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.

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.

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.

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.

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 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.

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

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.

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.