赖型钩端螺旋体及澳洲型钩端螺旋体的ompL1和flaB2抗原基因序列分析

目的：构建棘型钩端螺旋体017及澳洲型钩端螺旋体607株外膜蛋白抗原基因ompL1和内鞭毛抗原基因flaB2的重组质粒，并分别对ompL1及flaB2基因进行序列分析。方法：通过聚合酶链反应扩增ompL1及flaB2，并将其分别克隆到pcDNA3.1/Myc-His(＋）载体T7启动子下游，构建抗原基因表达质粒，进行序列测定分析。结果：序列分析显示赖型钩体017株与澳洲型钩体607株的ompL1相同碱基949个（98．85％），碱基变异11个（1．15％）；flaB2的相同碱基823个（96．94％），碱基变异26个（3．06％），呈很高的保守性。结论：赖型钩体017株与澳洲型钩体607株的ompL1及flaB2分别具有高度同源性。     

赖型钩体毒力相关基因ompL17的重组表达以及体外不同温度下的表达研究

目的:观察ompL17基因表达产物的免疫原性以及该表达蛋白在动物模型中的分布情况.方法:提取钩端螺旋体017株基因组DNA,扩增出ompL17基因,与表达栽体pGEX-4T-1重组,诱导表达OMPLl7蛋白,分析该基因在017株中体外不同温度的表达情况.结果:PCR和酶切分析证实构建成功了表达栽体,SDS-PAGE分析证实诱导表达出OmpL17蛋白;体外不同温度下发现该基因不表达蛋白.结论:成功表达出ompL17基因的蛋白,体外不同温度未发现该蛋白的表达,为赖型钩体的分子机制的阐明奠定了基础.     

中国钩端螺旋体强毒株017膜蛋白基因的质粒载体构？…

目的 构建表达我国钩体强毒赖型０１７株外膜蛋白的重组质粒。方法 用ＰＣＲ方法扩增并回收０１７株钩体外膜蛋白基因片段ＯｍｐＬ１，将其与质粒ｐＢＫ－ＣＭＶ重组，通过菌落颜色和酶谱分析筛选出重组质粒。     

中国强毒力赖型钩端螺旋体外膜蛋白基因的克隆表达及对大肠杆菌活力的影响

目的:构建表达致病性赖型 017株钩端螺旋体OmpL1外膜蛋白的真核-原核穿梭表达载体,并在原核细胞中表达出目的蛋白。方法:用PCR方法从 017株钩体基因组中钓出OmpL1蛋白基因,酶切纯化后与质粒pBK-CMV连接,通过电泳、限制性内切酶分析及PCR鉴定筛选出正确重组质粒。重组质粒在大肠杆菌中诱导表达后提取总蛋白以SDS-PAGE检测表达情况,同时监测目的蛋白表达前后各时段宿主菌OD600值的变化。结果:筛选出 5株带重组质粒菌,其中有 4株能在大肠杆菌中表达 37kD的特异蛋白,而且随着该外源蛋白的表达,宿主菌生长各时段的OD600值下降。结论:成功地构建了钩体OmpL1蛋白的穿梭表达质粒,并在大肠杆菌中表达出OmpL1融合蛋白,该异源蛋白的表达导致宿主菌的活力降低。本工作为OmpL1蛋白用于钩体病诊断、疫苗研制和致病机制的研究奠定了基础.     

问号状赖型钩端螺旋体LAg42膜蛋白基因的克隆及真核表达研究

目的:构建赖型钩端螺旋体lag42基因真核表达载体并转染哺乳动物细胞,为进一步研究奠定基础。方法:分别以问号状赖型钩体017株,56601株及双曲钩体PatocI株基因组为模板PCR扩增目的基因。构建lag42基因与质粒pcDNA3.1A 的重组真核表达质粒,克隆筛选并测序;通过脂质体介导将重组质粒转染入COS7细胞,用RT-PCR检测转染结果。结果:不同毒力赖型钩体均能扩增出约1100 bp的片段,而PatocI株则未能扩增出目的片段;PCR、双酶切及测序证实pcDNA3.1A -lag42构建成功;经RT-PCR检测证实重组质粒转染成功。结论:赖型钩体具有编码LAg42膜蛋白的基因,构建完成真核表达载体pcD-NA3.1A -lag42,并成功转染COS7细胞。     

钩端螺旋体外膜抗原基因ompL1在卡介苗中的克隆和初步 …

目的 将钩端螺旋体外膜基因ｏｍｐＬ１克隆到大肠杆菌－卡介苗（Ｅ．ｃｏｌｉ－ＢＣＧ）穿梭质粒载体ｐＹ６００２中，将重组质粒导入ＢＣＧ并对重组ＢＣＧ的表达进行初步表达研究。方法 采用ＰＣＲ技术直接从致病性钩端螺旋体（钩体）赖型０１７株基因组中扩增钩体外膜蛋白基因ｏｍｐＬ１，并克隆到Ｅ．ｃｏｌｉ－ＢＣＧ穿梭质粒载体ｐＹ６００２中，重组质粒经电转化导入ＢＣＧ。斑点杂交筛选重组ＢＣＧ，再通过免疫印迹对其表达     

问号赖型钩端螺旋体内鞭毛与外膜基因融合DNA疫苗载体的构建

目的为增强问号赖型钩端螺旋体(017株钩体)内鞭毛抗原(flaB)与外膜抗原(ompL1)的免疫保护作用,克服保护性免疫持续时间短的难题,应用DNA重组技术以pcDNA3.1为载体构建-表达flaB与ompL1基因的融合DNA疫苗载体,以期注入肌体后能延长保护期,激发机体长期免疫.在pcDNA3.1载体上,ompL1与flaB前后相连,前者较后者先获得递呈,与钩体天然递呈过程相似,并且双嵌合基因可诱导机体产生针对2个不同抗原表位的抗体,从而增加免疫应答能力.pcDNA3.1载体能表达融合蛋白,并且具有CpGmotifs,因而可发挥佐剂作用.方法提取017株钩体基因组DNA,参照钩体特有的高度保守的flaB与ompL1序列,设计二对四条引物P1、P2、P3、P4.flaB与ompL1已被证实是两个良好的钩体疫苗候选抗原.通过聚合酶链反应(PCR),P1、P2引物扩增017株钩体的ompL1抗原基因;P3、P4引物扩增钩体的flaB抗原基因,分别经双酶切,以pcDNA3.1载体,将ompL1与flaB顺次同时定向嵌入同一pcDNA3.1中,获得重组质粒(ompL1与flaB),并将其转入JM109宿主菌中.在设计引物时,分别在P2、P3引物中,采用多个结构简单又不易形成折叠的甘氨酸作为接头,以维持其空间构象,不影响自然折叠,保持天然活性.结果经酶切鉴定证实有一1.8kb片段插入载体,进一步经酶切证实这一1.8kb可被酶切为9.6kb及8.5kb的片段,与预期的片段大小一致.以嵌合重组质粒DNA为模板,经PCR证实P1、P2引物扩增出9.6kb片段;P3,P4引物扩增出一8.5kb片段.重组质粒经DNA序列检测,其中ompL1与flaB序列与文献报道的完全一致.结论表达017钩体flaB与ompL1抗原的融合蛋白的DNA疫苗载体构建成功,从而为下一步表达复合功能蛋白,进行钩体免疫保护性研究打下了基础.     

钩端螺旋体外膜抗原基因ompL1在卡介苗中的克隆和初步表达

目的将钩端螺旋体外膜基因ｏｍｐＬ１克隆到大肠杆菌－卡介苗（Ｅ．ｃｏｌｉ－ＢＣＧ）穿梭质粒载体ｐＹ６００２中，将重组质粒导入ＢＣＧ并对重组ＢＣＧ的表达进行初步表达研究。方法采用ＰＣＲ技术直接从致病性钩端螺旋体（钩体）赖型０１７株基因组中扩增钩体外膜蛋白基因ｏｍｐＬ１，并克隆到Ｅ．ｃｏｌｉ－ＢＣＧ穿梭质粒载体ｐＹ６００２中，重组质粒经电转化导入ＢＣＧ。斑点杂交筛选重组ＢＣＧ，再通过免疫印迹对其表达进行初步研究。结果在所得６个重组ＢＣＧ中，有３个表达了ｏｍｐＬ１基因产物，其中一个表达较强。结论本研究为发展新一代高效广谱的钩体基因工程疫苗打下了基础。     

感染人单核细胞THP-1前后钩端螺旋体外膜蛋白抗原表达差异性

目的 了解感染人单核细胞THP-1前后钩端螺旋体(简称钩体)外膜蛋白表达变化,为选择钩体基因工程疫苗候选抗原提供依据.方法 采用Triton X-114法提取感染THP-1细胞前后问号钩体黄疸出血群赖型赖株外膜蛋白.采用双向电泳技术分离钩体外膜蛋白,银染色法检测感染前后钩体外膜蛋白表达量及其差异.感染细胞后4个表达显著上调和4个表达显著下调的钩体蛋白点胰酶水解后,采用LC-MS/MS方法进行鉴定.应用生物信息学软件分析靶蛋白跨膜区和信号肽,采用实时荧光定量RT-PCR检测感染细胞前后靶基因mRNA水平变化.构建靶基因原核表达系统,采用钩体感染豚鼠模型了解重组靶蛋白的免疫保护作用.结果 感染THP-1细胞60 min后,问号钩体赖株外膜蛋白中Loa22、GroEL、F0F1 ATP合成酶α和β亚单位表达水平均显著升高(P＜0.05),FluB2、LigB、OmpA和OmpA家族蛋白表达显著下降(P＜0.05),实时荧光定量RT-PCR检测结果与之基本一致.生物信息学分析结果显示,上述8个外膜蛋白中,OmpA和OmpA家族蛋白为跨膜蛋白,其余均无跨膜结构,Loa22、LigB和OmpA家族蛋白含有信号肽.200 μg重组表达的靶蛋白 rLoa22或rGroEL对豚鼠的免疫保护率均为75.0％.结论 问号钩体赖株感染细胞时外膜蛋白表达谱可发生明显变化.感染后高表达的钩体外膜蛋白尤其是GroEL和Loa22,可作为钩体基因工程疫苗侯选抗原.     

赖型钩端螺旋体外膜蛋白LipL32基因真核表达载体的构建及表达的初步研究

目的:构建赖型钩端螺旋体外膜蛋白LipL32基因真核表达载体并在COS-7细胞中表达,为钩端螺旋体DNA疫苗的研究和开发奠定基础.方法:从赖型钩端螺旋体017株全基因组中PCR扩增出目的基因,双酶切构建重组质粒pcDNA3.1-LipL32.脂质体转染法将重组质粒转染COS-7细胞,通过RT-PCR、Western blot检测目的基因的表达.结果:成功构建了LipL32基因的真核表达载体,并在COS-7细胞中获得瞬时和稳定表达.结论:赖型钩端螺旋体外膜蛋白LipL32基因真核表达载体能在哺乳动物细胞内表达,为钩端螺旋体DNA疫苗的应用提供了实验依据.     

OmpL1 DNA vaccine cross-protects against heterologous Leptospira spp. challenge

Available leptospirosis vaccines made up of inactivated bacteria or their membrane components elicit immunity which is serovar specific and unsatisfactory immunological memory. A vaccine that protects across Leptospira serogroups/serovars, i.e. broad spectrum, and induces long-lasting memory is needed for both human and veterinary uses. In this study, a plasmid DNA vaccine was constructed from cloning gene encoding a transmembrane porin protein, OmpL1, of pathogenic Leptospira interrogans, serogroup Icterohaemorrhagiae, serovar Copenhageni into a mammalian expression vector pcDNA3.1(+). The protective efficacy of the ompL1-pcDNA3.1(+) plasmid DNA vaccine was studied by immunizing hamsters intramuscularly with three doses of the vaccine (100 microg per dose) at two week intervals. The empty pcDNA3.1(+) and PBS were used as mock as negative vaccine controls, respectively. All animals were challenged with the heterologous Leptospira interrogans, serogroup Pomona, serovar Pomona (10 LD50), at one week after the last vaccine booster. The ompL1-pcDNA3.1(+) plasmid DNA vaccine rescued some vaccinated animals from the lethal challenge and delayed death time, reduced morbidity, e.g. fever, and/or the numbers of Leptospira in the tissues of the vaccinated animals. While the results are encouraging, further studies are needed to optimize the immunization schedule, vaccine dosage and formulation in order to maximize the efficacy of the vaccine.     

中国钩端螺旋体强毒株017膜蛋白基因的质粒载体构建及表达

目的构建表达我国钩体强毒赖型０１７株外膜蛋白的重组质粒。方法用ＰＣＲ方法扩增并回收０１７株钩体外膜蛋白基因片段ＯｍｐＬ１,将其与质粒ｐＢＫ－ＣＭＶ重组,通过菌落颜色和酶谱分析筛选出重组质粒。然后从该重组质粒切下ＯｍｐＬ１基因片段,重新克隆至质粒ｐＢＶ２２０中,酶谱分析和ＤＮＡ杂交鉴定出正向重组质粒。将含有正向重组质粒的宿主菌诱导表达生长后,提取全菌总蛋白进行ＳＤＳ－ＰＡＧＥ分析。结果筛选出４个与ｐＢＶ２２０正向重组的质粒,其中一株带重组质粒ｐＢＬＭ１的菌株表达了一相对分子质量为３３．５×１０３的新蛋白。结论重组质粒ｐＢＬＭ１的构建和表达成功说明扩增得的ＯｍｐＬ１基因具有完整的阅读框架,并使简便获得大量天然的０１７株钩体ＯｍｐＬ１蛋白成为可能,为新型钩体疫苗的研究奠定了基础     

Chemotaxis of leptospires to hemoglobin in relation to virulence.

A guinea pig-lethal line of Leptospira interrogans serovar copenhageni strain Shibaura, but not an avirulent line of the same strain, moved in larger numbers toward hemoglobin than toward distilled water (control) in a U-shaped polypropylene tube. L. interrogans serovar lai strains 017 and KH-1, which were also guinea pig lethal, showed a similar move to hemoglobin. No such move toward hemoglobin was shown by 14 avirulent strains of L. interrogans (with one exception) or any of the 8 strains of L. biflexa tested.     

赖型钩端螺旋体外膜脂蛋白LipL41基因真核表达质粒的构建及表达

目的：构建赖型钩端螺旋体LipL41基因的真核重组表达质粒、并对其表达进行检测。方法： 以赖型钩端螺旋体017株基因组DNA为模板PCR扩增目的基因，克隆至原核表达质粒pGEX-4T-1上。测序分析后酶切，并连接至真核表达质粒pcDNA3上，构建真核重组表达质粒LipL41-pCDNA3。利用脂质体介导转染COS7细胞，提取细胞总RNA，RT-PCR检测其表达。结果： PCR扩增出1 011 bp大小的目的片段，序列分析显示它与Leptospira kirschneri的LipL41基因成熟肽序列同源性高达98%。酶切鉴定证实真核重组表达质粒LipL41-pCDNA3构建成功，RT-PCR检测显示，LipL41-pCDNA3转染组在大约1kb处出现特异的扩增带，而空质粒组无此条带。结论：LipL41的真核重组表达质粒构建成功，并可在哺乳动物细胞中表达。     

Immunological characteristics of the glycolipid antigen of Leptospira interrogans serovar lai.

The protective antigen (PAg), a glycolipid substance, was extracted from Leptospira interrogans serovar lai strain 017 with a chloroform-methanol-water (1:2:0.8 [vol/vol/vol]) solution and partially purified by silica gel column chromatography. The PAg was not detected by Coomassie brilliant blue staining in sodium dodecyl sulfate-polyacrylamide gel electrophoresis but was observed as a smearlike band, which corresponded to a 24- to 30-kilodalton standard protein, by silver staining. The outer envelope (OE) fraction showed the same band, suggesting that the PAg was one of the chemical components of the OE. The immunogenicity and protective activity of the PAg were compared with those of the OE. The PAg as well as the OE and whole cells was able to induce agglutinating antibody against L. interrogans. Furthermore, the immune sera exhibited opsonic activity against L. interrogans, as observed by measurement of chemical luminescence derived from reactive oxygen. The PAg exhibited protective activity in hamsters challenged with lethal doses of L. interrogans. Therefore, the antigen may be useful as a component vaccine against leptospiral infection.     

Protection against Leptospira interrogans sensu lato challenge by DNA immunization with the gene encoding hemolysin-associated protein 1

The use of DNA constructs encoding leptospiral proteins is a promising new approach for vaccination against leptospirosis. In previous work we determined that immunization with hemolysis-associated protein 1 (Hap1) (LipL32) expressed by adenovirus induced significant protection against a virulent Leptospira challenge in gerbils. To avoid the use of the adenovirus vector, we checked for clinical protection against lethal challenge by DNA vaccination. A DNA vaccine expressing Hap1 was designed to enhance the direct gene transfer of this protein into gerbils. A challenge was performed 3 weeks after the last immunization with a virulent strain of serovar canicola. Our results show that the cross-protective effect with pathogenic strains of Leptospira, shared by Hap1, could be mediated by the DNA plasmid vector. This finding should facilitate the design and development of a new generation of vaccines against bacteria, particularly Leptospira interrogans sensu lato.