The Bacillus anthracis S-layer protein, BslA, plays a crucial role in mammalian infection. BslA is required to mediate adherence between host cells and vegetative forms of bacteria and this interaction promotes target organs adherence and blood–brain barrier (BBB) penetration in vivo. This study attempts to identify the potential eukaryotic ligand(s) for B. anthracis BslA protein.

To investigate the surface display of the anthrax protective antigen (PA) on attenuated Bacillus anthracis, a recombinant B. anthracis strain, named AP429 was constructed by integrating into the chromosome a translational fusion harboring the DNA fragments encoding the cell wall-targeting domain of the S-layer protein EA1 and the anthrax PA. Crerecombinase action at the loxP sites excised the antibiotic marker. Western blot analysis, fluorescence-activated cell sorting and immunofluorescence analysis confirmed that PA was successfully expressed on the S-layer of the recombinant antibiotic marker-free strain. Notwithstanding extensive proteolytic degradation of the hybrid protein SLHs-PA, quantitative ELISA revealed that approximately 8.1 × 10(6) molecules of SLHs-PA were gained from each Bacillus cell. Moreover, electron microscopy assay indicated that the typical S-layer structures could be clearly observed from the recombinant strain micrographs.

AIM:To investigate the association between the tag single nucleotide polymorphisms (TagSNPs) of NOD1 and NOD2 and the risk of developing gastric cancer.METHODS:We conducted a hospital-based case-control study including 296 incident gastric cancer patients and 160 gastritis controls. Eight TagSNPs in the NOD1 and NOD2 genes were selected from the Hapmap database using the haploview software and genotyped by the Sequenom MassArray system. The serum levels of anti-Helicobacter pylori (H. pylori) IgG were measured by enzyme-linked immunosorbent assay to indicate H. pylori infection. The odds ratios (OR) and 95% confidence intervals (CI) were calculated by unconditional logistic regression, including sex and age as confounding factors.RESULTS:The NOD1 rs2907749 GG genotype showed a decreased risk for gastric cancer (OR 0.50, 95% CI: 0.26-0.95, P = 0.04) while the rs7789045 TT genotype showed an increased risk (OR 2.14, 95% CI: 1.20-3.82, P = 0.01). An elevated susceptibility to gastric cancer was observed in the subjects with H. pylori infection and the NaOD1 rs7789045 TT genotype (OR 2.05, 95% CI: 1.07-3.94, P = 0.03) or the NOD2 rs7205423 GC genotype (OR 2.52, 95% CI: 1.05-6.04, P = 0.04). Haplotype analysis suggested that the distribution of AGT (rs2907749, rs2075820 and rs7789045) in NOD1 between the cases and control groups was significantly different (P corrected: 0.04), and the diplotype AGT/AGT was associated with an elevated gastric cancer risk (OR 1.98, 95% CI: 1.04-3.79, P = 0.04). The association of the NOD1 rs7789045 TT genotype and the diplotype AGT/AGT was significant with H. pylori-related diffuse-type gastric cancer (OR 3.00, 95% CI: 1.38-6.53, P = 0.01; OR 4.02, 95% CI: 1.61-10.05, P < 0.01, respectively).CONCLUSION:Genetic polymorphisms in NOD1 and NOD2 may interact with H. pylori infection and may play important roles in promoting the development of gastric cancer in the Chinese population.

Objective: To study the immune response of BLAB/c mice to vaccination of recombinant bacterial ghosts cooperated with protein vaccine.Methods: The bacterial ghost displaying UreB epitope as well as recombi nant LTBKAT were constructed,and then they were mixed or separately orally administrated by BLAB/c mice.Re sults: The anti-UreB antibody titer of mice immunized with mixed vaccine was 1∶(681±76),which was significant ly higher than that immunized with single bacterial ghosts[1∶(239±23)].The anti-UreB antibody type of both groups was mainly IgG1.The anti-KatA antibody titer of mice immunized with mixed vaccine was 1∶(1800±400),which was lower than that immunized with single protein vaccine[1∶(2800±275)].The anti-KatA antibody type of mixed vaccination was IgG2a,which was very different from that of protein vaccination.The anti-UreB and an ti-KatA IgA antibody level of mixed vaccination were both higher than those of single immunization.Conclusion: Immunogenicity level of BLAB/c mice was improved,even immunogenicity type was changed.

Whether the immunogenicity of DNA vaccine can be further increased by novel deliver system-bacterial ghost is discussed. First, the bacterial ghost displaying ureB epitope by CS3 fimbriae was constructed, namely, plasmids pCSXureB harboring ureB epitope and plasmids pAcYclysis harboring lysis cassette were transformed into E. coli together. The recombinant bacterial ghosts were then enfolded the Helicobacter DNA vaccine pcDNAKAT by incubating at 24 degrees C for 30 min. It was confirmed by DNA agarose electrophoresis, fluorescent microscope that plasmids pcDNAKAT could bound to bacterial ghost unspecificly, successful loading also resulted in the fluorescence shift indicated by the MFI of flow cytometric analysis. The anti-katA antibody titer of mice immunized with packaged DNA vaccine was 1 : (520 +/- 54), which was significantly higher than that of control groups (P = 0.0058), which was indicated strongly that bacterial ghosts could deliver DNA vaccine effectively.

Extracellular antigen 1 (EA1), a major component of the Bacillus anthracis surface layer (S-layer), was used as a fusion partner for the expression of heterologous antigen. A recombinant B. anthracis strain was constructed by integrating a translational fusion harboring the DNA fragments encoding the cell wall–targeting domain of the S-layer protein EA1 and the 20-kDa N-terminal fragment of anthrax protective antigen (PA20) into the chromosome. A thermosensitive plasmid expressing Cre recombinase was introduced at a permissive temperature to remove the antibiotic marker. Cre recombinase action at the loxP sites excised the spectinomycin resistance cassette. The final derivative strains were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Western blot analysis, and immunofluorescence analysis. PA20 was successfully expressed on the S-layer of the recombinant antibiotic marker-free strain. Guinea pigs were immunized with the attenuated recombinant B. anthracis strain, and the bacilli elicited a humoral response to PA20. This antibiotic marker-free strain and the correlative experiment method may have potential applications for the generation of a live attenuated anthrax vaccine.

目的:应用生物信息学分析方法筛选幽门螺杆菌新的疫苗候选抗原。方法:从TIGRCMR下载幽门螺杆菌26695和J99株全基因组序列,应用生物信息学SignalP、PredTMBB、LipoP、TMHMM、Phobius、PSORT-B和SubLoc等分析软件,筛选幽门螺杆菌新的外膜蛋白和分泌蛋白疫苗候选抗原。结果:从幽门螺杆菌26695株筛选得到54个编码β-桶型跨膜蛋白、脂蛋白或分泌表达蛋白的疫苗候选蛋白抗原,从幽门螺杆菌J99株得到61个呈现上述表达方式的疫苗候选蛋白抗原;且这2株细菌的疫苗候选蛋白呈现良好的交集状况,即有43个候选疫苗蛋白是相同的。结论:用生物信息学分析方法可以从全基因组范围内快速筛选到保守的分泌或表面暴露的疫苗候选抗原,为疫苗抗原的快速筛选与鉴定奠定了基础。

Thrombin-like enzymes (TLEs) are studied widely because of their therapeutic potential in myocardial infarction and thrombotic diseases. We synthesized the DNA fragment encoding thrombin-like enzyme calobin from Agkistrodon caliginosus (Korean Viper) venom by fusion PCR and expressed it in Pichia pastoris. After induction by 0.5% methanol for 48 h, the expression level of recombinant calobin reached 3.5 g/L in medium. The recombinant calobin was purified by Q-Sepharose Fast Flow ion-exchange chromatography and Sephacryl-S-100 gel filtration chromatography. Purified sample had a molecular weight of 32 kD shown in SDS-PAGE. It hydrolyzed fibrinogen and formed a light white hydrolysis circle in fibrinogen plate. SDS-PAGE analysis showed that recombinant calobin cleaved Aalpha-chain of fibrinogen specifically, and produced an appropriately 40 kD new band. However, we failed to find its fibrin-clot formation activity.

Cre-LoxP homologous recombinant system was used to disrupt eag gene in B. anthracis AP422. To construct the recombinant vector, homologous regions and Spc(r) cassette with two loxP sites were amplified from the corresponding templates. The produced shuttle vector was then transformed into B. anthracis AP422. Under the pressure of temperature and antibiotic, homologous recombination occurred between the vector and genome of the host bacterium and the recombinants were selected by agar media with spectinomycin (Spc) and X-gal. To remove the SpC(r) cassette, a plasmid with Cre recombinase was introduced into the recombinant to delete the relevant DNA fragment, resulting in a single loxP site within the targeted genomic segment. The markerless mutant strains were detected by genome PCR, RT-PCR, total proteins SDS-PAGE analysis and Western blot. The results showed that the eag gene was successfully deleted.

Objective: To clone and evaluate the virulence and protection of suilysin(SLY), for developing a vaccine against the infective Streptococcus suis type 2(SS2). Methods: Sly gene was amplified from a SS2 strain isolated in Ziyang, Sichuan, and ligased into T-vector, then transferred into a refined pGEX-6p1 without LacIq gene. Protein SDS-PAGE electrophoresis and hemolysis test was used to check the expression and activity of SLY. Results: The SLY expression of recombinant strain was lower by degree for growth at 5 h, 10 h and 15 h, and transparent ring around recombinant strain were observed in electrophoresis and hemolysis test respectively. Conclusion: Secreted SLY was expressed in early stage and exhibited a distinctive β-hemolysis activity.

Bacterial spores are robust and dormant life forms with formidable resistance properties. Spores of the genus Bacillus have been used for a long time as probiotics for oral bacteriotherapy both in humans and animals. Recently, genetically modified B. subtilis spores and B. anthracis spores have been used as indestructible delivery vehicles for vaccine antigens. They were used as vaccine vehicles or spore vaccine for oral immunization against tetanus and anthrax, and the results were very exciting. Unlike many second generation vaccine systems currently under development, bacterial spores offer heat stability and the flexibility for genetic manipulation. At the same time, they can elicit mucosal immune response by oral and nasal administration. This review focuses on the use of recombinant spores as vaccine delivery vehicles.

The muramidase-released protein gene of Streptococcus suis serotypes 2 was isolated by PCR with the Streptococcus suis serotypes 2 strain from Sichuan in China,and ligated to the pMD18-T vector.The antibiotic resistance gene ampr of pMD18-T vector was destroyed by insertion of asd gene.Then the recombinant vector was transformed in Salmonella X4072 (asd-).The results of PCR,restriction enzyme digestion and SDS-PAGE indicated MRP was expressed by Salmonella X4072.

The biotechnology is a multidisciplinary specialty with bioscience,industry,and society.How to construct and develop is still controversial at present.Focusing on the discipline characteristics of the modern biotechnology and existent problems in current education of this specialty,we proposed training methodologies by emphasizing the characteristics,the cooperation of school and business,five years education mode,and continuous education mode from undergraduate to graduate.

The expression of a soluble protein on cell surface is often desirable for study of a functional protein, wide application of a protein or investigation of protein-protein interaction. The expression of a soluble protein on the surface of a cell is often achieved by genetically linking a protein to the extra-cellular fragment of a transmembrane partner. In this study, the myc epitope was linked with N terminal of transmembrane proteins either A2TM or deltaLNGFR amplified by overlapping PCR. The plasmids expressing fusion protein were transfected into 293FT cells and the expression of target proteins was evaluated by fluorescent microscope, flow cytometry and Western blotting. The results of flow cytometry revealed that both A2TM and deltaLNGFR were expressed on the cell surface, but A2TM could only be detected with high copy number. Western blotting showed that the expression level of deltaLNGFR was very high and protein was heavily glycosylated, by contrast the expression of A2TM was hardly detected. The results indicate that glycosylated deltaLNGFR is a good candidate partner for the expression of a soluble protein on the cell surface.

耳聋是人类最常见的遗传病之一.20世纪80年代以来,对遗传性聋的基因定位和基因克隆的研究发展飞速.

The solid CFA agar,liquid Luria Broth(LB),whole cell ELISA,and an oral Balb/c were used to estimate the CFA/I of Enterotoxigenic E.coli,the CS6,and an original immunity of vector bacillary dysentery LPS.The research results show that LB medium does not affect immune response of the ETEC antigens and shigellosis lipopolysaccharide.It,therefore,hints that LB is effective for production of vector vaccine against ETEC.

Keratinocyte growth factor-2 (KGF-2) is a member of the fibroblast growth factor family. The full-length human KGF-2 coding sequence, gained by synthesizing, was cloned into the pPICZαA vector in frame with the yeast α-factor secretion signal under the transcriptional control of the AOX promoter and integrated into Pichia pastoris strain GS115. In shake-flask culture induced with methanol, the rhKGF-2 content was about 17.5% of the total secreted proteins. Under the optimal conditions, stable production of rhKGF-2 around 1.0 g/l was achieved. The recombinant protein was purified by heparin affinity chromatography. A preliminary biochemical characterization of purified rhKGF-2 was performed both by Western blot analysis and biological activity analysis, and the result demonstrated that the recombinant KGF-2 was expressed successfully.

May-Hegglin anomaly is a rare inherited autosomal dominant platelet disorder,caused by a mutation of the MYH9 gene,which localized on chromosome 22q12.3-q13.2,and encoding non-muscle myosin heavy chain A.The most significant diagnosis is based on abnormally large platelets,leucocytic inclusions and thrombocytopaenia,which predisposes patients to bleeding disorders.The molecular basis for this genotype-phenotype relationship is not very clear.

Objective: To obtain the laminin α5 LG3 recombinant protein with 6×His tag by prokaryotic expression. Methods and Results: Total RNA of heart cell was prepared from a mature mouse and used in RT-PCR to amplify the laminin α5 LG3 cDNA fragment. The resulted fragment with the expected size was cloned into the vector of pET-28a to construct the recombinant plasmid pET-LG3. The plasmid pET-LG3 was subjected to restriction enzyme analysis and sequenced, the results showed that the laminin α5 LG3 gene sequence was 549 bp long and completely matched with the laminin α5 LG3 sequence in GenBank. The recombinant plasmid was transformed into E.coli BL21(DE3), and E.coli BL21(DE3)/pET-LG3 was induced by IPTG. The fusion protein was effective expressed and purificated. Conclusion: The target protein laminin α5 LG3 with 6×His tag was obtained.

AIM:To construct DNA vaccine targeted on antigen-presenting cells for the purpose of increasing the immunogenecity of Helicobacter pylori DNA vaccine. METHODS:DNA vaccine was constructed by combining the targeted DNA sequence with katA.Whether or not the DNA vaccine could be expressed in the mammalian cells was detected by indirect immunofluorescence assay of 293T cells transfected with DNA vaccine.The IgG, IgG1 and IgG2a antibody titers of BALB/c mice immunized with DNA vaccine were also deter- mined. RESULTS:It was indicated in the immuno- fluorescence assay of 293T cells transfected with DNA vaccine that the KatA protein could be expressed in these cells.Enzyme-linked im- munosorbent assay(ELISA)also showed that the transfected cells with pcDNAkathIgz had a higher affinity for IgG.The IgG antibody ti- ter of BALB/c mice immunized with targeted DNA vaccine was significantly higher than that of mice immunized with pcDNAkatA,and a shift form(Th2 response to Th1 response)was achieved in the mice immunized with DNA vac- cine. CONCLUSION:DNA vaccine targeted on anti- gen-presenting cells is constructed successfully, which can evoke a higher IgG antibody titer than non-targeted DNA vaccine.