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.

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.

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.

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.

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.

delineated individually based on CT or MRI to guarantee their coverage in radiotherapy.

Objective To compare the dose distribution of the three-dimensional conformal radiotherapy(3D-CRT)and 5-field or 7-field intensity modulated radiation therapy(IMRT), and to explore the value of IMRT in preoperative radiotherapy for rectal cancer.Methods Ten rectal cancer patients treated with preoperative combination radiotherapy and chemotherapy were enrolled in this study. 3D-CRT plan and the 5.field or 7-field IMRT plans were performed for each patient.The conformal index (CI),heterogeneity index(HI)of the planning target volume(PTV)and the dose of normal organs of 3D-CRT plan(3D-CRTp)and the 5-field or 7-field IMRT plans(IMRT5fp or IMRT7fp)were analyzed with the dose-volume histogram.Results The CI values of PTV were 0.91,0.87 and 0.78 in IMRT7fpIMRT5fp and 3D- CRT but with IMRT7fp>IMRT5fp>3D-CRTp(t=-5.69、-8.91,P<0.01),respectively.The HI values of PrV were 1.09,1.08 and 1.05 in IMRT5fp,IMRT7fp and 3D- CRTp but with IMRT5fp >IMRT7fp>3D- CRTp(t=3.41、-6.89,P<0.01),respectively.The ratio of dose volume were 0.08,0.10 and 0.19(t=2.79、3.52,P<0.05)in IMRT7fp,IMRT5fp and 3D- CRTp on the small intestine V50,with 0.07,0.10 and 0.19(t=2.58、3.40,P<0.05)in IMRT7fp,IMRT5fp and 3D-CRTp on the bladder V50 and 0.01,0.01 and 0.05(t=3.00、3.17,P<0.01)in IMRT7fp,IMRT5fp and 3D- CRTp on the fomoral head V45.The ratio of dose volume were 0.31 and 0.38(t=3.91,P<0.01)in IMRT7fp and IMRT5fp on the bone marrow V50,with 0.07 and 0.10 in IMRT7fp and IMRT5fp on bladder V45.Conclusions IMRT plan is superior to 3 D- CRT plan in dose conformal degrees of PTV with preoperative radiotherapy of rectal cancer and can significantly protect the normal tissues.The 7-field IMRT plan might be the optimal plan for dose conformal degree and dose uniformity compared with 5-field IMRT.

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.

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

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.