鼠疫耶尔森菌纤维蛋白溶解酶原激活因子的研究进展

鼠疫耶尔森菌纤维蛋白溶解酶原激活因子（Pla）是鼠疫菌特有的pPCP1质粒编码的细胞表面蛋白酶，介导了耶尔森菌外膜蛋白（YOPs）在体外的降解，具有温度依赖的纤溶酶原激活剂活性和凝固酶活性，可致纤溶酶原激活和α2抗纤溶酶失活，是鼠疫菌从皮下感染扩散到循环系统的重要毒力因子，是黏附素也是侵袭素，在鼠疫耶尔森菌致病过程中发挥重要作用。对Pla的研究将有助于进一步阐明鼠疫菌的致病机制，寻找更好的鼠疫诊断方法和防治措施。现对Pla的基因、活性、毒力研究近况进行综述。     

2013-2014年云南省剑川县野鼠鼠疫疫源地小肠结肠炎耶尔森菌的调查分析

目的 对剑川县野鼠鼠疫疫源地小肠结肠炎耶尔森菌分布情况调查分析。方法 从剑川采集鼠盲结肠标本进行小肠结肠炎耶尔森菌分离培养、表型鉴定和特异基因分析。结果 从采集的645份鼠类标本中,分离到34株小肠结肠炎耶尔森菌,分离率为5.27%(34/645);其中致病株1株、非致病株33株。鼠类标本分别是大绒鼠、贝氏树鼩、褐家鼠、齐氏姬鼠、小林姬鼠等,其中大绒鼠采集率和检出率最高(2.79%),其次为齐氏姬鼠(1.55%)。结论 剑川县野鼠鼠疫疫源地中,小肠结肠炎耶尔森菌的主要宿主与鼠疫菌主要宿主基本一致,推测小肠结肠炎耶尔森菌的存在与鼠疫的相对静息相关。     

耶尔森菌的分子分型

耶尔森菌属包括11个种,其中能引起人类疾病的有鼠疫耶尔森菌、小肠结肠炎耶尔森菌和假结核耶尔森菌.本文综述了目前耶尔森菌多种分子分型技术的研究现状.这些分子分型技术是对常规表型分型技术的补充和深入,使我们对所要研究的细菌有更加全面的了解.     

鉴定鼠疫耶尔森菌和假结核耶尔森菌的天冬酰脱氨酶试验

1975年,(?)根据假结核耶尔森氏菌不具有使天冬酰胺酸脱氨的作用,建议用天冬酰胺脱氨酶试验进行鼠疫耶尔森氏菌和假结核耶尔森氏菌的快速鉴别诊断,特别是对非典型的鼠疫耶尔森菌的鉴定。用接种环小心挑取经28℃培养48小时的培养物(不要触及培养基,以免产生非特异性反应)     

鼠疫耶尔森菌气溶胶发生与采样过程的生物活性损伤研究

目的研究鼠疫耶尔森菌气溶胶发生与采样过程中的生物活性损伤程度,探索减轻其损伤的方法。方法采用液体撞击式采样器对鼠疫耶尔森菌和粘质沙雷菌气溶胶进行采样和培养计数的方法,观察气溶胶发生与级联冲击采样测试过程中的生物活性损伤情况,以及不同采样液对生物活性的保护效果。结果鼠疫耶尔森菌气溶胶经AGI-10采样器采样10 min的生物活性损伤为98.63%,经发生器发生及AGI-10采样器采样10 min的生物活性损伤为98.80%,其采样和气溶胶发生采样过程生物活性损伤依次是粘质沙雷菌的160.36%和116.62%。PBS采样液在5 min采样时基本没有生物损伤,采样液加入吐温80和橄榄油可对30 min采样的鼠疫耶尔森菌活性提供有效的保护。结论鼠疫耶尔森菌在气溶胶发生与采样过程中生物活性损伤较大,其采样活菌数需进行校正,其气溶胶发生与采样方法需进行优化。该菌液体冲击式采样的优选采样介质为PBS+0.01%吐温80+0.005%橄榄油。     

浙江省温州市鼠疫静息期鼠类携带耶尔森菌调查分析

目的了解浙江省温州市鼠疫静息期鼠类耶尔森菌携带情况。方法采用日夹笼法捕鼠,无菌解剖捕获鼠,对盲肠末端内容物进行耶尔森菌分离。结果本次调查共捕获687只鼠,室内鼠占91.27%。共检到耶尔森菌81株,检出率为11.79%,以小肠结肠炎耶尔森菌为主,占79.01%,其次为中间型耶尔森菌、克氏耶尔森菌和假结核耶尔森菌,分别占11.11%、7.40%、2.47%。结论温州市鼠类耶尔森菌携带率较高,室内鼠类能携带各型耶尔森菌,野外鼠类只携带小肠结肠炎耶尔森菌。     

云南省鼠疫耶尔森菌多位点可变数目串联重复序列分析

目的 通过多位点可变数目串联重复序列分析(multiple-locus variable-number tandem-repeat analysis,MLVA)方法,对云南省鼠疫菌株进行基因分型。 方法 采用聚合酶链反应(PCR)和毛细管电泳,通过BioNumerics软件进行处理,分析云南省158株鼠疫耶尔森菌基因型。 结果 选取鼠疫菌的14个VNTR位点,5个VNTR位点对云南省鼠疫菌具有多态性分析意义,利用这5个位点对云南菌株进行分析,云南158株鼠疫菌可分为2个群,3个簇,5个基因型,野鼠鼠疫菌属于A簇,丽江玉龙鼠疫菌属于B簇,家鼠鼠疫菌属于C簇,与传统的生态分型结果吻合。 结论 本次试验筛选的5个位点可以用于云南省鼠疫菌的分型,云南家鼠鼠疫菌、野鼠鼠疫菌及玉龙鼠疫菌分属不同的基因簇,玉龙鼠疫菌与野鼠鼠疫菌同属一个群,聚类结果与实际的地理相关性很好。     

小肠结肠炎耶尔森菌感染性疾病

耶尔森菌属对人类致病的包括三个种：鼠疫耶尔森菌、假结核耶尔森菌、小肠结肠炎耶尔森菌,其中小肠结肠炎耶尔森菌是一种很重要的人类肠道致病菌,世界各大洲均有发现,近年来逐渐受到全球重视.它引起的临床症状十分广泛,从中度腹泻到肠系膜淋巴结炎,对一些免疫功能异常的病人则会出现严重的并发症（败血症）,在某些国家和地区（如芬兰）,小肠结肠炎耶尔森菌是一种引起慢性无菌性关节炎的常见因子.     

内蒙古鼠疫耶尔森菌差异区段分型特征研究

目的 研究分离自内蒙古不同鼠疫疫源地、不同宿主和媒介的鼠疫耶尔森菌的差异区段基因型分布特征。方法 选取内蒙古3个鼠疫疫源地分离的鼠疫耶尔森菌307株,采用差异区段分析法,设计24对引物进行DFR位点扩增分型,用BioNumerics软件分析。结果 通过差异区段分型方法共发现13种基因型,其中5种基因型(Gnm1~Gnm5)为本研究首次报道。长爪沙鼠疫源地有9种基因型,主要基因型为G11(71.8%,122/170);达乌尔黄鼠疫源地有6种基因型,主要为G10(44.1%,41/93)和G11(35.5%,33/93);布氏田鼠疫源地有4种基因型,主要为G14(90.9%,40/44)。3个疫源地均分布有G11型。结论 内蒙古鼠疫耶尔森菌差异区段基因型分布具有明显的地理分布特征,对分析内蒙古鼠疫的传播和遗传演变具有重要的分子流行病学意义。     

青藏高原喜马拉雅旱獭鼠疫疫源地鼠疫耶尔森菌遗传特征分析

目的 研究青藏高原喜马拉雅鼠疫疫源地那曲和比如地区鼠疫耶尔森菌（鼠疫菌）菌株的分子分型特征,确定具有该分型特征的鼠疫菌在青藏高原鼠疫疫源地的分布情况。方法 应用板块重排、差异区段分析、间区规律短回文重复、多位点可变数目串联重复序列分析方法对分离自青藏高原地区、四川省、云南省的98株鼠疫菌进行分析。结果 那曲和比如地区的鼠疫菌菌株中第57~60板块的排列方式与测序菌株Z176003一致,在其他实验菌株中未发现该重排特征。结论 分离自那曲和比如的鼠疫菌菌株具有独特的分子分型特征,该特征在本地区的菌株中普遍存在,且菌株多态性持续分化。     

Modification of cysteine residues in vitro and in vivo affects the immunogenicity and antigenicity of major histocompatibility complex class I-restricted viral determinants.

In studying the subdominant status of two cysteine-containing influenza virus nuclear protein (NP) determinants (NP39-47 and NP218-226) restricted by H-2Kd, we found that the antigenicity of synthetic peptides was enhanced 10-100-fold by treatment with reducing agents, despite the fact that the affinity for Kd was not enhanced. Reducing agents also markedly enhanced the immunogenicity of cysteine-containing peptides, as measured by propagation of long-term T cell lines in vitro. Similar enhancing effects were obtained by substituting cysteine with alanine or serine in the synthetic peptides, demonstrating that sulfhydryl modification of cysteine is responsible for the impaired antigenicity and immunogenicity of NP39-47 and NP218-226. We found similar effects for two widely studied, cysteine-containing peptides from lymphocytic choriomeningitis virus. The major modifications of cysteine-containing synthetic peptides are cysteinylation and dimerization occurring through cysteine residues. We demonstrate that both of these modifications occur in cells synthesizing a cytosolic NP218-226 minigene product and, further, that T cells specific for cysteinylated NP218-226 are induced by influenza virus infection in mice, demonstrating that this modification occurs in vivo. These findings demonstrate that posttranslational modifications affect the immunogenicity and antigenicity of cysteine-containing viral peptides and that this must be considered in studying the status of such peptides in immunodominance hierarchies.     

单纯疱疹病毒2型全长糖蛋白D抗原的原核表达及抗原性分析

目的:构建单纯疱疹病毒2型(HSV-2)全长糖蛋白D(gD)基因原核表达质粒,研究其编码的蛋白质在大肠杆菌中的表达,并鉴定重组蛋白的gD抗原性。方法:提取病毒DNA,PCR扩增出gD基因,克隆于原核表达载体pGEX-4T-1,并转化大肠杆菌BL21。PCR、双酶切及测序证实插入的gD基因序列正确后,IPTG诱导表达融合蛋白GST-gD,并进行免疫学鉴定。结果:获得了gDDNA。测序鉴定表明,该序列与Gen Bank中的序列一致,gD基因已正确插入到pGEX-4T-1中。重组融合蛋白表达载体pGEX-4T-gD经IPTG诱导后能在大肠杆菌中高效表达,Western Blotting证实,该蛋白具有天然gD抗原性。结论:成功构建了融合蛋白表达载体pGEX-4T-gD,在大肠杆菌中获得了有效表达,并证实融合蛋白具有gD免疫原性。为进一步研究gD蛋白的免疫学特性,制备gD亚单位疫苗和单克隆抗体奠定了基础。     

Development of miniaturized competitive immunoassays on a protein chip as a screening tool for drugs

BACKGROUND: Doping in sports has become a serious problem. Gas chromatography-mass spectrometry (GC-MS) serves as an effective reference method, but it is limited by low throughput and is therefore not suitable for large-scale screening. Use of protein chips for high-throughput screening of all athletes for prohibited substances could become an important complementary tool to GC-MS. METHODS: We developed a protein chip based on an aldehyde-activated glass slide containing 10 physically isolated arrays. The chip was used to screen urine from 1347 athletes for prohibited substances and to screen a negative control group consisting of 200 females and 120 males. Urine samples from 66 individuals known to be abusers, provided by the China Doping Control Center (CDCC), and 129 standard prohibited substances were tested as positive controls. RESULTS: All 1347 urine samples screened by means of the protein chips were also subjected to reference analysis by GC-MS at the CDCC. There was no qualitative difference between the results obtained with the two methods. The correlation coefficient (r(2)) for the quantitative results obtained with the protein chip and GC-MS was 0.991. CONCLUSIONS: The protein chip could be used to screen for a series of 16 prohibited drugs in urine samples. This system has the potential to become an effective screening method to test substances prohibited by the International Olympic Committee.     

结核分枝杆菌蛋白芯片检测结果判定和临床意义分析

目的 分析结核分枝杆菌(简称结核杆菌)蛋白芯片法检测结果的判定方法和临床意义.方法 以结核病患者及非结核病患者作为检测对象,评价结核杆菌蛋白芯片法对结核病的诊断性能.结果 不同指标的单独或组合检测具有不同的诊断性能.结论 结核杆菌蛋白芯片法诊断结核病具有较大的阴性预测意义,是临床辅助诊断结核病的参考方法.     

Heat shock protein 70-associated peptides elicit specific cancer immunity

Vaccination of mice with heat shock protein 70 (hsp70) preparations derived from the Meth A sarcoma, but not from normal tissues, renders the mice immune to a substantial challenge with Meth A sarcoma. The immunogenicity is dose dependent and tumor specific. Treatment of an antigenically active hsp70 preparation with ATP followed by removal of low-molecular weight material leaves hsp70 intact, as judged by SDS- PAGE but results in loss of antigenicity, as judged by tumor rejection assays. Separation of this low-molecular weight material on a C18 reverse-phase column shows a diverse array of peptides with molecular mass between 1,000 and 5,000 daltons. Our data indicate that antigenicity of hsp70 preparations derives, not from hsp70 per se, but from associated peptides. These observations may suggest a novel method of using the peptide-binding property of hsp70 for specific vaccination against cancer and infectious diseases.     

B7—PE40新型重组融合外毒素的分子生物学特性预测

为探讨构建的重组外毒素融合蛋白B7-1-Linker-PE40和B7-2-Linker-PE40及其接头设计的合理性,应用核酸和蛋白质序列分析软件系统GOLDKEY,对上述重组外毒素融合蛋白的柔性、抗原性,亲水性,表位和二级结构等分子生物学特性进行了计算机模拟预测。结果发现它们分别保留了B7-1,B7-2和PE40的表位特征,没有出现新的表位,其柔性接头处的抗原性也极低。与B7-1,B7-2和PE40的一、二级结构比较发现。这两种融合蛋白各有数个氨基酸残基改变,有的氨基酸改变可轻微影响融合蛋白的二级结构。原核表达的两个融合蛋白经Western印迹分析显示,它们能分别与B7-1,B7-2和PE40单克隆抗体发生特异性结合。表明它们较好地保留了B7和PE40的抗原性和空间结构。与预测的结果一致。提示预测结果会有助于我们研究该系列融合蛋白的体内,外生物学效应,以及设计构建新的其他融合蛋白。     

Coadsorption of HIV-1 p24 and gp120 proteins to surfactant-free anionic PLA nanoparticles preserves antigenicity and immunogenicity.

Biodegradable micro- or nanoparticles with surface adsorbed antigens represent a promising method for in vivo delivery of vaccines. Most vaccines, licensed or under development, are based on combined delivery of multiple antigens. Thus, we investigated the feasibility of combining two vaccine antigens, HIV-1 p24 and gp120 proteins, on the surface of surfactant-free anionic PLA nanoparticles obtained by an improved solvent diffusion method. The analysis of adsorption isotherms has shown that both proteins had similar and high affinities for the nanoparticles. Coadsorption of p24 and gp120 onto the same PLA particle was evidenced by sandwich ELISA, using antibodies directed against one protein for particle capture and the other one for detection. To assess structural integrity, the antigenicity of free and PLA-adsorbed antigens was compared by competition ELISA, using a set of 6 anti-p24 and 7 anti-gp120 antibodies, as well as soluble CD4. The antigenicity of proteins on the nanoparticle surface was well preserved, adsorbed either individually or in combination. Furthermore, both antigens maintained their immunogenicity, since high antibody titres (10(6) for p24 and 10(5) for gp120) were elicited in mice with monovalent and divalent PLA formulations. Taken together our results show that development of multivalent vaccines based on anionic PLA nanoparticles is possible. Moreover, coadsorption of a ligand for cell-specific targeting or of an immunostimulatory molecule will further extend the field of application of delivery systems based on charged micro- and nanoparticles.     

C-12多种肿瘤标志物蛋白芯片检测系统的临床评价

目的：研究C-12多种肿瘤标志物蛋白芯片检测系统的临床适用性。方法：用C-12多种肿瘤标志物蛋白芯片检测系统与2010电化学发光测定系统对血清标本进行肿瘤标志物对比测定，并比较与临床诊断的符合性。结果：两种方法测定结果符合性良好，与临床诊断相关性较好。结论：C-12多种肿瘤标志物蛋白芯片检测系统适用于健康人群的肿瘤普查，尤其适宜高危人群的肿瘤筛查。     

Rapid selection of aptamers based on protein microarray

We report a novel method for the efficient screening of aptamers from a complex ssDNA library based on a microarray chip, which was named Microarray-SELEX. In this method, the target protein (lactoferrin) and negative proteins (α-lactalbumin, β-lactoglobulin, bovine serum albumin, and casein) were each dotted and immobilized on a slide to form a protein microarray. Moreover, the library was added to this chip to interact with negative proteins, then with the target protein. The process of SELEX could be monitored on-line using a fluorescent microarray scanner and the whole process was performed in only six rounds. Finally, five aptamers (YFL-1, YFL-4, YFL-5, YFL-6 and YFL-7) were obtained, which showed good specificity towards lactoferrin in the presence of negative proteins. The equilibrium dissociation constants (K_d) of the aptamers were in the nanomolar range. Briefly, Microarray-SELEX is a rapid, easy, sensitive and efficient method for screening aptamers.

We report a novel method for the efficient screening of aptamers from a complex ssDNA library based on a microarray chip, which was named Microarray-SELEX.     

ANTIGENIC SPECIFICITIES ON MURINE SARCOMA CELLS : RECIPROCAL RELATIONSHIP BETWEEN NORMAL TRANSPLANTATION ANTIGENS (H-2) AND TUMOR-SPECIFIC IMMUNOGENICITY

Five methylcholanthrene-induced sarcomas were compared for their capacity to (a) absorb monospecific H-2 antisera, (b) induce tumor-specific immunity in syngeneic mice, and (c) metastasize early to the lungs. Comparison of the uptakes of monospecific H-2 antisera by the five different tumors showed that each of the tumors had a high, intermediate, or low surface representation of all of the seven specificities tested. No antigenic specificity was completely absent from any tumor, and no tumor had an unusually large or small amount of any individual specificity. The tumors could be placed in the sequence from one to five with respect to their H-2 antigenicity. The same five tumors were also ranked with respect to their capacity to induce a tumor-specific immune response in syngeneic mice. The tumor-specific immunogenicity had an inverse relationship to the H-2 antigenicity in that highly immunogenic tumors were those that had quantitatively less H-2 antigen on their surface and vice versa. Early metastases to the lung was associated with low levels of tumor-specific immunogenicity and high levels of H-2 antigenicity.