问号钩端螺旋体血清群LipL41基因型分析及其表达产物的免疫学鉴定

目的 确定我国15群15株问号钩端螺旋体(简称钩体)参考标准株和2群2株双曲钩体国际标准株携带LipL41基因情况，构建该基因的原核表达系统，鉴定表达产物的免疫原性。方法 常规酚—氯仿法提取上述17株钩体基因组DNA，高保真PCR扩增全长LipL41基因片段，T—A克隆后测序分型。构建LipL41基因原核表达系统，SDS-PAGE检测重组目的蛋白(rLipL41)表达情况。分别用钩体属特异性TP/patoe Ⅰ抗原、rLipL41兔抗血清的Western blot鉴定其免疫反应性和抗原性。分别用显微镜凝集试验(MAT)、钩体黏附J774A．1细胞模型检测兔抗rLipL41血清的交叉凝集效价和黏附阻断作用。结果 15株问号钩体均有LipL41基因，并可分为LipL41／1和LipL41／2两种基因型，2株双曲钩体则否。11个LipL41／1基因和4个LipL41／2基因克隆之间的核苷酸和氨基酸序列相似性分别为88．61％—88．67％和93．24％—97．18％。所构建的原核表达系统rLipL41／1和rLipL41／2的表达量分别占钩体总蛋白的30％和40％。rLipL41／1和rLipL41／2均能与TP/patoe Ⅰ抗血清发生结合反应，免疫家兔能产生抗体。rLipL41／1和rLipL41／2兔抗血清对上述15株问号钩体MAT效价为1：8，1：128、1：16～1：2．S6稀释时均能有效地阻断钩体对细胞的黏附。结论 我国主要的15群问号钩体代表株均有LipL41／1或LipL41／2基因。所构建原核表达系统能高效表达rLipL41／1和rLipL41／2。rLipL41／1和rLipL41／2是具有良好抗原性和免疫反应性、广泛存在于不同血清群问号钩体表面的蛋白抗原。     

问号钩端螺旋体rLTB/rCTB-LipL32/1免疫保护作用及野生株LipL32基因表达和患者抗体检测

目的构建LTB—LipL32／1和CTB—LipL32／1融合基因原核表达系统并鉴定其表达产物的免疫和佐剂活性及保护作用，了解问号钩端螺旋体(简称钩体)野生株LipL32基因携带、表达及钩体病人血清LipL32基因产物抗体产生频率。方法采用常规分子生物学技术构建LTB—LipL32／1和CTB—LipL32／1融合基因及其原核表达系统。采用SDS-PAGE检测目的重组蛋白rLTB-LipL32／1及rCTB-LipL32／1表达情况。分别采用Western blot和GM1-ELISA检测rLTB-LipL32／1及rCTB—LipL32／1的免疫反应性和佐剂活性。采用PCR和MAT分别检测97株问号钩体野生株LipL32基因及其表达情况。采用ELISA检测228例钩体病人血清LipL32基因产物的抗体。采用豚鼠保护试验检测重组蛋白的免疫保护作用。结果与报道的相关序列比较，LTB—LipL32／1和CTB-LipL32／1融合基因核苷酸序列相似性分别为99．12％～99．71％和98．54％～99．42％，氨基酸序列相似性分别为97．58％-99．63％和96．77％～99．63％。rLTB-LipL32／1和rCTB—LipL32／1表达产量均约为细菌总蛋白的10％，并主要以包涵体形式存在。rLTB—LipL32／1和rCTB-LipL32／1均分别能与LipL32／1兔抗血清和牛GMI结合。97．9％(95／97)问号钩体野生株含有LipL32基因，95．9％(93／97)问号钩体野生株与rLipL32／1和rLipL32／2兔抗血清的MAT结果阳性。97．4％(222／228)和94．7％(216／228)的病人血清rLipL32／1和rLipL32／2抗体阳性。rLTB-LipL32／1、rCTB-LipL32／1和rLipL32／1豚鼠保护率分别为75．0％、75．0％～87．5％、50．O％～62．5％。结论成功构建了LTB—LipL32／1和CTB—LipL32／1融合基因及其原核表达系统。rLTB-LipL32／1和rCTB-LipL32／1融合蛋白有良好的抗原性和佐剂活性及一定的免疫保护作用，具有成为问号钩体属特异性疫苗的良好前景。LipL32／1是不同问号钩体血清群中广泛存在、序列保守、高频率表达的基因。     

问号钩端螺旋体病人血清中LipL21抗体检测

寻找钩体属特异性保护抗原,对于研制通用型钩体疫苗及实验室诊断试剂盒均有重要价值。Cullen等首先报告了致病性问号钩体均具有属特异性lipL21脂蛋白基因。我们也曾证实我国15群15型问号钩体参考标准株均含有序列保守的lipL21基因。已知钩体病免疫保护作用主要依赖血清抗体为主的体液免疫。我们采用显微镜凝集试验(MAT)检测了156份钩体病人血清的凝集效价,并建立rLipL21-IgM/IgG-ELISAs对血清标本中LipL21的IgG和IgM型抗体进行了检测。     

我国主要问号钩端螺旋体血清群lipL21基因序列及其产物免疫反应性分析

目的 分析我国15群15型问号钩端螺旋体（简称钩体）参考标准株外膜脂蛋白lipL21基因序列，构建该基因原核表达系统并鉴定表达产物的免疫原性，了解lipL21基因自然表达状况。方法 高保真PCR扩增上述问号钩体株及双曲钩体Paloc型PalocⅠ株基因组DNA中全长lipL21基因片段，T-A克隆后测序并构建其原核表达系统。分别用钩体TR/PatocⅠ和rLipL21兔抗血清为一抗的Western blot鉴定目的重组蛋白rLipL21的免疫原性，显微镜凝集试验（MAT）检测rLipL21兔抗血清的交叉凝集效价。以盐变-去垢剂处理法提取钩体外膜蛋白，用SDS-PAGE和免疫印迹法检测上述钩体株lipL21基因自然表达情况。结果 上述钩体株均存在序列高度保守的lipL21基因，其核苷酸和氨基酸序列相似性分别为98．75％～99．82％和99．46％～100％。rLipL21能与TR/PatocⅠ及rLipL21兔抗血清发生结合反应。rLipL21免疫家兔能产生抗体，该抗体对上述15株问号钩体MAT效价为1：16～1：128。问号钩体外膜标本中均可检出LipL21，双曲钩体则否。结论 我国钩体群参考标准株均含有序列保守的lipL21基因并自然表达于外膜，双曲钩体PatocⅠ株虽含有lipL21基因但未表达。rLipL21具有良好的抗原性和免疫反应性，有可能作为新型钩体疫苗或检测试剂盒的候选属特异性表面抗原之一。     

问号钩端螺旋体血清群LipL32基因型分析及其重组蛋白的免疫学鉴定

目的　探讨 15群 15型问号钩端螺旋体 (简称钩体 )中国参考标准株及 2群 2型双曲钩体国际参考标准株是否均存在主要外膜蛋白 (MOMP)基因 (LipL32 ) ,克隆并构建该基因的原核表达系统 ,鉴定表达产物的免疫性。方法　常规酚 氯仿法提取上述钩体株基因组DNA ,高保真PCR扩增全长LipL32基因片段 ,T A克隆后测定核苷酸序列并构建表达系统 ,不同浓度IPTG诱导后用SDS PAGE检测rMOMP表达情况。分别用兔抗TR patocⅠ钩体全菌抗血清、rMOMPs免疫兔血清的Westernblot鉴定其免疫反应性和免疫原性 ,显微镜凝集试验 (MAT)检测rMOMPs免疫兔血清的交叉凝集效价 ,钩体细胞黏附模型检测抗体阻断效果。结果　上述 17株钩体均有LipL32基因 ,但可分LipL32 1和LipL32 2两种基因型。 13个LipL32 1和 4个Li pL32 2基因型之间核苷酸和氨基酸序列同源性分别为 95 .12 %～ 96 .6 0 %和 97.79%～ 98.16 %。IPTG诱导后rMOMP1和rMOMP2表达量分别占细菌总蛋白的 4 0 %和 10 %。rMOMP1和rMOMP2均能与兔抗钩体TR patocⅠ血清发生结合反应 ,免疫家兔可对上述 17株钩体产生 1∶2～ 1∶6 4MAT效价的凝集抗体。 1∶2～ 1∶16稀释的兔抗rMOMP1和rMOMP2血清均能有效地阻断钩体的黏附。结论　所有检测的钩体具有LipL32 1或LipL32 2基因。所     

问号钩端螺旋体T和B细胞表位联合基因的原核表达及其产物免疫性分析

目的 构建问号钩端螺旋体(简称钩体)LipL32、OmpL1和LipL21蛋白的优势T-和B-细胞联合表位融合基因及其原核表达系统,并对表达产物的免疫原性进行鉴定.方法 人工合成多表位联合基因并构建其原核表达系统.采用SDS-PAGE检测重组蛋白;采用MAT检测重组蛋白兔抗血清与我国钩体标准参考株的凝集效价;Western blot和ELISA检测重组蛋白的免疫原性.结果 获得了多表位融合基因并构建了原核表达系统.表达产物的相对分子质量约为23×103,且主要以可溶性形式存在;重组蛋白兔抗血清免疫双扩散效价为1∶8,该抗血清能与我国15群的钩体标准参考株发生凝集反应,ELISA证明该重组蛋白能检测不同群型钩体感染患者血清中的抗钩体抗体.结论 成功构建了包含钩体LipL32、OmpL1和LipL21蛋白的优势T和B细胞联合表位基因及其原核表达系统,表达产物具有良好的抗原性和交叉免疫反应性,可作为研制通用型问号钩体基因工程疫苗及血清学检测的抗原.     

问号钩端螺旋体ompA基因分析及其重组表达产物的免疫学鉴定

目的 了解我国15群15株问号钩端螺旋体(简称问号钩体)参考标准株携带ompA基因情况,重组表达OmpA(rOmpA)并鉴定rOmpA的免疫原性和免疫保护性.方法 采用酚-氯仿法提取问号钩体基因组DNA,PCR扩增全长ompA基因,T-A克隆后测序.构建问号钩体黄疸出血群赖型56601株ompA基因的原核表达系统,采用SDS-PAGE及Bio-Rad凝胶}冬{像分析系统检测rOmpA表达情况及其产鼍.rOmpA免疫家兔以获得抗血清,采用免疫扩散试验检测抗血清效价.采用Western blot检测rOmpA与其抗血清和问号钩体56601株全菌抗血清的免疫反应性,显微镜凝集试验(MAT)检测rOmpA抗血清对15株问号钩体的交叉凝集情况.分别采用问号钩体黏附J774A.1细胞模型和豚鼠感染模型,了解rOmpA兔抗血清黏附阻断及rOmpA免疫保护作用.结果 15株问号钩体均含有序列保守的ompA基因,双曲钩体Patocl株则否.rOmpA表达量约占细菌总蛋白的20%.rOmpA能诱导家兔产生抗体,其抗血清免疫扩散效价为1:4.兔抗血清及问号钩体56601株全菌抗血清均能与rOmpA产生阳性Western blot信号.rOmpA抗血清对15株问号钩体的MAT效价为1:20～1:320.1:10～1:160稀释的rOmpA抗血清均能阻断问号钩体黏附J774A.1细胞,100μg和200μg rOmpA对豚鼠的免疫保护率分别为50.0%和75.0%.结论 ompA基因仅存在于不同血清群致病性问号钩体基因组中.rOmpA具有较好的抗原性,多种免疫学方法 检测显示,有可能作为通用型问号钩体基因上程疫苗的候选抗原.     

问号钩端螺旋体属特异性外膜蛋白OmpL1和LipL21抗原表位预测及鉴定

目的 筛选问号钩端螺旋体(简称钩体)属特异性外膜蛋白OmpL1和LipL21有效T和B细胞联合抗原表位,为研制多抗原肽(multiple antigenic peptide,MAP)疫苗提供基础.方法 采用生物信息学方法预测OmpL1和LipL21分子中T和B细胞联合抗原表位.采用PCR扩增候选联合抗原表位片段并分别构建其噬菌体展示系统.分别以rOmpL1或rLipL21、黄疸出血群赖株、钩体患者抗血清为一抗,采用Western blot检测各抗血清与目的表位的免疫反应性及其强度.结果 通过抗原表位预测,选择了高分值的4个OmpLl和2个LipL21联合表位.经扩增获得了预期的各抗原表位片段,各目的表位序列均准确插入噬菌体PⅢ蛋白N端并有效表达.各抗血清均能识别上述6个联合表位.其中LipL21的97～112和176-184表位对任一抗血清均显示相似强度的杂交条带.综合4个OmpL1表位对3种抗血清的不同Western blot结果及其实际意义,杂交信号从强到弱依次为173～191、87～98、297～320和59～78表位.结论 所研究的6个联合表位均分别为LipL21和OmpL1的有效抗原表位,其中LipL21的97～112、176～184和OmpL1的87～98、173～191表位可应用于钩体MAP疫苗研制.     

钩端螺旋体多抗原肽的构建及免疫性分析

目的 构建问号钩端螺旋体(简称钩体)主要外膜蛋白OmpL1、LipL21和LipL32优势抗原表位的串联基因及其表达系统,了解该重组蛋白的免疫活性.方法 采用噬菌体M13KE表面展示技术结合Western blot分析,鉴定了OmpLl、LipL21和LipL32的优势抗原表位,人工合成优势抗原表位串联基因并构建其原核表达系统.SDS-PAGE检测重组蛋白的表达情况;Western blot及ELISA鉴定重组蛋白的免疫活性.结果 该合成基因在原核表达系统中得到了有效表达,且表达产物主要以可溶性形式存在.Western blot和ELISA结果 显示该重组蛋白能与兔抗钩体全菌抗体及不同血清群的钩体病人血清中的抗体产生免疫反应.结论 本研究成功构建了钩体多表位串联基因及其表达系统,所表达目的 蛋白具有良好的免疫活性,且对不同血清群型抗体之间均有免疫原活性.     

钩端螺旋体属特异性外膜蛋白抗原的分析

致病性问号钩端螺旋体(简称钩体)血清群众多,各群间交叉保护作用较弱或无,目前国内外均采用当地流行的钩体血清群来制备多价钩体疫苗,但对疫苗中未包含钩体血清群感染的保护作用极其有限,易造成钩体病的暴发流行。已知80℃灭活、生理盐水回收的腐生性双曲钩体PatocⅠ株全细胞抗原(TR PatocⅠ)能与所有钩体病人血清发生凝集反应,但其抗血清能否凝集不同血清群的问号钩端螺旋体尚不清楚。本研究中,我们采用TR PatocⅠ免疫的兔抗血清与我国15群17型问号钩体参考标准株、2群2型双曲钩体国际标准株进行了显微镜凝集试验(MAT) ,发现该抗血清能…     

Effect of Leptospira interrogans outer membrane proteins LipL32 on HUVEC

Leptospira cause disease through a toxin-mediated process by inducing vascular injury, particularly a small-vessel vasculitis. Breakdown of vessel endothelial cell integrity may increase vessel permeability which is correlated with the changes of tight junction and/or apoptosis in vessel endothelial cells. The specific toxin responsible remains unidentified. In this study, we amplified outer membrane protein LipL32 from the genome of Leptospira interrogans serovar Lai, and it was subcloned in pET32a(+) vector to express thioredoxin(Trx)–LipL32 fusion protein in Escherichia coli BL21(DE3). The protein was expressed and purified, and Trx–LipL32 was administered to culture with human umbilical vein endothelial cells (HUVEC) to elucidate the role of leptospiral outer membrane proteins in vessel endothelial cell. The purified recombinant protein was capable to increase the permeability of HUVECs. And the protein was able to decrease the expression of ZO-1 and induce F-actin in HUVECs display thickening and clustering. Moreover, apoptosis of HUVEC was significantly accelerated. But the fusion partner had no effect in these regards. It is possible that LipL32 is involved in the vessel lesions.     

LipL32 is an extracellular matrix-interacting protein of Leptospira spp. and Pseudoalteromonas tunicata

LipL32 is the major outer membrane protein in pathogenic Leptospira. It is highly conserved throughout pathogenic species and is expressed in vivo during human infection. While these data suggest a role in pathogenesis, a function for LipL32 has not been defined. Outer membrane proteins of gram-negative bacteria are the first line of molecular interaction with the host, and many have been shown to bind host extracellular matrix (ECM). A search for leptospiral ECM-interacting proteins identified the major outer membrane protein, LipL32. To verify this finding, recombinant LipL32 was expressed in Escherichia coli and was found to bind Matrigel ECM and individual components of ECM, including laminin, collagen I, and collagen V. Likewise, an orthologous protein found in the genome of Pseudoalteromonas tunicata strain D2 was expressed and found to be functionally similar and immunologically cross-reactive. Lastly, binding activity was mapped to the C-terminal 72 amino acids. These studies show that LipL32 and an orthologous protein in P. tunicata are immunologically cross-reactive and function as ECM-interacting proteins via a conserved C-terminal region.     

The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection

We report the cloning of the gene encoding the 32-kDa lipoprotein, designated LipL32, the most prominent protein in the leptospiral protein profile. We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment to design an oligonucleotide probe. A Lambda-Zap II library containing EcoRI fragments of Leptospira kirschneri DNA was screened, and a 5.0-kb DNA fragment which contained the entire structural lipL32 gene was identified. Several lines of evidence indicate that LipL32 is lipid modified in a manner similar to that of other procaryotic lipoproteins. The deduced amino acid sequence of LipL32 would encode a 272-amino-acid polypeptide with a 19-amino-acid signal peptide, followed by a lipoprotein signal peptidase cleavage site. LipL32 is intrinsically labeled during incubation of L. kirschneri in media containing [(3)H]palmitate. The linkage of palmitate and the amino-terminal cysteine of LipL32 is acid labile. LipL32 is completely solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL32 exclusively into the hydrophobic, detergent phase, indicating that it is a component of the leptospiral outer membrane. CaCl(2) (20 mM) must be present during phase separation for recovery of LipL32. LipL32 is expressed not only during cultivation but also during mammalian infection. Immunohistochemistry demonstrated intense LipL32 reactivity with L. kirschneri infecting proximal tubules of hamster kidneys. LipL32 is also a prominent immunogen during human leptospirosis. The sequence and expression of LipL32 is highly conserved among pathogenic Leptospira species. These findings indicate that LipL32 may be important in the pathogenesis, diagnosis, and prevention of leptospirosis.     

Major Surface Protein LipL32 Is Not Required for Either Acute or Chronic Infection with Leptospira interrogans

Leptospira interrogans is responsible for leptospirosis, a zoonosis of worldwide distribution. LipL32 is the major outer membrane protein of pathogenic leptospires, accounting for up to 75% of total outer membrane protein. In recent times LipL32 has become the focus of intense study because of its surface location, dominance in the host immune response, and conservation among pathogenic species. In this study, an lipL32 mutant was constructed in L. interrogans using transposon mutagenesis. The lipL32 mutant had normal morphology and growth rate compared to the wild type and was equally adherent to extracellular matrix. Protein composition of the cell membranes was found to be largely unaffected by the loss of LipL32, with no obvious compensatory increase in other proteins. Microarray studies found no obvious stress response or upregulation of genes that may compensate for the loss of LipL32 but did suggest an association between LipL32 and the synthesis of heme and vitamin B₁₂. When hamsters were inoculated by systemic and mucosal routes, the mutant caused acute severe disease manifestations that were indistinguishable from wild-type L. interrogans infection. In the rat model of chronic infection, the LipL32 mutant colonized the renal tubules as efficiently as the wild-type strain. In conclusion, this study showed that LipL32 does not play a role in either the acute or chronic models of infection. Considering the abundance and conservation of LipL32 among all pathogenic Leptospira spp. and its absence in saprophytic Leptospira, this finding is remarkable. The role of this protein in leptospiral biology and pathogenesis thus remains elusive.Leptospira interrogans is a zoonotic spirochete with a worldwide distribution. In the chronic carrier state, host animals such as rats do not exhibit overt disease but are colonized by Leptospira in their renal tubules and shed bacteria in their urine. Humans are incidental hosts that become infected through exposure to contaminated water, soil, or urine. In the acute form of leptospirosis, disease severity ranges from asymptomatic infection to multiple organ failure, pulmonary hemorrhage, and death (13).The cellular and molecular mechanisms of leptospiral pathogenesis remain unclear. One of the major sites of interaction with the host is the bacterial outer membrane (OM). Analysis of the L. interrogans OM has identified a number of proteins (11, 12), the most abundant of which is LipL32, a 32-kDa lipoprotein estimated to account for a remarkable 75% of the OM proteome (11). LipL32 is also the most abundant surface-exposed protein (12).LipL32 is found in all pathogenic species tested to date and is highly conserved, with average amino acid identity over 98%, but it is not found in saprophytic species (16, 17). LipL32 is expressed during both chronic and acute infection and is highly immunogenic (15, 16, 27). These features have generated interest in LipL32 as a potential diagnostic reagent in both PCRs (21) and enzyme-linked immunosorbent assays (14). There has also been much interest in the potential of LipL32 to generate heterologous immunity, overcoming the limitations of serovar-specific immunity. However, to date LipL32-based vaccines have met with limited success (5, 6).The abundance, conservation, unique presence in pathogenic species, and immunogenicity of LipL32 are consistent with an important role in pathogenesis. Available microarray data provide little insight into the role of LipL32 since gene expression is unchanged under conditions of different temperatures (22). Recent studies have shown that LipL32 may act as an adhesin binding to collagen, laminin, and fibronectin (18, 19) while LipL32 has also been associated with hemolysis (20). However, the precise role of LipL32 in pathogenesis remains unknown.In this study an lipL32 mutant was constructed by transposon mutagenesis. To our surprise, analysis of this mutant in the hamster model of acute infection and rat model of chronic infection showed that LipL32 is not required for causing either acute leptospirosis or renal colonization.     

Encoding gene for the outer membrane lipoprotein LipL32 as a genetic target for developing leptospira genotyping schemes

Based on the nucleotide sequence of the gene that encodes the LipL32 lipoprotein of the outer membrane of leptospiras, two primer pairs were designed, i.e., an outer one that flanks a fragment of 677 bp and an inner one flanking a 204-bp fragment. The distribution of the LipL32 gene among 79 strains of representatives of the Leptospiraceae family (77 of genus Leptospira, 1 of Leptonema and 1 of Turneria), using PCR-based testing systems, was studied using PCR-based testing systems. Both amplicons of predicted sizes were revealed in pathogenic representatives of Leptospira and L. interrogans (except L. inadai) but were absent from L. biflexa saprophytes. Only a fragment of 204 bp was found in representatives of L. inadai. Hence, the obtained results point indicate the possibility of differentiation between of pathogenic and saprophytic leptospiras according to the presence of this gene. The gene that encodes the lipoprotein of the LipL32 of the outer membrane suits all requirements for a genetic target for working out schemes of leptospiras genotyping (the distribution in a huge number of representatives of a given taxon and the presence of conservative and variable sites in its nucleotide sequence).     

Vaccination with Leptospiral Outer Membrane Lipoprotein LipL32 Reduces Kidney Invasion of Leptospira interrogans Serovar Canicola in Hamsters

The Leptospira interrogans vaccines currently available are serovar specific and require regular booster immunizations to maintain protection of the host. In addition, a hamster challenge batch potency test is necessary to evaluate these vaccines prior to market release, requiring the use of a large number of animals, which is ethically and financially undesirable. Our previous work showed that the N terminus of the outer membrane protein LipL32 was altered in Leptospira interrogans serovar Canicola vaccines that fail the hamster challenge test, suggesting that it may be involved in the protective immune response. The aim of this study was to determine if vaccination with LipL32 protein alone could provide a protective response against challenge with L. interrogans serovar Canicola to hamsters. Recombinant LipL32, purified from an Escherichia coli expression system, was assessed for protective immunity in five groups of hamsters (n = 5) following a challenge with the virulent L. interrogans serovar Canicola strain Kito as a challenge strain. However, no significant survival against the L. interrogans serovar Canicola challenge was observed compared to that of unvaccinated negative controls. Subsequent histological analysis revealed reduced amounts of L. interrogans in the kidneys from the hamsters vaccinated with recombinant LipL32 protein prior to challenge; however, no significant survival against the L. interrogans serovar Canicola challenge was observed compared to that of unvaccinated negative controls. This finding corresponded to a noticeably reduced severity of renal lesions. This study provides evidence that LipL32 is involved in the protective response against L. interrogans serovar Canicola in hamsters and is the first reported link to LipL32-induced protection against kidney invasion.     

Analysis of Multiple Leptospira interrogans Serovar Canicola Vaccine Proteomes and Identification of LipL32 as a Biomarker for Potency

The current batch potency test for Leptospira interrogans serovar Canicola vaccines requires the use of a large number of hamsters and has severe effects (i.e., hepatic and renal failure resulting in death); while this vaccine is effective, a safer, cheaper, more ethical replacement is desired. The aim of this study was to analyze vaccine proteomes and identify target molecules common to all L. interrogans serovar Canicola vaccines which could be used to design an in vitro potency test. Initial analysis of L. interrogans serovar Canicola vaccines (A to E) from different manufacturers, using the Limulus amebocyte lysate assay and silver-stained sodium dodecyl sulfate polyacrylamide gels, indicated that lipopolysaccharide was not present in all vaccines, preventing it from being a suitable target molecule. The protein contents of vaccines A to E were therefore determined by two-dimensional liquid chromatography mass spectrometry ([2D-LC/MS] 221 ± 31, 9 ± 8, 34 ± 4, 21 ± 5, and 34 ± 17 proteins [mean ± 1 standard deviation] found, respectively). The outer membrane protein LipL32 was established to be common to all and to be present at a significantly higher (P ≤ 0.05) relative spectral abundance in a batch of vaccine which passed the in vivo potency test than in one which had failed. Further analysis using multiple reaction monitoring revealed that the concentration of the N terminus of LipL32 was significantly lower (P ≤ 0.01) in failed batches (n = 2) of vaccine than in passed batches (n = 2); the concentration of the C terminus between the two batches was approximately the same. An in vitro Leptospira vaccine potency test, based on N-terminal amino acid quantification of LipL32, was subsequently developed.     

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.