衣原体噬菌体ФCPG1衣壳蛋白Vp1单克隆抗体的研制及临床应用初探

目的 获得衣原体噬菌体ФCPG1的Vp1蛋白,制备抗Vp1的单克隆抗体并检测临床分离的沙眼衣原体标本中是否存在噬菌体。方法 原核表达并纯化衣原体噬菌体ФCPG1的衣壳蛋白Vp1,通过杂交瘤技术获得单克隆抗体杂交瘤分泌株,利用ELISA、Western印迹等方法对单克隆抗体进行鉴定,采用动物体内诱生腹水的方法大量制备单克隆抗体并通过G蛋白亲和层析法纯化。采用免疫荧光法检测临床沙眼衣原体噬菌体。结果 获得纯化的Vp1蛋白和3株稳定分泌单抗的杂交瘤细胞株。杂交瘤细胞染色体分析发现染色体数目平均为96,结构上多数为端着丝点染色体,少数亚中部着丝点染色体。3株细胞分泌的单克隆抗体的免疫球蛋白类别均为IgG1。纯化后单克隆抗体效价可达1 ∶ 102 400。利用得到的单克隆抗体检测20份临床标本,结果未发现衣原体噬菌体。结论 成功获得重组的衣原体噬菌体ФCPG1 Vp1蛋白及抗Vp1蛋白的单克隆抗体,免疫荧光法检测临床沙眼衣原体株中噬菌体未发现阳性标本。     

沙眼衣原体噬菌体衣壳蛋白Vp1血清抗体的检测

衣原体噬菌体作为一种侵袭衣原体的病毒可使衣原体解体,具有抗感染的作用.目前已经发现6种衣原体噬菌体,其上的Vp1蛋白为衣原体噬菌体的衣壳蛋白.是主要的结构蛋白.该蛋白高度保守并可诱导机体产生抗体反应,适用于作为噬菌体的诊断抗原[1].沙眼衣原体是引起非淋球菌性尿道炎的主要病原体,其诊断困难、并发症严重.目前尚未在沙眼衣原体中发现噬菌体的存在.我们以豚鼠结膜炎衣原体(GPIC)噬菌体(PhiCPGI)的莺组Vp1纯化蛋白为抗原,通过ELISA法初步了解Ct感染者血清中针对Vp1蛋白的抗体,并用Western印迹法验证该抗体的特异性,推测Ct感染者血清中Vp1蛋白的存在,为沙眼衣原体与噬菌体的研究提供参考.     

沙眼衣原体感染血清学筛查候选蛋白组合的优化

目的 优化适用于沙眼衣原体感染血清学筛查的候选抗原蛋白组合.方法 收集天津医科大学总医院性病门诊经胶体金法检测沙眼衣原体感染者的血清和泌尿生殖道分泌物拭子各50份,胶体金法检测沙眼衣原体未感染者的血清和泌尿生殖道分泌物拭子各30份,此30份血清微量免疫荧光(MIF)法检测为阴性.以沙眼衣原体蛋白Hsp60和MOMP为参照,选择沙眼衣原体Pgp3、CPAF、CT143、CT101、CT694、CT875、CT813、IncA共8种免疫优势蛋白为抗原,检测血清中的相应抗体,将该结果与血清MIF检测和泌尿生殖道分泌物沙眼衣原体细胞培养两种传统金标准作比较,确定具有最高敏感性和特异性的抗原蛋白组合.结果 50份胶体金法阳性标本中,MIF检测阳性44份,阴性6份;细胞培养阳性14份,阴性36份.沙眼衣原体Pgp3、CT694和CT875这3种免疫优势蛋白组合的血清学实验结果与MIF阳性符合率达到97.73％(43/44).30份胶体金法检测沙眼衣原体阴性标本中,30份血清标本经MIF检测全为阴性,也未检出上述3种蛋白抗体.结论 Pgp3、CT694与CT875免疫优势蛋白组合用于沙眼衣原体感染的血清学初筛具有很好的敏感性和特异性,且操作简单,易于推广.     

衣原体噬菌体phiCPG1衣壳蛋白Vp1对豚鼠结膜炎衣原体及E型沙眼衣原体的抑制作用

目的 探讨豚鼠结膜炎衣原体（GPIC）噬菌体衣壳蛋白Vp1对GPIC及E型沙眼衣原体的抑制作用,为沙眼衣原体感染的治疗提供新的思路。 方法 用Vp1-pET30a（+）重组质粒菌表达Vp1蛋白,Western印迹法鉴定蛋白,透析袋纯化蛋白,BCA法测定蛋白浓度,将GPIC、E型沙眼衣原体分别与Vp1蛋白、Tris甘氨酸溶液、S蛋白及培养液室温孵育3 h,衣原体培养过程中分别 加入相同浓度的上述液体,72 h或48 h后,免疫荧光计数包涵体数。 结果 GPIC在Vp1蛋白组、Tris甘氨酸溶液组、S蛋白组及培养液组培养72 h,包涵体计数分别为5.0 ± 1.5、24 ± 1.2、25 ± 1.7及25 ± 1.5,各组包涵体数比较,差异有统计学意义（F = 476.632,P < 0.05）。Vp1蛋白组GPIC包涵体数显著低于Tris甘氨酸溶液组、S蛋白组及培养液组（P < 0.05）,而后3组之间差异无统计学意义（P > 0.05）。与阴性对照组（培养液组）相比,Vp1蛋白对GPIC的抑制率为（80.2 ± 3.99）%。此外,Vp1蛋白对E型沙眼衣原体的抑制率为（77.2 ± 1.79）%,t检验示Vp1对GPIC的作用与对E型沙眼衣原体的作用差异无统计学意义（t = 2.057,P > 0.05）。 结论 Vp1蛋白可明显抑制GPIC的感染,同时对E型沙眼衣原体有相似的抑制作用。     

四种检测泌尿生殖道沙眼衣原体方法的比较与评价

目的对胶体金免疫层析、酶免疫(EIA)、直接荧光抗体测定(DFA)和细胞培养四种检测泌尿生殖道沙眼衣原体的方法进行比较与评价。方法分别应用胶体金,EIA,DFA和细胞培养四种方法检测不同稀释倍数的沙眼衣原体E型标准株。35例临床疑为泌尿生殖道沙眼衣原体感染患者的宫颈或尿道拭子分别应用胶体金,EIA,DFA进行检测。结果能测出沙眼衣原体E型标准株为阳性的最大稀释倍数在胶体金,EIA,DFA和细胞培养法中分别为:104,106,108和104倍;35例临床标本的阳性率在胶体金,EIA和DFA法中分别为:11.43%,25.71%和40.00%,胶体金和DFA法之间差异有统计学意义(P<0.05),敏感性最高的为DFA法。结论 DFA法检测泌尿生殖道沙眼衣原体的敏感性较胶体金、EIA和细胞培养方法高,临床上可提倡应用。     

全自动酶免疫荧光分析仪检测泌尿生殖道沙眼衣原体感染的评价

近年来，欧美国家广泛采用全自动酶免疫荧光检测系统检测泌尿生殖道标本中的沙眼衣原体，标本采用男性尿液或尿道拭子、女性宫颈分泌物，临床使用方便，结果判断客观犤１－３犦。我们于２００１年４～６月，采用法国bioMérieux公司生产的miniVIDAS全自动酶免疫荧光分析仪作了男性尿液标本和女性宫颈拭子标本中的沙眼衣原体检测（VIDASCHL检测法），并与ClearviewChlamydia试验、衣原体培养及PCR（Roche公司试剂盒）检测进行了比较，现将结果报道如下。一、材料与方法（一）病例来源：１２３例患者均来自２００１年４～６月间我所性…     

生物信息学分析辅助合成、表达噬菌体Chp3 Vp1全基因的研究

目的取得在衣原体感染中具有重要意义且高度保守的衣原体噬菌体Chp3衣壳蛋白Vp1蛋白。方法以生物信息学技术分析设计、合成Chp3Vp1基因,并对Vp1基因进行克隆、表达、提纯。结果获得了经过测序鉴定的Vp1基因,其蛋白序列与Genebank相一致。诱导表达后,SDS-page和Western Blot均显示获得约55kDa处的衣原体噬菌体Chp3衣壳蛋白Vp1。结论顺利获得高度保守而特异的衣原体噬菌体Chp3衣壳蛋白Vp1。     

FQ—PCR与细胞培养检测女性HSV感染者宫颈排泌HSV的对照研究

目的：比较FQ—PCR与细胞培养方法检测无症状生殖器疱疹宫颈排泌HSV的阳性率。方法：用型特异性抗体-2型单纯疱疹病毒糖蛋白G—IgG（HSVgG2-IgG）筛选出阳性女性34例,每例连续2月每月在排卵期和月经前取宫颈拭子,共取材136个。每次取材的拭子分别使用细胞培养鉴定和荧光定量多聚酶反应（Fluorescent quantitation—polymerase chain reaction,FQ—PCR）检测宫颈分泌物中HSV。结果：FQ—PCR方法得到38个拭子阳性,均为HSV-2;细胞培养方法得到4个拭子阳性,经直接免疫荧光分型鉴定为HSV-2。即所有宫颈拭子HSV阳性标本均为HSV-2,无HSV-1。采用Mc-Nemer检验细胞培养与FQ—PCR检测结果阳性率的差异有统计学意义（P〈0．05）,FQ—PCR检测的阳性率比细胞培养法高。结论：对于女性无症状生殖器疱疹,FQ—PCR的方法检测宫颈排泌HSV优于细胞培养方法。     

沙眼衣原体噬菌体的研究进展

沙眼衣原体(chlamydia trachomatis,CT)是引起性传播疾病的主要病原体之一,能感染并裂解衣原体的一类细菌病毒称为衣原体噬菌体。从不同种类的衣原体中分离出的噬菌体共有6种:Chp1,Chp2,Chp3,Chp4,CPAR39和PhiCPG1。虽然至今未能从沙眼衣原体中成功分离出噬菌体,但关于噬菌体衣壳蛋白Vp1,Vp2及Vp3的研究提示沙眼衣原体噬菌体存在的可能性。有关沙眼衣原体噬菌体的研究可为沙眼衣原体感染的诊断和治疗提供重要的临床思路和方法。     

衣原体GPIC噬菌体衣壳蛋白Vp2的克隆、表达和鉴定

目的获得衣原体GPIC噬菌体衣壳蛋白Vp2基因及其蛋白。方法提取噬菌体φCPG1及其核酸,用PCR技术扩增Vp2基因片段,再将其定位插入到原核表达载体pET30a(+)中,构建重组表达质粒。然后将重组表达质粒转化入大肠杆菌(BL-21)中,并用酶切分析、PCR扩增及部分序列测定等方法对重组质粒进行了鉴定。然后诱导表达,用SDS-PAGE及蛋白印迹进行鉴定。结果所获得的Vp2基因片段经测序,长度为561bp,检索确认其序列与Genebank一致。对重组质粒进行诱导表达,SDS-PAGE和蛋白质印迹均显示获得相对分子质量约32kDa的衣原体GPIC噬菌体衣壳蛋白Vp2。结论成功表达了噬菌体衣壳蛋白Vp2,为进一步研究其作用机制和临床应用打下基础。     

Lipophagic panniculitis in re-excision specimens.

Lipophagic panniculitis consists of a macrophage infiltrate in the subcutaneous tissue. The macrophages transform into foam cells within the panniculus; they replace lipocytes and may form giant cells. Although those pathologic features have been described as diagnostic of Weber-Christian disease, we report the occurrence of lipophagic panniculitis in re-excision specimens. Among 252 re-excision specimens from previously biopsied skin tumors, 5 cases in which masses of lipophages were infiltrating and replacing the subcutaneous tissue were found. The infiltrate was localized to the deep dermis and superficial subcutaneous tissue below and beside the initial biopsy site. In 3 cases, suture or hair was detected within the tissue, and granulation tissue with foreign body giant cells was observed along the dermal suture line. In 4 cases there was evidence of phlebitis within or close to areas of infiltration. None of these patients developed symptomatic panniculitis. Lipophagia can be a normal response of wound healing in some patients.     

泌尿生殖道中阴道毛滴虫的检测

目的:探讨适合于检测男、女两性泌尿生殖道中阴道毛滴虫(TV)的取材方式和检验方法,以提高TV的阳性检出率。方法:对211例患者的尿道(阴道)分泌物和尿液采用湿片观察、培养法和湿片+培养法分别进行TV的对照检测。结果:在138例男性患者中泌尿生殖道共检出TV 18例(13.0％),在73例女性患者中泌尿生殖道共检出TV 49例(67.1％)。男性患者尿液和尿道分泌物培养法对TV的检出率差异无显著性(x2=133,P>0.05);女性患者阴道分泌物培养后TV检出率显著高于尿液的TV检出率(x2=19.4,P<0.005)。培养法、湿片+培养法与湿片观察相比,女性阴道分泌物中TV的检出率差异均有显著性(x2=4.17,P<0.05;x2=5.14,P<0.05)。女性尿液培养检测TV的敏感性和特异性最低(48.9％,96.2％)。结论:男性滴虫性尿道炎可采用尿液检测TV,在女性患者检测TV可采用湿片+培养法,该方法可作为性传播性疾病(STD)高危人群筛检TV感染的方法。     

Testing specimens for Chlamydia trachomatis

The introduction of NAATs has revolutionised chlamydial diagnostics and these tests are now the standard of care. However, as with all new technologies, they have also presented new challenges. This review attempts to answer some of the questions that have been raised, particularly by groups about to embark on implementing a screening programme. Laboratory tests are continually changing but it is hoped that the paper provides a useful update of the current situation.     

男性不育症患者精液标本中支原体检测与抗生素的耐药性分析

目的：分析男性不育症患者精液标本中支原体检测情况及其对抗生素的耐药性。方法：选取2001年1月－2012年6月的520例男性不孕症患者为研究对象,将所有患者精液标本中支原体感染情况进行检测,并将其耐药性进行统计分析及比较。结果：520例患者中支原体感染76例,感染率为14．62％,其中单纯解脲支原体比例最高,其次为解脲支原体与人型支原体混合感染和单纯人型支原体感染,且不同类型的耐药情况存在明显差异,P均〈0．05,均有显著性差异。结论：男性不育症患者精液标本中支原体检出率较高,且不同分型的对抗生素的耐药性也有差异。     

Methods of labeling skin surgical specimens

Background: Accurate pre‐operative or intra‐operative labeling of the skin is often necessary to mark exactly the surgical excision lines. Pre‐operative “unsterile” permanent skin labeling systems are needed for example for vein and sentinel lymph node surgery; here the dyes must resist two surgical skin disinfection procedures. In contrast, excision borders are labeled during surgery using a “sterile” skin marking system. Methods: Many commercial and non‐commercial pre‐ and intra‐operative skin‐labeling systems are available, such as autologous patient blood, fluorescence triphenylmethane dyes and commercial skin markers. The available skin marking systems have specific advantages and disadvantages. We review the different labeling systems, offering guidelines to help choose a cost‐effective system appropriate for a given surgical procedure. Results: The Edding® permanent markers 400 und 3000 are well suited for preoperative skin labeling and less expensive than commercial skin labeling systems. Autologous patient blood and eosin are well suited for intra‐operative labeling and are most cost effective. Eosin Y is widely used and well suited for labeling of dark skin, bone, cartilage, and muscle tissue and spares the expense of expensive commercial skin markers. Conclusion: Knowledge of the many commercial and non‐commercial pre‐ and intra‐operative skin labeling systems and their advantages and disadvantages helps to reduce the use of relatively expensive commercial skin markers.     

Genotyping of Chlamydia trachomatis from endocervical specimens in Brazil

BACKGROUND: There is no data concerning genotyping of Chlamydia trachomatis from Brazilian samples. GOAL: To characterize the genotype of C. trachomatis detected in women assisted at a STD public clinic and establish the prevalence of this infection in that population. STUDY DESIGN: Endocervical samples of a group of 100 women were tested for chlamydial infection with PCR directed to C. trachomatis cryptic plasmid. Genotyping of positive samples were done after omp1 amplification and sequencing. RESULTS: The overall prevalence of C. trachomatis infection was 19%, with the highest prevalence in women between 15 and 25 years old (68.4%). Four genotypes were found associated with endocervical infections: D, E, F, and K. Sequence analysis revealed a coinfection of genotypes D and E in 1 woman. CONCLUSIONS: To our knowledge this is the first study to characterize Brazilian C. trachomatis endocervical samples and Brazilian C. trachomatis genotype coinfection. Our results also emphasize the importance of routine diagnosis of C. trachomatis for the control of this STD.