6种沙门菌单克隆抗体的制备和效能评价

目的：制备稳定、特异、高亲和性的分别针对甲型副伤寒沙门菌、乙型副伤寒沙门菌、丙型副伤寒沙门菌、肠炎沙门菌、伤寒沙门菌和猪霍乱沙门菌的单克隆抗体。方法：用甲醛灭活的菌液抗原免疫BALB／c小鼠,取脾细胞与SP2／0骨髓瘤细胞融合;用灭活的菌液包被酶标板,ELISA筛选阳性克隆株,建立细胞系;选取高效分泌杂交瘤细胞,常规制备腹水并纯化,进行单抗特异性与亲和性评价。结果：筛选得到分泌6种沙门菌相应单克隆抗体的杂交瘤细胞株,获得高亲和性单抗;所有单抗与大部分病原菌（包括7种沙门菌、3株志贺菌、2株李斯特菌、4株致病性大肠杆菌、2株霍乱弧菌）无交叉反应,但由于同类型O抗原的广泛分布,抗乙型副伤寒沙门菌单抗与鼠伤寒沙门菌、抗伤寒沙门菌单抗与肠炎沙门菌有明显的交叉反应。结论：沙门菌单抗的制备,为感染性腹泻的监测、诊断奠定了基础。     

重组M2e-鞭毛蛋白流感疫苗在健康成人中的安全与免疫原性

<正>背景:基质蛋白2的胞外域(M2e)是一种有希望的具有广谱保护作用的A型流感疫苗候选制剂,因为它是高度保守的,而且抗M2e抗体在动物模型中具有保护作用。STF2.4x M2e(VAX102)是一种重组融合蛋白,即M2e抗原的4个串联拷贝与鼠伤寒沙门菌的鞭毛蛋白相连接而构成的重组融合蛋     

鼠伤寒沙门菌pStSR100质粒对生物膜形成的影响

目的:鼠伤寒沙门菌在多种表面形成的生物膜对其致病性和引起食物中毒等方面起着重要作用,本研究探讨鼠伤寒沙门菌pStSR100质粒对细菌在不同材质表面生物膜形成的影响。方法:用LB(Luria-Bertani,LB)培养基和TSB(Tryptose Soya Broth,TSB)培养基分别将携带pStSR100质粒的野生株在96孔板与放置无菌小圆玻片的24孔板中静态培养48 h,用结晶紫半定量法确定生物膜形成的适宜培养基。将野生株与消除质粒的突变株,用结晶紫半定量法和激光共聚焦显微镜(Confocal Laser scanning microscopy,CLSM)观察其在聚苯乙烯培养板和小圆玻片表面形成生物膜的差异。结果:用LB培养时细菌生物膜的形成能力高于用TSB培养,LB培养基更适宜生物膜形成;结晶紫半定量法结果表明野生株比突变株在小圆玻片表面形成生物膜的能力明显增强,而在聚苯乙烯培养板表面两者则无明显差异;CLSM观察发现,野生株在小圆玻片表面形成融合成片的大克隆,突变株仅形成较小克隆。结论:鼠伤寒沙门菌pStSR100质粒能促进该菌在亲水性材质表面生物膜的形成,但其对该菌在疏水性材质表面生物膜的形成未见明显影响,这一新发现为进一步研究鼠伤寒沙门菌生物膜形成的调控机制,研制抗感染材料提供了理论和实验依据。     

检测猪丹毒杆菌抗体重组SpaA ELISA的建立

【目的】利用表达纯化的猪丹毒杆菌表面保护性蛋白SpaA,建立检测猪丹毒杆菌抗体的间接ELISA方法。【方法】克隆扩增猪丹毒杆菌SpaA基因,并将SpaA基因与原核表达载体p GEX-6P-1连接,通过PCR、双酶切及测序鉴定后,将阳性重组质粒转化入受体菌E.coli Rosetta(DE3),并利用IPTG进行诱导表达,SDS-PAGE和Western blot鉴定表达产物。将SpaA重组蛋白按不同浓度包被酶标板,通过方阵滴定法确定最佳抗原包被浓度及血清稀释度,并对其他条件进行优化,最终建立检测猪丹毒杆菌抗体的间接ELISA方法。【结果】利用克隆表达的猪丹毒杆菌SpaA蛋白作抗原,通过方阵滴定法确定蛋白最佳包被浓度为1.0 mg/L,血清的最佳稀释度为1:100,建立了检测猪丹毒杆菌抗体的间接ELISA方法,批内及批间变异系数均小于10%,具有较好的重复性及特异性。用建立的间接ELISA方法检测猪丹毒疫苗免疫后的健康猪血清样品,检测结果与美国TSZ公司猪丹毒杆菌抗体检测试剂盒和Western blot鉴定结果进行对比,两者总符合率分别为92.20%、92.59%。【结论】试验利用原核表达的SpaA重组蛋白作抗原建立的检测猪丹毒杆菌抗体的间接ELISA方法,特异性强、重复性好、敏感性高,可用于猪丹毒杆菌的抗体检测及流行病学调查。     

布鲁菌核糖体蛋白L7/L12的表达纯化及生物活性鉴定

目的:原核表达系统表达布鲁菌核糖体蛋白L7/L12与GST的融合蛋白GST-L7/L12,并纯化蛋白L7/L12,建立检测特异性抗体的间接ELISA方法。方法:对含有L7/L12的原核表达载体pGEX-4T-1-L7/L12进行了原核表达。利用亲和层析柱分别纯化融合蛋白GST-L7/L12和蛋白L7/L12,并用SDS-PAGE及Western印迹分析鉴定。以L7/L12为抗原包被微量板,优化抗原包被浓度和羊抗鼠IgG-HRP稀释度,建立间接ELISA方法,并检测其特异性。结果:SDS-PAGE结果显示在相对分子质量为38000和12000处可见纯化蛋白的条带,Western印迹分析表明这2条带均能被免疫兔血清识别,表明获得了纯化的有生物活性的融合蛋白GST-L7/L12和蛋白L7/L12。间接ELISA方法的L7/L12抗原包被浓度为5μg/mL,羊抗鼠酶标二抗稀释度为1∶1000。小鼠免疫血清与L7/L12抗原出现阳性反应,而与布鲁菌融合蛋白OMP31、结核分枝杆菌抗原85b及牛血清白蛋白则呈阴性。结论:成功地对布鲁菌核糖体蛋白L7/L12进行了原核表达和纯化,以其为基础建立的间接ELISA方法稳定且特异。     

流行性乙型脑炎病毒E蛋白主要抗原域的原核表达与间接ELISA检测方法的初步建立

重组质粒pET-E转化宿主菌BL21,经1.0mmol/LIPTG诱导,外源基因以包含体的形式获得高效表达。通过Westernblotting检测证明表达产物具有良好的抗原性。以纯化后表达产物作为诊断抗原包被酶标板建立了检测JEV抗体的间接ELISA方法。结果表明,抗原的最佳稀释度为1:2000,血清的最佳稀释度为1:200,待检血清阳性标准初步定为:OD490nm>1.2,且待检血清与阴性血清的OD490nm比值大于2。     

伤寒沙门菌与甲、乙、丙副伤寒沙门菌质控血清的制备

应用免疫学原理,将伤寒沙门菌O901、H901和甲、乙、丙型副伤寒沙门菌分别制成全菌体抗原,免疫实验兔获取免疫血清。依据伤寒沙门菌和副伤寒沙门菌的抗原成分的异同性,选择适当的吸收菌除去免疫血清中的交叉反应抗体和类属凝集素,而保留其特异性的抗体。通过对诊断菌液的验证试验,证实吸收充分的免疫血清具有质控血清的特性。具备可靠性能的质控血清,适用于伤寒沙门菌与副伤寒沙门菌的菌种检定及其效价检测;亦有利于肥达氏诊断菌液的质量控制。     

鼠伤寒沙门菌pStSR100质粒对生物膜形成的影响

目的：鼠伤寒沙门菌在多种表面形成的生物膜对其致病性和引起食物中毒等方面起着重要作用,本研究探讨鼠伤寒沙门菌pStSR100质粒对细菌在不同材质表面生物膜形成的影响。方法：用LB（Lufia—Bertani,LB）培养基和TSB（TryptoseSoyaBroth,TSB）培养基分别将携带pStSR100质粒的野生株在96孔板与放置无菌小圆玻片的24孔板中静态培养48h,用结晶紫半定量法确定生物膜形成的适宜培养基。将野生株与消除质粒的突变株,用结晶紫半定量法和激光共聚焦显微镜（ConfocalLaserscanningmicroscopy,CLSM）观察其在聚苯乙烯培养板和小圆玻片表面形成生物膜的差异。结果：用LB培养时细菌生物膜的形成能力高于用TSB培养,LB培养基更适宜生物膜形成;结晶紫半定量法结果表明野生株比突变株在小圆玻片表面形成生物膜的能力明显增强,而在聚苯乙烯培养板表面两者则无明显差异;CLSM观察发现,野生株在小圆玻片表面形成融合成片的大克隆,突变株仅形成较小克隆。结论：鼠伤寒沙门菌pStSR100质粒能促进该茵在亲水性材质表面生物膜的形成,但其对该菌在疏水性材质表面生物膜的形成未见明显影响,这一新发现为进一步研究鼠伤寒沙门菌生物膜形成的调控机制,研制抗感染材料提供了理论和实验依据。     

流行性乙型脑炎病毒E蛋白主要抗原域的原核表达与间接ELISA检测方法的初步建立

重组质粒pET-E转化宿主菌BL21,经1.0mmol/LIPTG诱导,外源基因以包含体的形式获得高效表达.通过Western blotting检测证明表达产物具有良好的抗原性.以纯化后表达产物作为诊断抗原包被酶标板建立了检测JEV抗体的间接ELISA方法.结果表明,抗原的最佳稀释度为12000,血清的最佳稀释度为1200,待检血清阳性标准初步定为OD490nm＞1.2,且待检血清与阴性血清的OD490m比值大于2.     

中和性流行性感冒病毒基因工程抗体在昆虫细胞中的全基因表达、纯化及鉴定

将中和性流行性感冒 (流感 )病毒基因工程抗体IV 2、IV 6的轻链和重链Fd段基因 ,分别克隆入全抗体表达载体 pAC L Fc ,构建成杆状病毒表达载体pAC L Fc Ⅳ 2和 pAC L Fc Ⅳ 6 ,转染昆虫Sf9细胞 ,利用杆状病毒 /昆虫细胞系统实现抗体的分泌型表达 ,表达产物进行亲和层析分离纯化。SDS PAGE电泳和Westernblot法证实有完整免疫球蛋白的表达 ,免疫印迹法证实它们能与流感病毒血凝素蛋白特异性结合。经间接竞争性抑制ELISA法测定 ,抗体与流感病毒抗原结合的解离常数KD 值分别为 2 5× 10 -9M和 3 0× 10 -9M。流感病毒基因工程全抗体经在昆虫细胞中的表达、纯化和抗体特性鉴定 ,获得了两株纯化的全抗体 ,可用于以后的动物模型呼吸道粘膜被动免疫抗感染的研究。     

Cloning and characterization of the gene encoding the z66 antigen of Salmonella enterica serovar Typhi

Z66 antigen-positive strains of Salmonella enterica serovar Typhi change flagellin expression in only one direction from the z66 antigen to the d or j antigen, which is different from the phase variation of S. enterica serovar Typhimurium. In the present study, we identified a new flagellin gene in z66 antigen-positive strains of S. enterica serovar Typhi. The genomic structure of the region containing this new flagellin gene was similar to that of fljBA operon of biphasic S. enterica serovars. A fljA-like gene was present downstream of the new flagellin gene. A rho-independent terminator was located between the new flagellin gene and the fljA-like gene. Hin-like gene was not present upstream of the new flagellin gene. We generated a mutant strain of S. enterica serovar Typhi, which carries a deletion of the new flagellin gene. Western blotting revealed that the 51-kDa z66 antigen protein was absent from the population of proteins secreted by the mutant strain. Southern hybridization demonstrated that the z66 antigen-positive strains of S. enterica serovar Typhi carried the new flagellin gene and fliC on two different genomic EcoRI fragments. When z66 antigen-positive strains were incubated with anti-z66 antiserum, the flagellin expression by S. enterica serovar Typhi changed from z66 antigen to j antigen. The new flagellin gene and the fljA-like gene were absent in the strain with altered flagellin expression. These results suggested that the new flagellin gene is a fljB-like gene, which encodes the z66 antigen of S. enterica serovar Typhi, and that deletion of fljBA-like operon may explain why S. enterica serovar Typhi alters the flagellin expression in only one direction from the z66 antigen to the d or j antigen.     

A gold nanoparticles based immuno-bioprobe for detection of Vi capsular polysaccharide of Salmonella enterica serovar Typhi

A rapid and sensitive gold-nanobioprobe based immunoassay format has been presented for the detection of capsular Vi polysaccharide of Salmonella enterica serovar Typhi (surface antigen) using anti-Vi antibodies. The Vi antigen was extracted from serovar Typhi cells, under the optimised growth conditions for its over-expression. Anti-Vi antibodies were produced and conjugated with gold nanoparticles (GNPs) of definite size (~30 nm), which served as the nano-bioprobe in the detection system. A sandwich immunoassay was developed using nitrocellulose dot blot comb (8/12 wells) membranes immobilized with anti-Salmonella antibodies at the optimal concentration (43 ng spot(-1)). The Vi antigen in the clinical isolates, spiked samples and also in the standard strain (serovar Typhi Ty2) was detected by measuring the colour intensity of GNPs and correlating it with the concentration of serovar Typhi in samples. Using this developed immunoassay technique Vi positive serovar Typhi strains could be detected with a sensitivity of up to 10(2) cells mL(-1) in the clinical isolates as well as in the spiked samples. The developed immunoassay technique could be useful for the detection of typhoid fever and may be important from an epidemiological point of view.     

Transcriptional expression of fljB:z66, a flagellin gene located on a novel linear plasmid of Salmonella enterica serovar Typhi under environmental stresses

A previous study identified that z66+ strain of Salmonella enterica serovar Typhi contains two different flagellin genes, the fliC encoding d or j antigen in chromosome and the fljB-like gene encoding z66 antigen in a novel linear plasmid, respectively. The promoter of fljB:z66 is different from that of fliC:d/j and z66+ strain alters flagellin expression in only one orientation, from z66 to d orj antigen, raising the suspicion that z66+ strain is a special biphasic strain. To clarify the expressional characteristics of flagellin genes of z66+ strain, expression patterns of fljB:z66 and fliC were investigated by RT-PCR under a series of environmental stresses during infection, such as acidic stress, osmotic stress, bile acid stress and oxidative stress. Results showed that the expression level of fljB:z66 is over 10-fold higher than the level of fliC in low and middle osmotic conditions before stresses. Only the expressional regulatory tendency of fljB:z66 in response to bile acid stress is similar to that of fliC. Differential expressional patterns between fljB:z66 and fliC of S. enterica serovar Typhi were seen under osmotic stress, bile acid stress and oxidative stress. These results support the hypothesis that the z66+ strain is a special biphasic strain of S. enterica serovar Typhi.     

Precise excision of the large pathogenicity island, SPI7, in Salmonella enterica serovar Typhi

The large pathogenicity island (SPI7) of Salmonella enterica serovar Typhi is a 133,477-bp segment of DNA flanked by two 52-bp direct repeats overlapping the pheU (phenylalanyl-tRNA) gene, contains 151 potential open reading frames, and includes the viaB operon involved in the synthesis of Vi antigen. Some clinical isolates of S. enterica serovar Typhi are missing the entire SPI7, due to its precise excision; these strains have lost the ability to produce Vi antigen, are resistant to phage Vi-II, and invade a human epithelial cell line more rapidly. Excision of SPI7 occurs spontaneously in a clinical isolate of S. enterica serovar Typhi when it is grown in the laboratory, leaves an intact copy of the pheU gene at its novel join point, and results in the same three phenotypic consequences. SPI7 is an unstable genetic element, probably an intermediate in the pathway of lateral transfer of such pathogenicity islands among enteric gram-negative bacteria.     

Screening method for Salmonella enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones by PCR-restriction fragment length polymorphism

Salmonella enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones (MICs of ciprofloxacin, 0.25 to 2 microg/ml) have a mutation at codon either Ser-83 or Asp-87 of gyrA gene. A screening method by PCR-restriction fragment length polymorphism (PCR-RFLP) was designed to screen the mutations at codon Ser-83 and Asp-87 of the gyrA gene of S. enterica serovar Typhi and serovar Paratyphi A clinical isolates. This method successfully screened the gyrA mutations of S. enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones.     

Diversity of genome structure in Salmonella enterica serovar Typhi populations

The genomes of most strains of Salmonella and Escherichia coli are highly conserved. In contrast, all 136 wild-type strains of Salmonella enterica serovar Typhi analyzed by partial digestion with I-CeuI (an endonuclease which cuts within the rrn operons) and pulsed-field gel electrophoresis and by PCR have rearrangements due to homologous recombination between the rrn operons leading to inversions and translocations. Recombination between rrn operons in culture is known to be equally frequent in S. enterica serovar Typhi and S. enterica serovar Typhimurium; thus, the recombinants in S. enterica serovar Typhi, but not those in S. enterica serovar Typhimurium, are able to survive in nature. However, even in S. enterica serovar Typhi the need for genome balance and the need for gene dosage impose limits on rearrangements. Of 100 strains of genome types 1 to 6, 72 were only 25.5 kb off genome balance (the relative lengths of the replichores during bidirectional replication from oriC to the termination of replication [Ter]), while 28 strains were less balanced (41 kb off balance), indicating that the survival of the best-balanced strains was greater. In addition, the need for appropriate gene dosage apparently selected against rearrangements which moved genes from their accustomed distance from oriC. Although rearrangements involving the seven rrn operons are very common in S. enterica serovar Typhi, other duplicated regions, such as the 25 IS200 elements, are very rarely involved in rearrangements. Large deletions and insertions in the genome are uncommon, except for deletions of Salmonella pathogenicity island 7 (usually 134 kb) from fragment I-CeuI-G and 40-kb insertions, possibly a prophage, in fragment I-CeuI-E. The phage types were determined, and the origins of the phage types appeared to be independent of the origins of the genome types.     

Inversions over the terminus region in Salmonella and Escherichia coli: IS200s as the sites of homologous recombination inverting the chromosome of Salmonella enterica serovar typhi

Genomic rearrangements (duplications and inversions) in enteric bacteria such as Salmonella enterica serovar Typhimurium LT2 and Escherichia coli K12 are frequent (10(-3) to 10(-5)) in culture, but in wild-type strains these genomic rearrangements seldom survive. However, inversions commonly survive in the terminus of replication (TER) region, where bidirectional DNA replication terminates; nucleotide sequences from S. enterica serovar Typhimurium LT2, S. enterica serovar Typhi CT18, E. coli K12, and E. coli O157:H7 revealed genomic inversions spanning the TER region. Assuming that S. enterica serovar Typhimurium LT2 represents the ancestral genome structure, we found an inversion of 556 kb in serovar Typhi CT18 between two of the 25 IS200 elements and an inversion of about 700 kb in E. coli K12 and E. coli O157:H7. In addition, there is another inversion of 500 kb in E. coli O157:H7 compared with E. coli K12. PCR analysis confirmed that all S. enterica serovar Typhi strains tested, but not strains of other Salmonella serovars, have an inversion at the exact site of the IS200 insertions. We conclude that inversions of the TER region survive because they do not significantly change replication balance or because they are part of the compensating mechanisms to regain chromosome balance after it is disrupted by insertions, deletions, or other inversions.     

Virulence-defective strains of Salmonella enterica serovar Typhi as candidates for education at level 2 facilities

The use of biosafety level 3 pathogens is an essential element of education and training at medical schools. We previously reported on invasion-defective strains of Salmonella enterica serovar Typhi, GTC 3P408 (DeltainvA, DeltasipB) and GTC 3P409 (DeltainvA, DeltasipB, and DeltaviaB), as candidates for use in educational programs. Vi negative strains of S. enterica serovar Typhi became extremely sensitive to complement attack but showed increased invasiveness. Therefore, this study was conducted to construct two virulencedefective strains, GTC 3P460 (DeltainvA, DeltasipB, and DeltarpoS) and GTC 3P461 (DeltainvA, DeltasipB, DeltaviaB, and DeltarpoS), of S. enterica serovar Typhi by deleting rpoS from the GTC 3P409 and GTC 3P408 strains. Stress tests demonstrated that GTC 3P460 and GTC 3P461 are sensitive to conditions of starvation, acid stress and oxidative stress. These results suggest that these virulence-defective strains have difficulty surviving in the gastric environment and in macrophages, characteristics that make them ideal candidates for education at level 2 facilities. Colony morphology and conventional biochemical features of these strains are identical to the parent strain S. enterica serovar Typhi GIFU 10007.     

Composition,acquisition, and distribution of the Vi exopolysaccharide-encoding Salmonella enterica pathogenicity island SPI-7

Vi capsular polysaccharide production is encoded by the viaB locus, which has a limited distribution in Salmonella enterica serovars. In S. enterica serovar Typhi, viaB is encoded on a 134-kb pathogenicity island known as SPI-7 that is located between partially duplicated tRNA(pheU) sites. Functional and bioinformatic analysis suggests that SPI-7 has a mosaic structure and may have evolved as a consequence of several independent insertion events. Analysis of viaB-associated DNA in Vi-positive S. enterica serovar Paratyphi C and S. enterica serovar Dublin isolates revealed the presence of similar SPI-7 islands. In S. enterica serovars Paratyphi C and Dublin, the SopE bacteriophage and a 15-kb fragment adjacent to the intact tRNA(pheU) site were absent. In S. enterica serovar Paratyphi C only, a region encoding a type IV pilus involved in the adherence of S. enterica serovar Typhi to host cells was missing. The remainder of the SPI-7 islands investigated exhibited over 99% DNA sequence identity in the three serovars. Of 30 other Salmonella serovars examined, 24 contained no insertions at the equivalent tRNA(pheU) site, 2 had a 3.7-kb insertion, and 4 showed sequence variation at the tRNA(pheU)-phoN junction, which was not analyzed further. Sequence analysis of the SPI-7 region from S. enterica serovar Typhi strain CT18 revealed significant synteny with clusters of genes from a variety of saprophytic bacteria and phytobacteria, including Pseudomonas aeruginosa and Xanthomonas axonopodis pv. citri. This analysis suggested that SPI-7 may be a mobile element, such as a conjugative transposon or an integrated plasmid remnant.