAB-? fusion protein in E. coli JM109 at high level. These results provide the foundation for future research.

Construction of recombinant plasmid of Pseudomonas aeruginosa exotoxin A and its secreting expression in E. coli / 解放军医学杂志

Objective To clone and to express the full-length Pseudomonas aeruginosa exotoxin A gene and to purify the expressed protein using affinity chromatography. Methods Exotoxin A gene was amplified from the recombinant plasmid pSK/PEA-T vector and subcloned into the pMAL-P2X vector. Then the recombinant plasmid pMAL- PEA was transformed to E.coli BL21. After induction with IPTG, the expressed protein was analyzed by SDS-PAGE and purified with affinity chromatography. Results The recombinants expressed the fusion protein at a level of about 20% of total cell proteins, and 80% of the fusion protein was secreted into the supernatant. Conclusion Successful expression and purification of PEA are of significance for in-depth study of the pathogenic mechanism of Pseudomonas aeruginosa and preparing immunotoxin.

CONSTRUCTION OF THE MUTANTS OF HUMAN PEPTIDE ANTIBIOTICS hPAB-? AND ITS RECOMBINANT BACULOVIRUS / 解放军医学杂志

The aim was to determine the antimicrobial activities of peptide antibiotics hPAB ? and construct the recombinant baculovirus of its mutants. Four mutants of hPAB ? were designed based on molecular autosyndetic modeling and its genes were cloned by PCR. The transfer plasmid and recombinant baculovirus were constructed. The results showed that the hPAB ? cloned previously, is a good peptide antibiotics. Four mutant genes of hPAB ? were inserted into pFAST HTa plasmid and the recombinant pFAST hPAB ? were screened by restriction enzymes analysis and DNA sequencing. The recombinant baculovirus was obtained after transforming pFAST hPAB ? into Escherichia coli DH10Bac. Our work lays a good foundation for further research.