Porphyromonas gingivalis is a specific causative agent of human chronic periodontitis. This anaerobe produce collagenase PrtC encoded by prtC. To constructed the prokaryotic expression system for the prtC gene from P.gingivalis so as to be used to explore antigenicity and immunoreactivity of prtC gene product as well as the relationship between the local level of PrtC and the periodontitis damage, the entire length of prtC genes fragment from the ATCC-33277 and 47A-1 strains of P.gingivalis was amplified by PCR. After T-A cloning and sequencing, the prokaryotic expression system for prtC was constructed by using pET32a plasmid and E.coli BL21DE3 strain. The expression of the target recombinant PrtC protein was induce with different concentrations of IPTG. Western blot assay was used to detect the antigenicity and immunoreactivity of PrtC protein, and ELISA assay was used to detect the PrtC level in the subgingival plaque samples of patients with chronic periodontitis. The experimental results showed that the nucleotide sequences of the prtC genes from ATCC-33277 and 47A-1 strains of P.gingivalis were entirely identical, and the sequence similarities of nucleotides and amino acids were 98.46% and 99.07% respectively. Under the induction of different concentration of IPTG, the output of recombinant expressed product PrtC may reach up to 50% of the total bacterial proteins. It was also proved that the recombinant PrtC could bind with antibody against the whole cell of P.gingivalis, and could induce the production of specific antibodies in rabbit. In 91.39% of the subgingival plaque samples, the PrtC could be detectable, in which the level of positive rates of detection was higher in severe cases of chronic periodontitis than that in the mild cases. So far, a prokaryotic expression system for the prtC gene from P.gingivalis with high expression efficiency was successfully constructed in the present study, and the expressed product PrtC possesses well antigenicity and immunoreactivity, suggesting the possibility to be used as the candidate antigen for developing the serological kit and P.gingivalis vaccine.

ObjectiveTo generate a prokaryotic expression system of series predominant epitopes (UreB322 and UreB527) of Helicobacter pylori UreB protein,and to synthesize a multiple antigenic peptide (MAP) vaccine by linking both the two epitopes with a peptide carrier (Poly-Asp-Lys),and to determine the immunogenicity and immunoprotection of the MAP vaccine.MethodsLinking primer PCR was performed to generate an enterokinase(EK) site-containing series UreB322 and UreB527 epitope encoding gene for construction of its prokaryotic expression system.The expressed target recombinant fusion protein 8 ×[rEK-UreB322-EK-UreB527-EK] was hydrolyzed with EK and then rUreB322-EK and rUreB527-EK epitope peptides were extracted using a Sephadex G-25 column.rUreB322-EK,rUreB527-EK and Poly-Asp-Lys were linked using carbodiimide method to produce a MAP vaccine (MAP-rUreB322/B527).The antigenicity and immunoreactivity of each of the two epitope peptides and MAP-rUreB322/527 were determined by ELISA and Western blot assay.An animal H.pylori strain SS1-infected model in BALB/c mice was used to detect the immunoprotection of MAP-rUreB322/527.ResultsAn octuple-repeated series UreB322-UreB527 encoding gene and its prokaryotic expression system were obtained.The yield of target fusion protein 8×[rEKUreB322-EK-rUreB527-EK] was as high as 48％ of the total bacterial proteins.EK hydrolyzed the target fusion protein completely into rUreB322-EK and rUreB527-EK peptides.The linking ratio of rUreB322-EK,rUreB527-EK and Poly-Asp-Lys was as high as 92.5％.The antibody against whole cell of H.pylori and rUreB-IgG could recognize and combine with the rUreB322-EK,rUreB527-EK or MAP-rUreB322/527.The specific serum antibody level in MAP-rUreB322/527-immunized mice was significantly higher than that in rUreB-immunized mice (P＜0.05).The immunoprotective rates(83.3％ and 91.7％ ) by immunization with50 or 100 μg MAP-rUreB322/527 in the H.pyloristrain SSI-infected mice were significantly higher that those(d1.7％ and 50.0％ ) by immunization with equal rUreB(P＜0.05).ConclusionAn gene composed for encoding a repeated series predominant epitopes of H.pylori UreB protein and its prokaryotic expression system are successfully generated in this study.MAP-rUreB322/527,the multiple antigenic peptide vaccine based on the two predominant epitopes of UreB,can noticeably increase the immunoprotection in H.py/or/infected mice.

Objective To screen and identify the predominant T-and B-cell ( T-B) combined an-tigenic epitopes on the outer membrane protein Loa22 of pathogenic Leptospira interrogans ( L.interrogans) stains and to further analyze their immunogenicity.Methods PCR analysis was used to detect loa22 gene in L.interrogans strains belonging to eight different serogroups or serovars prevalent in China.The PCR prod-ucts were sequenced after T-A cloning.A prokaryotic expression system for loa22 gene of L.interrogans sero-group Icterohaemorrhagiae serovar Lai strain Lai was constructed.The expressed recombinant protein rLoa22 was extracted by Ni-NTA affinity chromatography.The rabbit anti-rLoa22 serum samples and IgG were pre-pared.The T-B combined antigenic epitopes on Loa22 protein were predicted by using professional bioinfor-matic softwares.Phage display in combination with Western blot assay and ELISA were performed to identify the immunogenicity of the recombinant phage PⅢ protein-displayed and artificially-synthesized T-B com-bined antigenic epitopes, respectively.MTS assay and ELISA were performed to detect the activation of T cells and the expression of IL-2, IL-4 and IFN-γinduced by T-B combined antigenic epitopes, respectively. Results All of the tested pathogenic Leptospira strains were positive for loa22 gene, sharing 85.5%-99.8%homologies in nucleotide sequences and 93.9%-99.5%homologies in amino acid sequences.The construc-ted prokaryotic expression system for loa22 gene efficiently expressed the rLoa22 protein.Among four T-B combined antigenic epitopes (Loa22-77, Loa22-90, Loa22-125 and Loa22-157), only Loa22-90 epitope presented a strong positive band in Western blot analysis.The proliferation of CD4+T cells and the expres-sion of IL-2 ( Th1 ) and IL-4 ( Th2 ) were significantly enhanced by the stimulation with Loa22-90 epitope peptide (P<0.05).Conclusion Loa22 protein is a sequence-conserved genus-specific outer membrane protein of L.interrogans.The Loa22-90 epitope is the predominant T-B combined antigenic epitope of Loa22 protein, which might be used as a candidate antigenic epitope in the development of multiple antigenic pep-tide ( MAP) vaccines against Leptospira infection.

Objective To determine the role of flagellar motor protein MotA in the pathogenesisassociated chemotaxis and colonization of Campylobacter jejuni. Methods The motA gene as well as Kan~r gene and plus-motA gene segments for motA gene knock-out were amplified by PCR and the target amplification fragments were sequenced after cloning. A suicide plasmid(pBlueskrit-Ⅱ-SK~(motA-kan)) and a motA gene knock-out mutant (motA~-) were constructed based on homologious recombination. By using semisolid plate migration test, hard agar plus (HAP)-based chemotactic test towards sodium deoxycholate (SDC) in vitro, and jejunal colonization test in BALB/c-ByJ mice were performed to determine the differences of flagellar motility, chemotaxis towards SDC and colonization in murine jejunum between motA~- mutant and wild-type strain. Results The nucleotide and amino acid sequences of the cloned motA gene were 100% identical to the reported corresponding sequences. The results of PCR, sequencing and continuous passage culture in antibiotics-contained medium demonstrated that both suicide plasmid and motA~- mutant were successfully generated. The diameters of clonies on semisolid plate and 0.2 mol/L SDC-induced chemotactic tings in HAP as well as the bacterial numbers adhering to the surface of murine jejunal mucosa and in jejunal content of motA~- mutant were significantly less than those of wild-type strain(P<0.05). Conclusion A motA gene knock-out mutant of C. jejuni was successfully constructed in this study, motA is an essential gene for flagellax motility, pathogenesis-associated chemotaxis and colonization of C. jejuni.

Objective To generate a prokaryotic expression system of Salmonella paratyphi A nmpC gene that encoding an outer membrane protein(OMP),and to determine immunogenicity and immonuprotection of the recombinant expressed product rNmpC and carrying and expression frequencies of the nmpC genes in isolates of S.paratyphi A.Methods A nmpC gene clone was obtained from a clinical S.paratyphi A strain JH01 by PCR and T-A cloning method,and then a prokaryotic expression system of the gene clone was generated.SDS-PAGE and Bio-Rad Agarose Image Pattern Analysis System were applied to examine the expression and yield of rNmpC.Antigenicity and immunoreactivity of rNmpC were determined by immunodiffusion test,Western blot assay and micro-Widal's test.The carrying and expression rates of nmpC genes in 98 S.paratyphi A isolates were detected by PCR and ELISA.By a mouse infection model,the immunoprotective effect of rNmpC against the lethal challenge of S.paratyphi A was determined.Results All the cloned nmpC genes had 100% nucleotide and putative amino acid sequence identities compared to the reported sequencing data.The expression yield of rNmpC was approximately 30% of the total bacterial proteins.rNmpC could efficiently induce rabbits to produce specific antibody and present positive Western hybridization signals with S.paratyphi A antiserum.All the tested S.paratyphi A strains have nmpC gene as well as express NmpC protein,but no nmpC gene could be detectable in S.typhi,S paratyphi B and S paratyphi C.Immunization with 100 μg and 200 μg rNmpC contributed 41.7%(5/12) and 66.7%(8/12) immunoprotective rates in mice,respectively.The sera from rNmpC immunized mice and survival mice in the NmpC is an unique OMP antigen of S.paratyphi A with conserved sequence,extensive distribution and natural expression.This OMP can be used as one the candidate antigens for developing multiple-valence genetic engineering vaccine of S.paratyphi A based on its fine immunogenicity and certain immunoprotection.

Objective To generate the spaO-ompA fusion gene of Salmonella paratyphi A and its prokaryotic expression system,and to determine the immunoprotection of the recombinant expression product rSpaO-OmpA.Methods A flexible peptide sequence was used to link spaO and ompA genes and a prokaryotic expression system of spaO-ompA fusion gene was subsequently generated.SDS-PAGE and Bio-Rad Agarose Image Analyzer were applied to examine the expression as well as the yield of the target recombinant protein rSpaO-OmpA.The antigenicity and immunoreactivity of rSpaO-OmpA were determined using immunodiffusion test,Western Blot assay and micro-Widal's test.By a mouse infection model,the immunoprotection of rSpaO-OmpA against the lethal challenge of S.paratyphi A was determined.In the animal protective test,the recombinant expressed SpaO (rSpaO) and OmpA ( rOmpA ) were used as the controls.Results The generated spaO-ompA fusion gene had 100％ nucleotide and amino acid sequence identities compared to the single spaO or ompA gene.The constructed prokaryotic expression system IPTG E.coli BL21DE3pET42a-spaO-ompA expressed the recombinant protein rSpaO-OmpA.rSpaO-OmpA combined with the antiserum against wholecell of S.paratyphi A to present positive hybridization signal and induced specific antibody in the immunized rabbits.Immunization with 100 or 200 μg rSpaO-OmpA contributed 66.7％ (8/12) or 83.3％ (10/12) immunoprotective rates in mice when the animals were attacked with S.paratyphi A.The immunoprotective rates produced by rSpaO-OmpA were significantly higher than that of equal rSpaO or rOmpA( P＜0.05 ).The sera from rSpaO-OmpA immunized mice presented 1∶5-1∶40 agglutination titers to the H antigens of different S.paratyphi species,and 1∶1-1∶16 immunodiffusion titers to rSpaO,rOmpA and rSpaO-OmpA proteins,respectively.Conclusion The artificially fusion antigen,rSpaO-OmpA,has more powerful immunogenicity and immunoprotection that the equal rSpaO or rOmpA.

Objective To clone fliH, fliⅠ, fliY and fliN genes that encoding flagellum-associated proteins of L. interrogans for construction of their prokaryotic expression systems, and to determine the loca- tions of Flirt, FliⅠ, FIiY and FIiN. Methods The fliH, fliⅠ, fliY andfliN genes were amplified by PCR and sequenced after T-A cloning. Prokaryotie expression systems of the target genes were constructed subsequently. Expression of target recombinant proteins were demonstrated by SDS-PAGE and BioRad Gel Image Analyzer, and Ni-NTA affinity chromatography was performed to extract the target expression prod- ucts. Rabbits were subcutaneously immunized with the four recombinant proteins respectively to obtain anti- sera. ELISA was performed to measure the titers of antisera and Western blot assay was used to determine the immunobinding abilities among the antisera and their antigens. Immunoelectron microscopy was selected to locate the position of FliH, FliⅠ, FliY and FIiN. Results Segments of fliH, fliⅠ, fliY andfliN genes with 924, 1365, 1065 and 318 bp in size were successfully obtained by PCR. Similarities of nucleotide and puta- tive amino acid sequences from the four genes were 100% compared with the reported sequences. The con- structed prokaryotic systems efficiently expressed rFliH, rFliⅠ, rFliY and rFliN with the outputs of approxi- mate 20% of the total bacterial proteins. The rabbits immunized by rFliH, rFliⅠ, rFliY or rFliN could pro- duce antibody. The antisera had the titers above 1:100 000, and could recognize the corresponding recombi- nant proteins and membrane proteins of L interrogans to display positive Western hybridization bands. Flirt, FliⅠ, FliY and FliN were found to distribute on the external surface of inner envelope, the internal surface of outer envelope or the interspaces between the two layers of L. interrogans envelope. Conclusion The pro- karyotic expression systems was successfully constructed in this study, which could efficiently express flagel-lum-associated proteins FliH, FliⅠ, FliY and FliN of L. interrogans. The antisera with high titers to recognize their protein antigens were also obtained. Flagellum-associated proteins Flirt, FliⅠ, FIiY and FIiN are the inner envelope proteins and/or outer envelope proteins of L. interrogans.

Objective To construct a prokaryotic expression vector of Salmonella paratyphi A ompA gene, and to determine immunogenieity and immonuprotection of the recombinant expressed product rOmpA and carrying frequency of ompA gene in S. paratyphi A isolates. Methods ompA gene was amplified from a clinical S. paratyphi A strain JH01 by PCR, cloned into T-A cloning vector, and then the prokaryotic expression vector was constructed. The rOmpA expression was determined by SDS-PAGE. Antigenicity and immunoreactivity of rOmpA were determined by immunodiffusion test, Micro-Widal's test and Western blot assay, ompA gene exiting in 98 S. paratyphi A isolates was detected by PCR. By using immunoprotective effect of rOmpA against the challenge of S. paratyphi A strain 50001 in mice were determinded. Results Both the nucleotide and putative amino acid sequence identities of the cloned ompA gene were 100% com-pared to the reported corresponding sequences. The expression yield of rOmpA was approximately 65% of the total bacterial proteins, rOmpA was able to induce specific antibody in rabbits, and reacted efficiently with rabbit antisortm or the antiserum against whole cell of S. paratyphi A detected by Western blot. 94.9% (93/98) of the S. paratyphi A isolates had ompA gene. 41.7% (5/12) and 58.3% (7/12) of the mice im-murtized with 100 and 200 μg rOmpA were survival after lethal challenge with S. paratyphi A strain 50001. The titer of antibody to the H antigens of S. paratyphi spp in the sera from rOmpA immunized mice and sur-viral mice in the S. paratyphi A challenge detected by Micro-Widal's test was 1:5-1:40. Conclusion ompA gene has an extensive distribution in clinical isolates of S. paratyphi A. rOmpA possesses an immunogenicity and a certain immunoprotective effect, and can be used as the candidate antigen in genetic engineering vaccine.

OBJECTIVETo investigate therapeutic effects of vacuum sealing drainage (VSD) in the treatment of soft tissue defect combined with tendon and bone exposure.

METHODSFrom October 2007 to February 2011, 397 patients (412 feet) with open ankle fracture and dislocation combined with soft tissue defected were treated by VSD. There were 301 males and 96 females with an average age of 36 years (ranging age from 20 to 73 years). According to AO classification, 74 feet were type I, 211 feet were type II, 108 feet were type III and 19 feet were type IV. The mean time from injury to operation was 5.6 h ( 2 to 12 h). The mean treatment time of was 10 months (4 to 19 months).

RESULTSOne hundred and forty-one patients were primarily healed, 97 patients were sutured at stage II. Split-thick skin grafting was performed at stage II was performed in 103 patients; free flap transplantation was performed in 25 patients. Three of the 34 patients with infection were removed steel plate; Eviscerate flap coverage wound was performed in 14 patients caused by the first metatarsal bone exposure; Toe amputation were performed in 22 cases caused by toes necrosis. Tarsometatarasl joints perforators' surgery was performed in 10 patients with forefeet necrosis. Thirty hundred and six patients were followed up from 3 to 20 months (averaged 10 months). The wounds healed well.

CONCLUSIONVSD for soft tissue defects caused by ankle injury is a simple and effective method, but can not replace debridement and transfer flap.

Adult ; Aged ; Ankle Fractures ; Debridement ; Drainage ; methods ; Female ; Humans ; Joint Dislocations ; surgery ; Male ; Middle Aged ; Skin Transplantation ; Treatment Outcome ; Vacuum ; Young Adult

Objective To understand the differences in killing ability and mechanism of human mononuclear macrophages and neutrophils against Leptospira interrogans.Methods Human THP-1 and HL-60 cell lines were respectively pretreated with PMA (phorbol 12-myristate 13-acetate) and ATRA (all-trans retinoic acid) to induce their differentiation into macrophages and neutrophils.Confocal microscopy was used to detect the changes in total ROS (reactive oxygen species) and NO (nitric oxide) levels as well as free Ca2+ concentration ([Ca2+]i) in THP-1 monocyte-derived macrophages and HL-60 cell-derived neutrophils after infection with Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai.Fluorospectrophotometry was applied to analyze the differences in killing ability between THP-1 monocyte-derived macrophages and HL-60 cell-derived neutrophils against intracellular leptospires before and after treatment with total ROS and NO inhibitors and intracellular free Ca2+ chelator.Results The total ROS and NO levels and [Ca2+]i in THP-1 monocyte-derived macrophages and HL-60 cell-derived neutrophils were significantly increased after infection with the spirochete (P<0.05).Moreover,the total ROS and NO levels and [Ca2+]i in the former were significantly higher than those in the latter (P<0.05).The THP-1 monocyte-derived macrophages had stronger killing ability against intracellular leptospires than the HL-60 cell-derived neutrophils (P<0.05).Inhibiting total intracellular ROS,NO or free Ca2+ could result in decreased killing ability of THP-1 monocyte-derived macrophages against intracellular leptospires,but not affect the killing ability of HL-60 cell-derived neutrophils.Conclusion Mononuclear macrophages rather than neutrophils act as the main phagocytes eliminating Leptospira interrogans.High levels of total intracellular ROS,NO and free Ca2+ are closely associated with the ability of mononuclear macrophages to kill Leptospira interrogans.