幽门螺杆菌Lpp20真核表达重组载体的构建及鉴定

目的以幽门螺杆菌外膜脂蛋白Lpp20为目的基因,构建H.pylori Lpp20基因的真核表达重组载体pcD-NA3.1( )-Lpp20,为H.pylori核酸疫苗的研制奠定实验基础。方法用Pri mer5.0软件分析GenBank中H.pylori外膜脂蛋白Lpp20基因序列,设计合成相应特异性引物,引入BamHⅠ和XhoⅠ酶切位点,以H.pylori26695株基因组为模板,PCR扩增Lpp20目的基因片段,定向插入真核表达载体pcDNA3.1( )多克隆酶切位点中,构建重组体pcDNA3.1( )-Lpp20;通过酶切分析,PCR鉴定及测序鉴定,筛选阳性重组体。结果以H.pylori26695株基因组DNA为模板扩增出特异的Lpp20基因片段,大小约为528bp;双酶切及测序鉴定证明成功构建了H.pylori真核表达重组载体pcD-NA3.1( )-Lpp20。结论PCR扩增得到了大小约为528bp的H.pylori Lpp20目的基因片段;并成功构建了pcD-NA3.1( )-Lpp20真核表达重组载体。     

运动对慢性疲劳综合症小鼠NK细胞活性和IL-2水平的影响

目的建立慢性疲劳综合症（CFS）小鼠模型，研究运动对C聆小鼠NK细胞活性和IL-2水平的影响。方法选取40只小鼠随机分为4组，每组10只。I组为正常对照组，Ⅱ组为CFS对照组，Ⅲ组为CFS运动1组，Ⅳ组为CFS运动2组。用力竭性游泳试验建立CFS小鼠模型。不同强度运动后，计算脾脏指数，MTT法和ELlSA分别检测小鼠脾脏中NK细胞活性与IL-2含量。结果脾脏指数试验组明显低于CFs对照组（P〈0．05）；每天运动两次可明显升高CFS小鼠NK细胞活性和增加脾脏IL-2诱生量，与CFS对照组比较，差异有显著性（P〈0．05），且随运动量增加可增强NK细胞活性、提高IL-2水平。结论适当运动能提高CFS小鼠的NK细胞活性和IL-2含量。     

多聚酶链反应诊断生殖支原体感染的研究

生殖支原体（Ｍｇ）与急、慢性非淋菌性尿道炎（ＮＧＵ）、慢性前列腺炎、宫颈炎或盆腔炎、不育症等疾病［１］有关。Ｍｇ的生长极其缓慢,初代生长时间长,需要的营养成分复杂,从临床标本中分离培养Ｍｇ难度大,阳性率很低。血清学诊断因与肺炎支原体（Ｍｐ）有交叉反应而特异性差。为评价聚合酶链反应（ＰＣＲ）方法检测Ｍｇ的可靠性,了解Ｍｇ在衡阳地区高危人群中的感染状况,我们在ＭｇＰａ基因上选择了３对不同的引物,用ＰＣＲ方法对泌尿生殖道感染患者的标本进行了Ｍｇ检测,现将结果报告如下。材料和方法一、标准菌株生殖支原体（…     

六种抗菌药物体外对解脲支原体与人型支原体的抗菌作用

采用琼脂对倍稀释方法，测定了６种国产抗菌药物对３０株解脲支原体（Ｕｕ）、３０株人型支原体（Ｍｈ）体外抗菌作用效果。结果显示：乙酰螺旋霉素、氟哌酸和强力霉素有较强的抗Ｕｕ与Ｍｈ作用。乙酰螺旋霉素抗 Ｕｕ的 ＭＩＣ５０与 ＭＩＣ９０为０． ２５ｍｇ／Ｌ与１ｍｇ／Ｌ，抗Ｍｈ为０． ５ｍｇ／Ｌ与１ｍｇ／Ｌ。氟哌酸抗 Ｕｕ与Ｍｈ作用相同，ＭＩＣ５０与ＭＩＣ９０为０．５ｍｇ／Ｌ与１ｍｇ／Ｌ。强力霉素抗Ｕｕ的ＭＩＣ５０与ＭＩＣ９０为０．５ｍｇ／Ｌ与１ｍｇ／Ｌ，抗Ｍｈ为０．２５ｍｇ／Ｌ与２ｍｇ／Ｌ。Ｍｈ对红霉素有高度抵抗作用，ＭＩＣ５０与ＭＩＣ９０为６４ｍｇ／Ｌ与１２８ｍｇ／Ｌ。６株 Ｕｕ、４株Ｍｈ对四环素高度耐药，ＭＩＣ５０与 ＭＩＣ９０分别为６４ｍｇ／Ｌ与１２８ｍｇ／Ｌ和１６ｍｇ／Ｌ与３２ｍｇ／Ｌ，但对其它抗菌药物敏感。小檗硷抗Ｕｕ与Ｍｈ作用相同，ＭＩＣ５０与ＭＩＣ９０为４ｍｇ／Ｌ与８ｍｇ／Ｌ，对耐四环素的Ｕｕ与Ｍｈ也有相同的抗菌作用。     

中国南方部分城市泌尿生殖道沙眼衣原体基因分型研究

目的 了解中国南方4个城市性病高危人群中沙眼衣原体(Ct)基因型的分布特征。方法 从衡阳、上海、广州、江门四个城市的性病门诊收集1180份泌尿生殖道标本,经质粒PCR筛选后,阳性者用巢式PCR扩增Ctomp1基因片段,酶切omp1扩增产物,分析Ct各临床株omp1基因的限制性片段长度多态性(RFLP),将临床株分型。结果 Ct质粒PCR阳性者301份,用于分型者286份。通过RFLP分型,共检出10个基因型,其中常见的是E(30.8%)、J(23.8%)、D(17.5%)、F(10.5%)、G(6.2%)型,并且不同城市间基因型的分布差异无统计学意义(χ²=0.981,P>0.05)。结论 4个城市存在Ct多种基因型,并且基因型的分布大致相同。     

肺炎支原体P1蛋白片段免疫学活性及黏附功能的研究

目的 了解肺炎支原体(Mycoplasma pneumoniae,Mp)P1蛋白第1125 ～1395氨基酸片段(P1C蛋白)的免疫学活性及其细胞黏附作用.方法 构建用于表达重组P1C片段(rP1C)的原核表达载体pGEX6p-2/p1c,采用SDS-PAGE和Western blot鉴定rP1C.采用基于GST的亲和层析法提纯rP1C,提纯的rP1C免疫BALB/c小鼠,ELISA检测小鼠抗rP1C血清的效价.采用Western blot检测rP1C对Mp感染患者血清的免疫反应性.采用间接免疫荧光法检测rP1C黏附HeLa细胞及其免疫血清黏附抑制作用.结果 所构建的原核表达系统能有效表达相对分子质量约为66×103的可溶性rP1C.rP1C免疫小鼠后,其抗血清ELISA效价高达1∶64000.rP1C能被Mp感染者血清及小鼠抗rP1C血清识别并与之结合.rP1C能黏附HeLa细胞,其抗血清可阻断Mp对HeLa细胞的黏附,该黏附阻断作用随抗血清浓度增高而增强.结论 rP1C具有良好的免疫原性和免疫反应性及黏附细胞功能,可作为Mp疫苗及血清学检测的候选抗原.     

肺炎嗜衣原体CPAF重组蛋白致小鼠肺组织炎症及对炎症细胞因子的影响

目的:研究肺炎嗜衣原体(Chlamydophila pneumoniae,Cpn)蛋白酶样活性因子(CPAF)的181~400aa基因(CPAFm)的重组蛋白在BALB/c小鼠体内引起的肺部炎症改变及对炎症细胞因子TNF-α和IL-6的水平的影响,为进一步探索CPAF在Cpn感染中的致病作用提供实验依据。方法:将纯化的CPAFm重组蛋白于鼻内或尾静脉注射BALB/c小鼠,HE染色观察肺组织病理形态学改变,计小鼠外周血与支气管肺泡灌洗液(Bronchoalveolar lavage fluids,BALF)中白细胞总数,ELISA方法检测血清和BALF中的TNFα-和IL-6的水平。结果:实验组BALB/c小鼠的肺组织出现中性粒细胞、淋巴细胞等炎症细胞浸润,对照组与正常组BALB/c小鼠肺组织未见明显病理改变。鼻内滴入重组蛋白实验组BALB/c小鼠BALF中白细胞总数明显高于GST对照组(P<0.05)、PBS对照组(P<0.05)和正常组(P<0.01);鼻内滴入和尾静脉注射重组蛋白实验组BALF中炎症因子IL-6、TNF-α的水平明显高于GST、PBS对照组与正常组(均P<0.01);鼻内滴入重组蛋白实验组外周血中炎症因子IL-6和TNF-α水平显著高于GST、PBS对照组和正常组(P<0.05);尾静脉注射重组蛋白实验组外周血中炎症因子IL-6的水平明显高于相应的对照组(均P<0.01)。结论:CPAFm重组蛋白有致病性,能引起BALB/c小鼠肺组织的炎症损伤和炎症因子TNF-α、IL-6的水平的升高,肺损伤与BALB/c小鼠体内炎症细胞增多和炎症因子TNF-α、IL-6的水平的升高相关。     

黏膜佐剂大肠埃希菌不耐热肠毒素B亚单位优化的淋病奈瑟菌孔洞蛋白B核酸疫苗的构建及其诱导小鼠的免疫应答

目的 分析黏膜佐剂大肠埃希菌不耐热肠毒素B亚单位(LTB)辅佐的淋病奈瑟菌孔洞蛋白B(PorB)核酸疫苗诱导小鼠的免疫应答水平.方法 PCR法扩增目的 基因porB、ltB、ltB-porB,分别构建3种相应的peDNA3.1(-)真核重组表达载体,经PCR、双酶切及基因测序鉴定后转染Hela细胞,用细胞免疫荧光法鉴定质粒的蛋白表达.将核酸疫苗经鼻饲免疫雌性BALB/c小鼠,检测体液免疫及细胞免疫应答水平,同时用免疫组织化学法检测porB、ltB、ltB-porB基因在小鼠鼻黏膜内的表达.组间均数比较采用方差分析.结果 构建的真核重组质粒均能在Hela细胞内及小鼠鼻黏膜组织内表达.核酸疫苗免疫组小鼠的生殖道灌洗液PorB特异性sIgA及血清porB特异性IgG水平明显高于对照组(P＜0.01;P＜0.05),且ltB-porB融合基因组特异性sIgA明显高于porB组(P＜0.05);pcDNA3.1(-)/porB组小鼠脾淋巴细胞培养上清液中IFN-γ、IL-4和脾淋巴细胞刺激指数(SI)分别为(170.04±23.89)pg/mL、(114.68±14.27)pg/mL和1.68±0.19,pcDNA3.1(-)/ltB-porB组分别为(161.42±27.50)pg/mL、(124.16±19.04)pg/mL和1.73±0.28,均高于对照组pcDNA3.1(-)和PBS,差异均有统计学意义(P＜0.01;P＜0.05),高于对照组pcDNA3.1(-)/ltB,差异均有统计学意义(均P＜0.05),但两核酸疫苗免疫组间差异无统计学意义(均P＞0.05).结论 所构建的核酸疫苗分别在Hela细胞及小鼠鼻黏膜组织内获得了表达,经黏膜途径免疫能诱导小鼠产生较高水平的特异性体液免疫和细胞免疫应答,尤其是黏膜免疫应答;证实黏膜佐剂LTB可辅佐PorB诱导小鼠产生高水平的生殖道黏膜免疫.

Abstract：

Objective To investigate the specific humoral immune response and cellular immune response induced by DNA vaccine with Neisseria gonorrhoeae porin B (PorB) fused with B subunit of Escherichia coli heat-labile enterotoxin B (LTB) in mice. Methods Target genes of porB, ltB and ltB-porB were amplified by polymerase chain reaction (PCR) and cloned into eukaryotic vector pcDNA3.1(-). The recombinants were identified by PCR, enzyme digestion and DNA sequencing.The vectors were transfected into Hela cells, and expressed proteins were checked by cytoimmunofluorescence. Female BALB/c mice were intranasally immunized with recombination vectors. The humoral immune response and cellular immune response were detected by enzyme linked immunosorbent assay (ELISA) and methyl thiazolyl tetrazolium (MTT) colorimetric assay. The expressions of recombination vectors in intranasal mucosal tissues of the immunized mice were detected by immunohistochemistry. The means between groups were compared by analysis of variance. Results All the three recombinants were expressed in Hela cells and intranasal mucosal tissues. The PorB specific IgG in serum and sIgA in vaginal secretions in DNA vaccine immunized mice were significantly higher than those in controls (P＜0.01 ; P＜0.05). Moreover, the sIgA level in pcDNA3.1 (-)/ltB-porB group was higher than that in peDNA3, 1(-)/porB group (P＝0. 002). The levels of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) in the supernatants and stimulation index (SI) of spleen lymphocyte culture in pcDNA3, 1(-)/porB group were (170.04±23.89) pg/mL, (114.68±14.27) pg/mL and 1. 68±0.19, respectively; and those in pcDNA3, 1(-)/ltB-porB group were (161.42±27.50) pg/mL, (124.16±19.04) pg/mL and 1.73±0.28, respectively; which were both higher than those in pcDNA3.1(-)/ phosphate buffered saliae (PBS) group (P＜0. 01; P＜0.05) and pcDNA3.1 (-)/ltB group (all P＜0.05), while there was no significant difference between pcDNA3.1 (-)/ltB-porB group and pcDNA3. 1 (-)/porB group (0. 998, 0. 696, 0. 994; all P＞0.05). Conclusions The constructed DNA vaccines are all successfully expressed in Hela cells and murine intranasal mucosal tissues. The mucosal immunization of the vaccines [pcDNA3. 1 (- )/porB and pcDNA3.1 ( -)/ltBporB] could induce humoral immune response and cellular immune response, especially mucosal immune response. It is confirmed that mucosal adjuvant LTB could promote PorB to induce higher level of mucosal immune response in mice.     

改革医学微生物学实验教学 培养创新型医学人才

医学微生物学实验准备工作质量的高低与实验教学效果有密切的关系。本文对实验技术人员自身素质、实验准备计划的制定、实验预做、实验技术人员和实验带教教师的关系、实验方法改革、实验准备模式等方面进行了改革和探索,为提高实验教学质量,培养医学生创新能力提供参考和借鉴。