我国4个钩端螺旋体血清群lipL21基因序列分析及其表达产物的鉴定

目的本文采用高保真PCR从我国主要流行的问号钩体黄疸出血群赖型56601株、波摩那群波摩那型56608株、流感伤寒群临型56609株及腐生性双曲钩体参考标准株三宝垄群patoc型PatocⅠ株基因组DNA中扩增了全长lipL21基因片段。序列分析结果表明,所克隆的4株钩体lipL21基因与已报道的相应序列(GenBankNo.:AY187271)比较,其核苷酸和氨基酸序列相似性分别高达99.64～99.82%和99.46～100%。所构建的问号钩体黄疸出血群赖型56601株lipL21基因原核表达系统在IPTG诱导下,能有效地表达目的重组蛋白rLipL21,其产量约为细菌总蛋白的10%。Westernblot结果证实,兔抗钩体TR/patocⅠ属特异性抗原血清能识别rLipL21并与之结合。上述实验结果提示,lipL21基因序列非常保守,其表达产物有良好免疫原性,可作为研制通用型钩体基因工程疫苗的候选抗原。     

重组钩端螺旋体基因疫苗与不同毒力钩体交叉免疫研究

目的　欲了解赖型钩端螺旋体 (简称钩体 )基因多肽疫苗与我国主要 7群不同毒力致病钩体间的交叉免疫情况。方法　常规制备不同毒力钩体超声变性抗原及钩体基因疫苗兔抗血清和全钩死疫苗 (WCV)兔抗血清。以WCV抗血清与各株钩体反应为阳性对照 ,以非致病钩体、PT7- 7(多肽疫苗保护性抗原基因DNA片段质粒载体 )与WCV抗血清反应为阴性对照 ,进行EIA测定。结果　 (1)多肽基因疫苗抗血清与强毒力钩体 (黄疸出血型、秋季热型、澳洲型、巴叶赞型 )具良好的免疫交叉反应(效价为 1/ 5 2 42 88～ 1/ 12 80 0 ;WCV抗血清效价为 1/ 2 6 2 144～ 1/ 6 40 0 )。 (2 )多肽基因疫苗抗血清与弱毒力钩体 (犬型、流感伤寒型、七日热型、波摩拿型 )也具良好的免疫交叉反应 (效价为 1/ 2 5 6 0 0～ 1/ 12 80 0 ;WCV抗血清效价为 1/ 2 5 6 0 0～ 1/ 6 40 0 )。 (3)多肽基因疫苗抗血清与非致病钩体 (PatocI)、PT7— 7质粒载体阴性对照无明显免疫交叉反应 (效价分别为 1/ 16 0 0 ,1/ 2 5 6 ;WCV抗血清效价分别为 1/ 32 0 0 ,1/ 5 12 )。结论　赖型钩体基因重组疫苗抗血清与不同毒力致病钩体具良好的免疫交叉反应。提示 :该基因重组多肽疫苗有可能对我国主要流行之 7群钩体感染具完全交叉保护 ,从而弥补了全钩死疫苗仅对部分钩体有     

恙虫病东方体Gilliam株56kDa外膜蛋白基因的克隆与表达

目的　表达恙虫病东方体 (Orientiatsutsugamushi)Gilliam株 5 6kDa外膜蛋白 ,探索其作为诊断抗原的可能性。方法　采用PCR方法 ,从Ot Gilliam株菌种基因组DNA中扩增出 5 6kDa外膜蛋白基因片段 ,将目的基因片段定向插入原核表达载体pQE30 ,再用重组质粒转化大肠杆菌 ,最后用IPTG诱导转化菌 ,SDS -PAGE和Western -blotting检测重组质粒目的基因的表达。结果　 (1)获得长约 15 0 0bp的Ot Gilliam株 5 6kDa外膜蛋白基因 ,SDS -PAGE检测表达产物 ,在相对分子量5 6× 10 4处有表达带 ;(2 )DNA测序证明重组质粒 pQE30 / 5 6中插入的基因片段的序列与已报告的Ot Gilliam株 5 6kDa外膜蛋白基因序列基本一致。 (3)诱导表达菌体经超声破碎后 ,显示目的蛋白主要以包涵体形式存在 ;(4 )免疫印迹显示该重组质粒转化的大肠杆菌有一相对分子量约为 5 6× 10 4的独特蛋白带 ,该蛋白约占细菌蛋白总量的 2 9 4 %。结论　Ot Gilliam株5 6kDa外膜蛋白基因在大肠杆菌中获得了高效表达 ,表达的重组蛋白具有免疫反应性 ,与Ot Gilliam株感染的小鼠血清在免疫印迹中呈阳性反应。     

芯片法筛选钩端螺旋体外膜蛋白抗原表位

目的 通过芯片法筛选钩端螺旋体主要外膜蛋白中抗原表位,为后续研制有广泛交叉保护作用的通用钩体疫苗提供科学依据。方法 采用PCR及测序法检测主要外膜蛋白编码基因在不同血清群钩体中的分布情况及其序列相似性。采用生物信息学软件预测钩体外膜蛋白抗原表位。采用抗体纯化磁珠试剂盒纯化大鼠抗不同血清群钩体抗血清IgG。采用抗原芯片法检测各抗原肽的免疫反应性,确定优势抗原表位。结果 候选抗原蛋白编码基因均广泛存在于不同血清群的致病性钩体中且序列保守。综合生物信息学软件结果预测出30个候选T-B联合抗原表位。合成短肽结合芯片检测方法共筛选出9个优势抗原表位,各抗原肽对不同血清群钩体抗血清IgG反应性基本一致。结论 芯片法可高效筛选钩体外膜蛋白优势表位,为表位肽疫苗的研制提供支持。     

恙虫病东方体Karp株56kDa与47kDa外膜蛋白基因的嵌合和嵌合基因在大肠杆菌的表达

目的　将恙虫病东方体 (Orientiatsutsugamushi,Ot)Karp株的 5 6kDa和 4 7kDa外膜蛋白基因嵌合 ,并使嵌合基因在大肠杆菌细胞内表达 ,产生双抗原融合蛋白。方法　采用PCR方法 ,从OtKarp株基因组DNA中扩增 5 6kDa蛋白基因片段 ,将该片段分别与原核表达载体pQE30及 4 7kD蛋白基因重组质粒 pQE30 / 4 7连接 ,构建 pQE30 / 5 6及pQE30 / 5 6 - 4 7重组质粒 ;用IPTG诱导转入大肠杆菌内的重组质粒的目的基因表达。结果　SDS -PAGE显示 ,pQE30 / 5 6转化的大肠杆菌产生一约 4 5kDa的融合蛋白和 pQE30 / 5 6 - 4 7转化大肠杆菌产生一约 90kDa融合蛋白 (5 6 - 4 7融合蛋白 ) ,免疫印迹分析显示两融合蛋白均与OtKarp株感染鼠血清产生特异性反应 ,5 6 - 4 7融合蛋白分别与 4 7kDa、5 6kDa重组蛋白免疫的小鼠血清产生特异性反应 ,以及 5 6 - 4 7融合蛋白免疫血清与 5 6kDa和 4 7kDa重组蛋白反应。结论　OtKarp株的 5 6kDa与 4 7kDa外膜蛋白基因嵌合后在大肠杆菌细胞内实现了表达 ,表达的融合蛋白具有 5 6kDa和 4 7kDa外膜蛋白的抗原特性。     

3株钩端螺旋体外膜蛋白基因的序列分析

目的 探讨钩端螺旋体外膜蛋白基因在钩体遗传分类中的作用。方法 对我国的2株问号钗体赖型56601株和爪哇型56602株的外膜蛋白基因Ompl1进行PCR扩增,得到外膜蛋白基因并进行了全基因序列分析,测序的2株钩体与流感伤寒型RH52株进行了核苷酸序列比较和推导的氨基酸序列比较。结果 赖型56601和爪哇型56602株钩体Ompl1基因核苷酸序列与流感伤塞型RH52株的同源性分别为89％和80％,的外膜蛋白氨基酸序列的同源性分别为92％和88％。由此可以看出56601株、56602株与RH52株分别属于不同的血清群,这与Yasuda的遗传分类相。结论 我国的赖型56601株和爪哇型56602株及流感伤寒型RH52株Ompl1基因的核苷酸序列存在一定差别,利用Ompl1基因可对钩端螺旋体进行分类和鉴定。     

Asia1型口蹄疫病毒分离株P1基因的原核表达及其多克隆抗体的制备

目的原核表达口蹄疫病毒Asia1/HeB株衣壳蛋白前体P1基因并纯化。制备其兔多克隆抗体并进行鉴定。方法从重组克隆载体pGEM-P1中扩增编码P1区结构蛋白的基因片段。并克隆入原核表达载体pET-28a(+)中。原核表达与蛋白纯化后,免疫新西兰兔,制备P1蛋白抗血清。抗体效价及特异性检测分别用间接ELISA和Westernblot法鉴定。结果在大肠杆菌中成功表达了分子量约为80.475kDa的P1蛋白。ELISA法检测抗血清的效价可达到1∶25600,Westernblot分析抗血清可与原核表达的P1蛋白特异性结合。结论在大肠杆菌中成功表达了口蹄疫病毒Asia1/HeB株衣壳蛋白前体,并制备了P1衣壳蛋白前体的抗血清,为进一步研究P1区结构蛋白的结构、功能以及抗原表位的确定奠定了基础。     

光生物素标记Patoc 1株DNA探针鉴别双曲和问号钩端螺旋体的初探

本文以光生物素标记的双曲钩体Ｐａｔｏｃ １株全ＤＮＡ作为探针，对不同血清型的问号构体与双曲钩体在硝酸纤维素膜上和微量反应板中进行ＤＮＡ－ＤＮＡ杂交试验。结果发现标记探针只能识别双曲钩体ＤＮＡ，而对问号钩体ＤＮＡ无一发生杂交，表明该探针具有高度特异性和敏感性，可用于鉴别双曲钩体和致病性钩体。     

恙虫病东方体Gilliam株56kDa蛋白部分基因的原核表达及初步鉴定

目的　对恙虫病东方体Gilliam株 5 6kDa蛋白部分基因进行原核表达 ,以获得高效表达、有生物活性目的蛋白 ,为恙虫病诊断试剂的研制打下基础。方法　根据Gilliam株东方体 5 6kDa蛋白基因序列设计特定引物 ,用PCR法扩增出编码5 6kDa抗原富含亲水区及同源性高的C端一段长约 70 0bp的DNA ,插入原核载体PGEX - 4T - 2 ,转化大肠杆菌TGI ,抽提质粒 ,酶切鉴定 ,含插入片段的重组质粒进行序列分析 ,再转化表达宿主菌大肠杆菌BL2 1(DE3) ,IPTG诱导表达 ,用SDS -PAGE及Western -blot进行分析鉴定。结果　SDS -PAGE检测表明该截短片段以融合蛋白的方式得到成功表达 ,在相对分子量 5 8kDa处有表达条带 ,经薄层扫描分析 ,目的蛋白条带占全菌蛋白的 30 5 %。Western -blot证实该融合蛋白能被恙虫病患者阳性血清识别。结论　表达产物初步鉴定具有生物活性 ,有望应用于恙虫病诊断试剂的研制     

抗波摩那型109株钩端螺旋体外膜抗原单克隆抗体的研制

本文用波摩那型109株钩体外膜抗原免疫的BALB/c鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,获得3株分泌抗波摩那型钩体外膜抗原单克隆抗体的杂交瘤细胞株,定名为LS_(15)、LS_(22)和LS_(28)。经酶标(ELISA)和显微镜凝集试验(MAT)证实LS_(22)和LS_(28)McAbs为波摩那型109株钩体特异的抗体;LS_(15)McAb为针对存在于一定钩体群型中共同抗原的非凝集抗体。杂交瘤细胞注入同系小鼠腹腔可诱生含高效价McAbs的腹水。经鉴定LS_(15)、LS_(22)和LS_(28)McAbs均属IgG_2亚类。     

Use of locally isolated saprophytic Leptospira strain for serological testing of human leptospirosis.

A saprophytic Leptospira isolate recovered from tap water was utilized for serological testing. One hundred-twenty Serum samples comprising 55 cases from PUO/febrile jaundice and 65 samples from apparently healthy individuals were tested by MAT and HA using this environmental saprophytic strain and the results compared with that of Leptospira biflexa semaranga patoc, the standard saprophytic strain commonly employed for sero-diagnosis of leptospirosis. The MAT data showed 96.4 per cent correlation between the two strains. Similarly, the HA results were matching to the extent of 94.5 per cent. Results, therefore, suggest that local saprophytic Leptospira strain may serve as a substitute to serovar patoc for serodiagnosis of leptospirosis.     

贵州省钩端螺旋体血清群特异性PCR鉴定结果与分析

目的 采用血清群特异性PCR技术了解贵州省近年钩端螺旋体(钩体)流行菌群,为贵州省钩体病的防控提供技术手段和科学依据。方法 采用致病性钩体特异性PCR方法(G1/G2-PCR)对来自贵州省近年的钩体分离菌株进行鉴定,进一步应用基于致病性钩体O抗原基因的血清群特异性PCR(O-PCR)对贵州省近年的58株钩体分离株进行血清群鉴定,并采用显微凝集试验(MAT)对O-PCR方法的检测结果进行验证。结果 G1/G2-PCR检测结果显示58株菌株均为致病性钩体,O-PCR将58株致病性钩体菌株鉴定为黄疸出血群,与传统的MAT法鉴定结果一致。结论 O-PCR技术可作为我省钩体快速分群鉴定可靠的实验室诊断技术手段,贵州省近年的钩体流行菌群为黄疸出血群。     

Characterization of Leptospira strains HY-1, HY-2, and HY-10 isolated in Korea by means of monoclonal antibodies and restriction endonuclease DNA analysis.

To analyze the characteristics of Leptospira strains HY-1, HY-2, and HY-10, which were isolated from patients with leptospirosis in Korea in 1985, 12 monoclonal antibodies (MAbs) against strain HY-1 and six MAbs against serovar lai strain 017 were produced, and their properties were determined by the microscopic agglutination test. Genetic relationships among the leptospires were determined by restriction endonuclease DNA analysis. Three MAbs reacted with all strains of serogroup Icterohaemorrhagiae, but did not react with any strains of the other 10 serogroups. All MAbs reacted with strains 017, HY-1, HY-2, and HY-10 at nearly identical titers. Two MAbs reacted only with these four strains. These four strains also had the same restriction endonuclease cleavage patterns. Based on these results, strains HY-1, HY-2, and HY-10 were identified as serovar lai, which is one of the common serovars in China. It is suggested that serovar lai is one of the prevalent serovars in Korea, and that the Mabs produced in this study are useful for the accurate and rapid identification of this serovar.     

Antigens involved in the human antibody response to natural infections with Leptospira interrogans serovar copenhageni.

Serum samples from patients with leptospirosis were screened by the microscopic agglutination test (MAT), ELISA and by immunoblotting. The latter two tests were performed with L. interrogans serovar copenhageni isolated from human blood culture. Immunoblotting with patients' sera revealed antibodies recognizing several leptospiral components in the molecular weight range 14.5-105 kDa of both IgM and IgG response. All patients' serum samples presented IgM antibodies reacting with a diffuse band of mol. wt of 14.8-22 kDa proteinase-K resistant and most reacted with bands of 26.5-28.7, 38-39 and 43-43.5 kDa. The IgG response appeared to be at variance. Examination of sequential serum samples obtained over a 4-8-month period revealed little change in the profile of antigen recognized after the 40th day of infection. Sera from healthy individuals presented IgM antibodies reacting with several leptospiral antigens, but lacked response against those of diffuse band of 14.8-22 kDa.     

Leptospirosis among rice mill workers of Salem,South India

Leptospirosis is not commonly reported from the Salem district in central Tamil Nadu in India. In October 2000, a rice mill worker, who had fever and jaundice, tested positive in leptospiral IgM enzyme-linked immunosorbent assay (ELISA). Microbiological and serological investigations were performed on the patient, a sample population of the rice mill workers, and the animal and rodent populations living in the same premises. Leptospira was isolated from the patient about 2 weeks after the onset of symptoms when he had recovered from illness following a course of doxycycline. The isolate was serovar Icterohaemorrhagiae of serogroup Icterohaemorrhagiae. The patient also showed a fourfold rise in titers in microscopic agglutination test (MAT) and IgG ELISA. The rice mill workers had a seroprevalence rate of 68.3%, which was significantly higher than that among a control group consisting of persons engaged in other occupations. Serological studies conducted among cattle, dogs, cats, and rats showed seroprevalence rates of 52.9%, 50.0%, 66.6%, and 52.1%, respectively. Leptospires were isolated from two rats, but the isolates were lost during subculturing and could not be characterized. The most predominant serogroup identified by MAT was Autumnalis for rice mill workers and all animal populations. The other serogroups that reacted in MAT were Icterohaemorrhagiae, Australis, Grippotyphosa, and Patoc. Although Australis and Grippotyphosa showed agglutination in the case of human samples, none of the animals had detectable titers to these serogroups. The rice mills of Salem, having large rodent populations, various animals living in close proximity, a wet environment, and unprotected exposure of the workers to the environment, constitutes an ideal setting for transmission of leptospirosis and could be an epidemiological niche of leptospirosis.     

Leptospira borgpetersenii from free-living white-eared opossum (Didelphis albiventris): First isolation in Brazil

Leptospirosis is a zoonotic disease that occurs all over the world, caused by bacteria of the genus Leptospira. Marsupial and didelphidae families are considered susceptible to infection caused by a wide range of Leptospira serovars for which they serve as reservoirs. Thirty-three free-living white-eared opossums (Didelphis albiventris) were captured in Southern Brazil and bodily fluids were collected. From the urine samples it was possible to obtain an isolate identified as Leptospira borgpetersenii by rpoB gene sequencing and belonging to serovar Castellonis by Multilocus Variable-Number Tandem-Repeat Analysis. This is the first report of the isolation of Leptospira spp. from the white-eared opossum in Brazil. In addition, the new strain was also virulent in the hamster model of lethal leptospirosis. The microscopic agglutination test (MAT) was used for detecting the presence of antibodies against Leptospira spp. in white-eared opossum, human, cattle and canine sera using a panel of 59 Leptospira strains that included the new isolate. The inclusion of the new strain in the MAT battery increased the MAT sensitivity for canine sera. These findings suggest that the white-eared opossum is an important reservoir of pathogenic Leptospira spp.     

Occurrence of severe leptospirosis in a breeding colony of squirrel monkeys.

Although experimental leptospirosis has been studied in various species of monkeys, the occurrence of acute leptospirosis in a population of nonhuman primates is uncommon. We report on a number of severe cases of icterohemorrhagic leptospirosis that appeared in the squirrel monkey (Saimiri sciureus) colony of 109 animals at the Institute Pasteur in French Guiana. Initially, 11 animals had acute illness, with jaundice and a hemorrhagic syndrome, leading to 10 deaths. Two Leptospira interrogans strains were isolated from blood cultures of sick monkeys, and one was isolated from the urine of a rat trapped in the breeding park. All three belonged to serovar copenhageni, and tests using monoclonal antibodies showed that these three strains were extremely similar. In the following weeks, five pregnant female monkeys had miscarriages; two of them had antibodies against the Icterohaemorrhagiae serogroup. An epidemiologic study conducted on the 93 remaining animals demonstrated a seropositivity rate of 26% (microagglutination test [MAT] titer greater than or equal to 100) primarily for the Icterohaemorrhagiae serogroup, but also for the Ballum, Grippotyphosa, Sejroe, and Panama serogroups. In addition, 12% showed lower MAT titers (50) for the same serogroups. Lastly, recently trapped feral squirrel monkeys were shown to have agglutinins against the Grippotyphosa and Sejroe serogroups. A vaccine, which was prepared from one of the strains isolated, was used in addition to antibiotic prophylaxis to control the enzootic disease. This confirms that the squirrel monkey is highly susceptible to icterohemorrhagic leptospirosis and is probably receptive to other serogroups, and that this animal may be useful in studying experimental leptospirosis and for testing new human vaccines.     

我国新型钩端螺旋体参考菌株的国际复核结果

本文报道了由美国CDC和荷兰RTI对我国分离发现的36株新型钧端螺旋体（钩体）参考菌株的国际复核检定结果。有24株钩体各实验室检定结果相一致,各自代表一个新的国际血清型,分属14个血清群24个血清型。有12株钩体检定结果互有差异,对此进行了分析,并提出了建议。因此,目前我国至少存在有14个血清群26个国际新血清型钩体存在。