应用羟基磷灰石层析分离纯化鼠疫菌Pla蛋白的研究

目的应用CHT陶瓷羟基磷灰石层析柱分离纯化鼠疫耶尔森氏菌纤溶酶原激活因子（Pla）。方法优化层析条件,对采用超声破碎结合硫酸铵盐析得到的Pla粗提样品进行纯化,收集纯化中各洗脱峰样品,进行SDS-PAGE。结果上样缓冲液中添加钙离子,可促进Pla与层析柱的结合,经纯化后的Pla,去除了大部分杂蛋白,得到主要由相对分子质量约为31×103、35×103、37×103 Da和3条蛋白带为主的Pla,经软件分析计算,占蛋白总量约80%。结论 CHT陶瓷羟基磷灰石层析能实现对Pla的基本纯化。     

高效液相色谱分离纯化鼠疫菌Pla蛋白

目的用高效液相色谱分离纯化鼠疫菌纤溶酶原激活因子。方法优化离子交换、凝胶过滤,并将2种层析组合实现纯化。结果用高效液相色谱纯化的Pla主要由相对分子质量约31×10^3、35×10^3、37×10^3的3条蛋白带组成,占蛋白总量80％以上。结论Pla经高效液相色谱分离可以达到基本纯化。     

鼠疫耶尔森氏菌Pla蛋白的分离纯化与质谱分析

目的建立一种从鼠疫耶尔森氏菌疫苗株EV76中分离纯化纤维蛋白酶原激活因子（Pla）的方法。方法采用超声破碎与硫酸铵盐析结合的方法初步提取Pla,经CHT陶瓷羟基磷灰石层析分离纯化,所获取的Pla进行质谱分析及Western blot实验。结果菌悬液超声破碎后上清以0~10%饱和硫酸铵沉淀,获得含有相对分子质量（Mr）约为31 000、35 000、37 000的3条Pla蛋白带,约占蛋白总量33%;CHT柱层析后,3条带约占蛋白总量80%。经质谱分析,在Mr约为31 000、35 000的条带中含有Pla,Pla单克隆抗体能特异性地识别该蛋白。结论采用超声破碎结合硫酸铵盐析的方法可从鼠疫菌中提取Pla,CHT柱层析可达到基本纯化,获得的样品经质谱分析和Western blot实验证实为Pla蛋白。     

鼠疫耶尔森菌纤维蛋白溶解酶原激活因子蛋白及其特异性片段的原核表达

目的 重组表达鼠疫耶尔森菌纤维蛋白溶解酶原激活因子(Plasminogen activator,Pla)蛋白及其特异性片段并检测其免疫反应性.方法 以鼠疫EV76株为模板,PCR扩增pla、pla-c基因,并与质粒原核表达载体(prokaryotic expression vector,pET)32a(+)连接,将pET32a(+)-pla、pET32a(+)-pla-c分别转化E.coli BL21(DE3)诱导表达;产物经镍离子金属螯合亲和层析纯化,免疫印迹(Western blot)法鉴定其免疫反应性.结果 所表达的Pla蛋白相对分子质量(Mr)为52.8×103,以包涵体形式存在;Pla-c蛋白Mr为24.0×103,以可溶形式存在;这两个蛋白质均可与鼠疫免疫血清产生特异结合反应.结论 所表达的鼠疫菌Pla及其特异性片段具有免疫反应性,为辅助诊断鼠疫提供了选择.     

冠心病患者血浆纤维蛋白溶解系统活性测定及与脂蛋白(a)水平的关系

为探讨冠心病患者血浆纤维蛋白溶解酶（简称纤溶酶）活性及与脂蛋白（a）水平的关系，对37例冠心病患者血浆组织型纤溶酶原激活刘、纤溶酶原激活剂抑制物、纤溶酶原和血清脂蛋白（a）水平进行了测定，并与13名健康人对照。结果发现．冠心病患者血浆组织型纤溶酶原激活剂活性低于健康人（P＜0．05）。纤溶酶原激活剂抑制物活性显著高于健康人（P＜0.001），纤溶酶原活性低于健康人（P＜0．05）．血清脂蛋白（a）水平除急性心肌梗塞患者高于健康人（P＜0．05）外，其他患者与健康人相比．差异无显著性。结果还发现：急性心肌梗塞患者血浆组织型纤溶酶原激活刻的活性与血清脂蛋白（a）水平呈负相关（r=-0.898．P＜0．001）。以上结果提示，测量血浆纤溶系统活性和脂蛋白（a）水平，对于冠心病的诊治具有参考价值。     

改良溶解圈纤溶酶原激活物测定法及其在革兰氏阴性菌中的应用

本研究建立的测定纤溶酶原激活物（Ｐｌａ）的纤维蛋白溶解圈法，不仅简单易行，而且具有很高的敏感性和特异性。检测灵敏度较原来的普通溶解圈法提高了大约５００倍，能检出２．５×１０－５ＩＵ的尿激酶（ＵＫ）活力。本方法特别适用于低Ｐｌａ含重的定性定量检测，如革兰氏阴性菌纤溶酶原激活物的测定。应用此方法，本研究首次证明了伤寒和鼠伤寒杆菌具有纤溶酶原激活物的活性，１０Ｄ鼠伤寒菌的Ｐｌａ活性相当于４×１０－２ＩＵＵＫ；１ＯＤ伤寒菌的Ｐｌａ活性相当于５×１０－３ＩＵＵＫ。     

从表达菌培养液中提取鼠疫菌重组F1抗原

目的从表达鼠疫耶尔森氏菌F1蛋白的诱导培养液中提取纯化重组F1并检测其免疫反应性。方法收集诱导培养后离心去除菌体的培养液,采用硫酸铵沉淀法和PEG浓缩法提取F1蛋白,产物经镍离子金属螯合亲和层析纯化,免疫印迹(Western blot)鉴定其免疫反应性。结果硫酸铵沉淀法和PEG浓缩法均能从培养液中提取到重组F1蛋白,产物经纯化后可与鼠疫疫苗株免疫兔血清和既往鼠疫患者血清发生特异性结合反应。结论该表达菌表达效能较高,在诱导培养液中存在大量具有免疫学活性的重组F1蛋白。     

胰岛素对血管平滑肌细胞增殖和组织型纤溶酶原激活剂及抑制物活性的影响

以体外培养的猪主动脉平滑肌细胞为实验对象，研究了不同浓度胰岛素对细胞增殖和组织型纤溶酶原激活剂及抑制物活性的影响。细胞增殖检测采用氚标脱氧胸腺嘧啶核苷掺入法，组织型纤溶酶原激活剂及其抑制物活性测定采用底物显色法。结果发现胰岛素促进血管平滑肌细胞增殖，刺激纤溶酶原激活剂抑制物活性增加，均呈剂量依赖性。而组织型纤溶酶原激活剂活性在猪血管平滑肌细胞体外培养液中不存在，且与胰岛素的刺激与否及作用浓度、时间均无关。此结果提示高胰岛素血症可能是致动脉粥样硬化主要危险因素之一。     

反义纤溶酶原激活物抑制剂1 RNA对家兔动脉粥样硬化形成的影响

为探讨纤溶酶原激活物抑制剂1反义RNA对家兔血浆纤溶活性及纤溶酶原激活物抑制剂1的表达、血脂及对动脉粥样硬化斑块形成的影响,通过聚合酶链反应扩增纤溶酶原激活物抑制剂1第二外显子,将聚合酶链反应产物纯化克隆后连入真核细胞表达载体pcDNA3.1,构建纤溶酶原激活物抑制剂1 反义RNA重组质粒.将pcDNA3.1-反义纤溶酶原激活物抑制剂1重组质粒注射到哈尔滨大白兔腹部皮下组织.通过发色底物法测定家兔血浆组织型纤溶酶原激活物及纤溶酶原激活物抑制剂1活性变化,通过免疫组织化学方法检测组织中纤溶酶原激活物抑制剂1表达的改变.测定家兔血脂变化,病理检测其动脉粥样硬化程度.结果显示,应用反义纤溶酶原激活物抑制剂1 RNA重组质粒的家兔血浆纤溶酶原激活物抑制剂1活性降低,组织型纤溶酶原激活物活性升高, 纤溶酶原激活物抑制剂1蛋白表达于内皮细胞,而在平滑肌细胞中未表达 (动脉粥样硬化对照组中内皮细胞、平滑肌细胞和泡沫细胞内均有表达);应用反义纤溶酶原激活物抑制剂1 RNA重组质粒的家兔胆固醇和甘油三酯明显低于动脉粥样硬化对照组(96±42 mg/L比123±12 mg/L, 15±10 mg/L比46±29 mg/L),且动脉粥样硬化程度亦轻于后者.以上提示,反义纤溶酶原激活物抑制剂1 RNA重组质粒的皮下注射能有效阻断家兔体内纤溶酶原激活物抑制剂1蛋白的合成,减轻动脉粥样硬化程度.     

纤溶活性与2型糖尿病及其大血管病变

组织型纤溶酶原激活剂(t-PA)和纤溶酶原激活物抑制剂-1(PAI-1)是纤溶系统的主要调控因子.2型糖尿病患者高血糖、高胰岛素血症、脂代谢紊乱、炎症反应、肥胖等多种因素可引起血浆t-PA水平降低、PAI-1水平升高.纤溶活性的降低参与了大血管病变的发生、发展.提高纤溶活性的综合治疗将对防治糖尿病大血管并发症起一定作用.     

Tuberculin peptide from culture filtrate of Mycobacterium tuberculosis

A highly purified tuberculin peptide, named TPH71U, was isolated from the heated culture filtrate of Mycobacterium tuberculosis strain H37Rv. This peptide was prepared by precipitation with ammonium sulfate and by treatment with diethylaminoethyl-Sephadex, Sephadex G-75, and preparative gel electrophoresis in the presence of a high concentration of urea. The presence of urea remarkably increased the sensitivity of the gel analysis and the efficiency of the preparation procedure. The isolated peptide, TPH71U, was homogeneous in the gel electrophoretic analysis, and the molecular weight was estimated to be 5,800 daltons. The amino acid composition was analyzed. The peptide elicited strong tuberculin cantaneous reactions in guinea pigs sensitized with Mycobacterium tuberculosis strains H37Rv and Aoyama B, and Bacille Calmette Guèrin, and weak reactions in those sensitized with Mycobacterium intracellulare, Mycobacterium kansasii, and Mycobacterium phlei. Based on the cutaneous reactions to this peptide, its potency was estimated to be approximately one twentieth of that of the standard tuberculin purified protein derivative.     

日本血吸虫腺苷酸激酶基因的表达及其重组蛋白免疫反应性的评价

目的　将亚克隆入 pET3 2a( +)质粒的日本血吸虫腺苷酸激酶 (adenylatekinase ,AK )基因进行表达及蛋白纯化 ,并对表达产物的免疫反应性进行评价。　方法　将重组表达质粒 pET3 2a( +) AK转入宿主菌大肠埃希菌BL2 1,异丙基 β D 硫代半乳糖苷 (IPTG)诱导表达后超声裂菌 ,离心 ,取上清进行十二烷基硫酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE) ;将SDS PAGE后的蛋白转移至聚偏二氟乙烯 (PVDF)膜上 ,分别用 6 组氨酸 ( 6 His)抗体、日本血吸虫尾蚴感染6wk的兔血清及紫外照射减毒尾蚴免疫的兔血清为一抗进行蛋白质印迹试验 (Westernblotting) ;用金属Ni螯合物亲和层析树脂 (Ni NTA)层析柱纯化目标蛋白 ;以纯化后的融合蛋白为包被抗原 ,酶联免疫吸附测定 (ELISA)检测正常兔血清和血吸虫感染兔血清。　结果　重组菌用IPTG诱导表达后进行SDS PAGE ,于相对分子质量 (Mr) 40 0 0 0处见一特异表达带 ,与预期表达的融合蛋白大小相符 ;Western印迹分析显示该重组的融合蛋白带有预期的 6 His基团 ,且能与尾蚴感染兔血清及紫外照射减毒尾蚴免疫的兔血清发生特异性反应 ;ELISA结果显示纯化的重组AK蛋白可以特异识别血吸虫感染兔血清。　结论　AK基因在工程菌中以可溶性融合蛋白的形式得到表达 ,该重组蛋白与血吸虫感染兔血清及紫外照射减毒尾蚴免疫的兔血清具有特异的免疫反应性。     

Immunostimulatory mouse granuloma protein.

Earlier studies have shown that from subcutaneous talc-induced granuloma in mice, a fraction could be extracted that fully protected mice against Listeria monocytogenes. Using standard biochemical procedures--i.e., ammonium sulfate fractionation, preparative electrophoresis, gel filtration chromatography, isoelectric focusing, and preparative polyacrylamide gel electrophoresis--we have now purified an active factor to homogeneity. A single band was obtained in NaDodSO4/polyacrylamide gel with an apparent Mr of 55,000. It migrated with alpha 1-globulins and the isoelectric point was 5 +/- 0.1. The biological activity was destroyed with Pronase but not with trypsin and a monospecific polyclonal rabbit antiserum was obtained. The intravenous injection of 5 micrograms of this "mouse granuloma protein" fully protects mice against a lethal inoculum of L. monocytogenes. Moreover, after their incubation with 10 nM mouse granuloma protein, mouse peritoneal cells became cytostatic against Lewis carcinoma cells.     

Evidence for monomeric and oligomeric hormone-binding domains in affinity-purified gonadotropin receptor from rat ovary

Rat ovarian lutropin/choriogonadotropin receptor was purified from a Triton X-100-solubilized membrane preparation by affinity chromatography with Affi-Gel 10 coupled to purified human choriogonadotropin. The affinity-purified receptor preparations contained a single class of high-affinity binding sites for 125I-labeled human choriogonadotropin, with an equilibrium dissociation constant (Kd) of 2.5 x 10(-9) M, which is comparable to the Kd values for membrane-bound and solubilized receptors. The purified receptor appeared as two dominant bands with molecular weights of 135,000 and 92,000 after sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) under nonreducing conditions. These two bands were also detected in subsequent direct ligand blotting analysis when the purified receptor was electrophoretically transferred to a nitrocellulose membrane after SDS/PAGE under nonreducing conditions. When the individual affinity-purified receptor bands were electroeluted from the gel and analyzed again by SDS/PAGE under nonreducing conditions, both the Mr 92,000 and the 135,000 proteins retained their original molecular form even when 8 M urea was included in the gel. However, when the electrophoretically purified Mr 92,000 and 135,000 bands were subjected to SDS/PAGE under reducing conditions, the Mr 135,000 species was almost completely converted to a Mr 92,000 band, but the Mr 92,000 species did not undergo any alteration in molecular weight. The results suggest that the lutropin/choriogonadotropin receptor from rat ovary exists in two molecular forms, and the higher molecular weight form appears to be composed of disulfide-linked Mr 92,000 subunit, which comprises the hormone-binding domain.     

Absence of plague in certain mammals from Java and Kalimantan (Borneo).

Antibodies against plague were lacking in 237 wild mammal sera from Java and 103 from Kalimantan. Wild mammal spleens, 114 from Java and 18 from Kalimantan were negative for plague bacilli. A variety of mammalian species and areas was examined.     

双重式聚合酶链反应检测印鼠客蚤鼠疫菌的观察

为建立蚤类感染鼠疫的快速诊断方法，应用双重式聚合酶链反应（PL－FI－PCR）技术，对感染鼠疫菌的40只印鼠客蚤进行检验，同时单体拉胃培养作比较。结果显示细菌培养阳性22只，双重式聚合酶链反应阳性40只，阳性率后明显高于前，且特异性扩增带比较清晰、典型、稳定。提示双重式聚合酶链反应适用于蚤类等微小标本染带鼠疫菌的检测。     

青藏铁路沿线鼠疫菌质粒的研究

目的 检测青藏铁路沿线鼠疫菌携带的质粒种类及相对分子质量.方法 采用碱裂解,酚一氯仿抽提法提取鼠疫菌质粒,经琼脂糖凝胶电泳进行检测并分析质粒的相对分子质量.结果 所检测的18株鼠疫菌具有6×106,45×106,52×106,65×106,92×106质粒,其中大质粒的变化范围在52×106～92×106.结论 青藏铁路沿线鼠疫菌除具有规范的质粒图谱外,鼠疫菌最大质粒的变化有一定的规律性,具有分类属性,对研究鼠疫自然疫源地空间结构和鼠疫菌的遗传学特性具有重要意义.