4株抗旋毛虫单克隆抗体细胞系的建立与初步特性鉴定

为了筛选旋毛虫保护性抗原,建立了4株分泌抗旋毛虫单克隆抗体的细胞株。其中2G_3和2C_(10)的靶抗原为旋毛虫幼虫表膜。免疫球蛋白类型及亚类鉴定表明2G_3和2C_(10)均为IgG_1,1D_5为IgG_3,1C_9为IgM。除了2C_(10)和IC_9对伊氏锥虫可溶性抗原呈现阳性反应外,未见对猪圆线虫、猪囊尾蚴、伊氏锥虫表膜抗原和正常猪肉反应、4株细胞连续培养已达30余代,仍     

人巨细胞病毒感染对小鼠脑组织同源框基因表达的影响

目的　研究小鼠脑组织同源框基因 (homeoboxgene ,Hox)的表达状态及人巨细胞病毒(humancytomegalovirus ,HCMV)感染对小鼠脑组织Hox基因表达的影响 ,以探讨HCMV致畸的分子机制。方法　昆明系小鼠 4 8只 ,随机分为 :实验对照组 (16只 )和病毒感染组 (32只 ) ,病毒感染为脑内注射。在感染后 7、15、30、6 0d分别以病理学方法观察病理损伤 ,以免疫组化方法检查HCMV LA(latean tigen) ,PCR检测小鼠脑组织中HCMV DNA。在建立HCMV脑部感染的小鼠模型基础上 ,用RT PCR测定Hox基因在病毒感染组和实验对照组小鼠脑组织中的表达 ,并用同位素标记的Hox寡核苷酸探针进行Northernblot检测相应的表达状况。结果　 (1)接种HCMVAD16 9株的小鼠脑组织发生了病理改变 ,免疫组化和PCR方法在神经细胞内查到了HCMV LA和DNA ;(2 )RT PCR和Northernblot发现 :对照组的小鼠脑组织表达Hoxa2、Hoxb1和Hoxb2 ,但不表达Hoxb5、Hoxb8;HCMV感染后 ,小鼠脑组织被诱导表达Hoxa1,与对照组比较差异有显著性意义 (P <0 .0 1) ,且于感染后的 15d达到高峰 ,而Hoxb1的表达下调 (P <0 .0 5 ) ;Hoxa2和Hoxb2无明显变化 ;Hoxb5和Hoxb8仍旧不表达。结论　HCMVAD16 9株可感染小鼠神经系统。HCMV感染可诱导小鼠神经系统发育基因Hox基因表达改变 ,这对     

MAGE-B3基因在多株胃肠道肿瘤细胞系中的表达

从转录和翻译两个水平检测MAGE B3基因在胃肠道肿瘤细胞系中的表达 ,以分析其在临床胃肠道肿瘤免疫治疗中的可行性。提取胃肠道肿瘤细胞株的总RNA后 ,RT PCR方法检测MAGE B3基因转录水平表达 ,采用间接免疫荧光法检测细胞中MAGE B3抗原。在转录水平除肠癌细胞株LOVO阴性外 ,其余 7株胃肠道癌细胞株MAGE B3基因表达均为阳性 ,而在蛋白水平所检测 8株胃肠道癌细胞株MAGE B3均显阳性表达。基于MAGE B3基因和抗原在胃肠道肿瘤细胞株中的高表达 ,可利用该基因或抗原作为靶分子进行免疫治疗。因而上述细胞株可作为良好的研究工具     

转染MHCII类基因对卵巢癌细胞株免疫特性的影响

建立稳定表达CIITA基因的人卵巢癌细胞株HO CIITA ,分析转染CIITA基因对T细胞体外抗肿瘤免疫应答的影响。以CIITA基因逆转录病毒 (pLXSN/CIITA )转染并筛选获得HO CIITA。用FACS和RT PCR分析HLA以及抗原加工递呈基因表达水平。以磁珠法分离得到正常人外周血CD4 +/CD8+T细胞 ,分别进行混合淋巴细胞反应及细胞因子测定 (ELISA和RT PCR )。结果显示 ,转染CIITA基因后 ,使HO细胞HLAII类分子和LMP7基因表达增高 ,而Ii基因表达由阳性转为阴性 ,未检测到TAP1表达 ;HO和HO CIITA细胞刺激CD4 +T细胞分泌IL 4含量两者有显著差异。刺激 4 8h后达到顶峰 ,前者分泌量约为后者的 1/2 ,但分泌IFN γ无差异 ,RT PCR与ELISA两种测定结果一致。表明转染CIITA基因可增加肿瘤细胞表面MHCII类分子的表达 ,该作用与IFN γ具有协同效应 ;并能诱导CD4 +T细胞表达IL 4。     

fgfr3基因敲除载体的构建及中靶ES细胞的筛选与鉴定

目的构建成纤维细胞生长因子3型受体(nbroblast growth factor receptor-3,FGFR3)基因敲除载体,并对中靶ES细胞进行筛选与鉴定,为最终建立FGFR3条件性敲除小鼠模型或FGFR3功能减低小鼠,研究FGFR3在小鼠骨骼等器官发育、损伤修复过程中的作用打下基础。方法在分析小鼠fgfr3基因组DNA序列结构的基础上,设计并构建了针对小鼠fgfr3外显子9、10的条件性基因敲除载体,采用电穿孔法转染ES细胞,用G418与Gancyclovir对电转的ES细胞进行正负筛选,最后对ES细胞克隆做Southern与PCR鉴定。结果对ES细胞正负筛选后共挑取180个克隆;用5′端探针进行Southern杂交鉴定后发现2株阳性克隆;经PCR检测发现这两株克隆fgfr3的8号内含子内的LoxP序列丢失。结论得到两株fgfr3基因中只含有neo基因的ES细胞克隆,fgfr3的表达是降低的,可用来建立FGFR3功能降低的小鼠。     

旋毛虫新生幼虫T668重组抗原对小鼠的保护性免疫

目的研究旋毛虫新生幼虫T668重组抗原的免疫原性,制备基因工程疫苗。方法以旋毛虫新生幼虫期特异性基因T668在大肠杆菌中的表达蛋白为抗原免疫小鼠,每间隔10d免疫1次,共免疫3次。末次免疫后10d,每只小鼠攻击感染200条旋毛虫感染性肌幼虫,感染后5周用消化法检查肌幼虫(ML)负荷。结果T668重组抗原免疫组肌幼虫减虫率明显高于佐剂组和对照组。结论T668重组抗原能诱导小鼠产生一定程度的保护性免疫,且可激发特异性体液免疫。     

旋毛虫感染性童虫全抗原制备及三种血清抗体检测方法比较

<正> 有关旋毛虫(Trichinalla Spiralis)抗原组成分析,单克隆抗体制备,不同虫期抗原成份的差异及其在虫体内分布,近年来已有不少研究。但旋毛虫病的血清学诊断国内多采用血凝、对流电泳及环蚴试验等方法。我室根据硝酸纤维素膜(NC)对蛋白质及核酸等具有很强吸附作用的特点建立了旋毛虫抗     

寄生虫学

弓形虫不同地理株致密颗粒蛋白基因的比较研究；zs株弓形虫p22基因片段在巨噬细胞中的表达；急性弓形虫感染所致昆明鼠不孕的实验研究；弓形虫p24基因敲除转染质粒pGB／P5-P3的构建；大鼠天然抗体相关的弓形虫抗原基因的免疫筛选与克隆；弓形虫膜抗原SAG3基因打靶载体的构建及筛选；大鼠卡氏肺孢子虫肺炎的实验病理学研究；卡氏肺孢子虫病鼠氧化损害与中药防治的实验研究；肾移植后并发卡氏肺孢子虫肺炎12例临床研究；人蛔虫Ⅱ期幼虫提取物诱导人肺上皮细胞凋亡；旋毛虫新生幼虫cDNA文库的免疫筛选；广州管圆线虫成虫cDNA文库抗原基因的筛选；用组织化学方法鉴别日本血吸虫细胞来源的研究；日本血吸虫EST序列的电子延伸及结果分析。     

白纹伊蚊(广州株)唾液Aalb_56 ku基因克隆表达与免疫原性分析

探讨Aalb_56 ku基因在白纹伊蚊(广州株)不同组织中的表达差异;对唾液腺中Aalb_56 ku基因进行克隆表达并探索其免疫原性。采用实时荧光定量RT-PCR法对雌蚊中肠、脂肪体、唾液腺不同组织中Aalb_56 ku基因的表达差异进行分析。针对Aalb_56 ku基因序列设计特异性引物,以唾液腺RNA为模板获得56 ku全长基因序列,构建重组质粒并测序,将测序序列进行生物信息学和抗原表位预测分析。IPTG诱导表达,亲和层析法纯化重组蛋白;制备小鼠特异性抗Aalb_56 ku蛋白抗血清。结果显示,Aalb_56 ku在白纹伊蚊(广州株)唾液腺(SG)中高表达。克隆所获唾液腺56 ku基因全长1 593 bp,可编码530个氨基酸,信息学分析提示该基因含有信号肽序列,等电点为8.40,抗原表位预测提示含有23个表位。p ET30a(+)-56 ku重组质粒可表达约56 k Da大小的融合蛋白,且呈包涵体形式表达,Western blotting鉴定具有His标签。纯化蛋白免疫Balb/c小鼠后可获得1∶100效价的特异性抗血清,提示该重组蛋白具有一定的免疫原性。     

小鼠内源性逆转录病毒Gag抗原在胰岛的表达及单克隆抗体的制备

目的 探究一种新发现的与1型糖尿病(T1D)相关的T细胞抗原[内源性逆转录病毒(ERV)的组特异性抗原(Gag)]与T1D发病的关系。方法 首先对一个从非肥胖糖尿病(NOD)模型小鼠胰岛获得的Gag抗原(Gag 194)的氨基酸序列进行分析[多肽位置：193-210(VSRLRGRRDPPAVDSTTS)],确定Gag 194多肽作为靶点，制备单抗；然后，再利用ELISA和Western blot检测单抗的特异性；最后，在免疫组织化学实验中检测不同年龄的T1D敏感的NOD小鼠与抗性的C57BL/6小鼠胰腺Gag抗原表达的差异性。结果 成功制备了5株(1F4C8、2D4C6、3C3B9、4D6B3、5F9E4)IgG2b亚型的小鼠ERV Gag单抗；5株单抗的敏感性及特异性具有差异；其中，生物素化后的3株单抗(1F4C8、2D4C6、5F9E4)能特异识别NOD小鼠胰腺中表达的ERV Gag抗原且该抗原的表达可在3周龄的NOD小鼠检测到，Gag抗原多表达在NOD小鼠胰岛β细胞区域内，而非淋巴细胞浸润的区域。此外，C57BL/6小鼠的胰腺Gag抗原的表达为阴性。结论 在NOD小鼠幼年时期E...     

Monoclonal antibodies raised in Btk(xid) mice reveal new antigenic relationships and molecular interactions among gp53 and other Trichinella glycoproteins

Tyvelose-bearing glycoproteins or Trichinella spiralis Group 1 antigens (TSL-1 antigens) are thought to be key molecules in the immunobiology of Trichinella. In the present study, we investigated the binding characteristics of several mAbs produced in Btk(xid) immunodeficient mice that recognise gp53 and some other minor glycoproteins of this parasite. The data obtained reveal the existence of an O-glycan/peptide epitope (recognised by mAb US8) common to all TSL-1 glycoproteins, as well as a specific interaction between the TSL-1 antigen gp53 and other unknown Trichinella glycoproteins in the 35-40 kDa range (these latter react with mAbs US8 and US9, but not with mAb US5). Some of the epitopes recognised by our mAbs are differentially expressed in Trichinella species: the epitope recognised by mAb US5 on gp53 (another O-glycan/peptide epitope) is present only in T. spiralis, whereas those recognised by mAbs US8 and US9 (peptide epitopes) are present in encapsulated Trichinella species. The data obtained also reveal that gp53 is synthesised and glycosylated in beta-stichocytes only. The possible relevance of these findings is discussed.     

Identification of diagnostic antigens fromTrichinella spiralis

The Western blotting technique was used to determine the antigens ofTrichinella spiralis muscle larvae that were recognized by antibodies in sera from humans and pigs displayingT. spiralis infections. This resulted in the identification of several antigens that were recognized by all sera. Some of these antigens, notably those that were recognized during the early stage of infection, cross-reacted with antibodies to other parasites. This cross-reactivity was caused by the presence of phosphorylcholine on these antigens. A large portion of the antigens that were recognized by antibodies from infected humans and pigs were found to share a singleTrichinella-specific determinant. TheTrichinella-specific antigen population could be isolated from phosphorylcholine-containing antigens by a simple two-step affinity chromatography procedure using monoclonal antibodies to both determinants. The resulting preparation consisted primarily of a single antigen showing an apparent molecular weight of 45 kDa that corresponded to a mamor constituent of excretory-secretory (ES) products of muscle larvae. When tested in an enzyme-linked immunosorbent assay (ELISA), this antigen displayed diagnostic specificity that was comparable with the ES fraction and diagnostic sensitivity comparable with the crude muscle-larvae extract.     

Antigens of Toxoplasma gondii with a diagnostic and potential immunoprophylactic value: new strategies of identification

Toxoplasma gondii is an ubiquitous protozoan parasite which induces severe pathology in in utero infected children and in immunosuppressed patients (particularly in the case of AIDS). Previous work that focused on toxoplasma somatic antigens failed to demonstrate an efficient protection against highly virulent T. gondii strains. The authors therefore first studied the role of parasite excreted-secreted (ES) antigens in the immune response. They describe here the preparation of excreted-secreted antigens in cell-free medium from tachyzoites, the intracellular proliferative stage present during acute infection. Major ES antigens have Mr of 108 K, 97 K, 86 K, 57 K, 42 K, 39 K, 28.5 K, 27 K and 21 K. The protective role of ES antigens has been demonstrated using congenitally athymic (Nu/Nu) rats that are highly sensitive to T. gondii infection (+/+ Fischer rats are resistant). The humoral and cellular components of this protection have been studied by the passive transfer either of sera or of T lymphocytes from ES-immunized +/+ Fischer rats into Nu/Nu rats. Adoptive transfers were carried out 24 hours before infection with the highly virulent T. gondii RH strain. Based on the concept of concomitant immunity, the authors have characterized antigens from tachyzoites and bradyzoites (the encysted stage persisting during chronic infection) which share common epitopes. Four tachyzoite antigens, P63, GP43, P39 and GP 28.5 have been shown by immunoprecipitation to cross-react with bradyzoite antigens. Two monoclonal antibodies raised against ES antigens permitted to demonstrate the localization of the 28.5 K and 27 K antigens inside the dense granules of tachyzoites and bradyzoites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of major antigenic proteins of six strains of the human granulocytic ehrlichiosis agent by Western immunoblot analysis.

The etiologic agent of human granulocytic ehrlichiosis (HGE) is an obligate intracellular bacterium. In 1996, blood specimens from 53 patients suspected of having HGE were examined by indirect fluorescent antibody (IFA) testing with the HGE agent no. 13 isolate as the antigen, by nested PCR, and by culture. All patients resided in Westchester County, N.Y. Twelve patient specimens were positive for IFA (titer > or = 1:40). Seven of these were also positive by PCR. Of the seven specimens positive by both IFA testing and PCR, the HGE agent was isolated from four (no. 2, 3, 6, and 11) and continuously cultured in HL-60 cells. These were confirmed as the HGE agent by sequencing of 16S rDNA. Both purified whole-cell organisms and the outer membrane fractions of the new isolates were compared with no. 13 isolate and a tick (USG) isolate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot analysis. No. 11 and 13 isolates had identical SDS-PAGE patterns with respect to 49- and 47-kDa proteins. No. 3 and USG isolates lacked the 47-kDa protein, and no. 6 isolate lacked the 49-kDa protein. Both 49- and 47-kDa bands were absent in no. 2 isolate. Western blot results with seven different sera, including five convalescent-phase sera from these patients, one dog anti-USG isolate, and one horse anti-BDS isolate, showed that all major antigens in six isolates were recognized by all sera. However, the molecular sizes and the numbers of major antigens recognized varied among the six isolates. Overall, HGE agent no. 3, 6, 11, and 13, and USG isolates had similar patterns, with 1 or 2 major antigens with molecular masses of around 49 and 47 kDa. No. 2 isolate was quite distinct in having a major antigen of 43 kDa. This indicates that although these antigenic epitopes are all cross-reactive among strains, the HGE agent has a strain pleomorphism in its major antigenic proteins. The major antigen profiles of the outer membrane protein fractions and of whole organisms of six HGE agent isolates were similar, suggesting that 49- and 47-kDa major antigens are the outer membrane proteins of the HGE agent.     

Expression of a new human THY-1 related antigen in Ewing's sarcoma and peripheral neuroectodermal tumors

Ewing's sarcoma (ES), peripheral neuroectodermal tumor (PNET) and Askin's tumor of the chest wall share a reciprocal chromosomal translocation between the long arms of chromosomes 11 and 22 (q23-24; q12). In the absence of other distinguishing features this specific translocation is regarded as marker of a common and neuroectodermal origin for these rare tumors. A monoclonal antibody (HBA-71) developed in our laboratory has been found to recognize an unique ES and PNET associated antigen, which is also expressed in some normal tissues, including thymus, bone marrow, islets of Langerhans, ependyma and adenohypophysis. It is shown in this study that this HBA-71 antigen is closely related to the murine THY-1 antigens, major cell surface glycoproteins of thymocytes and brain in mice and rat. Both antigens have similar molecular ratios (18,000), amino acid compositions and sensitivity to tryptic digestion, show high cell surface expression, and binding of the appropriate antibodies to HBA-71 antigen triggers proliferation in thymocytes. The HBA-71 epitope may represent a primitive neuroectodermal marker of ES/PNET, or its expression may be directly linked to the reciprocal translocation invariably associated with HBA-71-positive ES and PNET tumors, which maps to the same region of chromosome 11 (q23-24) as the human Thy-1 gene.     

Expression of mucin antigens and Lewis X-related antigens in carcinomas and dysplasia of the pharynx and larynx

Recent studies have Identified that much antigens and Lewis X (Le^x)-related antigens behave like oncodevelopmental tumor-asnoclated antigens in several human adenocarcino-mas. However, the expression of these antigens In pharyn-geal and latyngeal squamous cell carcinomas (SCC) and in the precursor lesion is not fully elucidated yet. In the present study, the expression of mucin core protein antigens associated with the MUC1 gene product (DF3 antigen, mammary-type apomucin) and the MUC2 gene product (intestinal-MRP antigen, Intestinal-type apomucin) much carbohydrate antigens that are associated with the earliest steps in mucin glycosylation (Tn, sialyl-Tn and T), and Le^x -related antigens (Le^x, Leγ and slayl Le^x-1) In biopsy or resected specimens from 26 normal squamous epithelia (NSE), 49 dysplastic squamous epithelia (DSE) and 51 SCC were examined. The DF3 antigen was not expressed In NSE (0%), whereas it was expressed in 20 DSE (41%) and in 31 SCC (61%). The intestinal-MRP antigen showed no expression in NSE, DSE or SCC. The Tn antigen showed no expression in NSE, but showed low expression rates In DSE (14%) and in SCC (16%). The sialyl-Tn and T antigens were expressed in NSE, as well as in DSE and SCC. The T antigen expression increased with progression from NSE to DSE to SCC, while the sialyl-Tn antigen did not show such a tendency. Any of the three Le^x-related antigens showed no characteristic expression in DSE and SCC. In the eight antigens examined, only DF3 antigen was an effective marker for DSE and SCC in the pharyngeal and laryngeal region. Cytoplasmic expression of DF3 and slalyl- Tn antigens were more frequently seen In SCC than in DSE, and might be useful to differentiate SCC from DSE.     

Expression of cancer-testis antigens in endometrial carcinomas using a tissue microarray.

Cancer-testis (CT) antigens are expressed in a variety of malignant tumors, but in normal adult tissue, they are only expressed in testicular germ cells. Owing to this tumor-associated expression pattern, these antigens are of major interest as potential targets for immunotherapy and possibly for diagnostic purposes. This study was performed to analyze the expression of four CT antigens, NY-ESO-1, MAGE-A3, MAGE-A4, and CT7/MAGE-C1, in endometrial carcinoma using immunohistochemistry, and to correlate expression with histologic subtypes, grade, and expression of WT1 and p53. Formalin-fixed paraffin-embedded tissues of 130 endometrial carcinomas of the following types and grades were analyzed using a tissue microarray: 85 endometrioid carcinomas (FIGO grade 1, 39; grade 2, 11; and grade 3, 35), 18 papillary serous carcinomas, 12 clear cell carcinomas, 13 malignant mixed mullerian tumors, one mucinous adenocarcinoma, and one undifferentiated carcinoma. The following anti-CT monoclonal antibodies/antigens were studied by immunohistochemistry: monoclonal antibody ES121/NY-ESO-1, monoclonal antibody M3H67/MAGE-A3, monoclonal antibody 57B/MAGE-A4, and monoclonal antibody CT7-33/CT7. The CT expression data were compared to WT1 and p53 protein expression as analyzed in a previous study. Positive staining with anti-CT monoclonal antibodies was graded as follows: focal, <5% positive cells; 1+, 5-25% cells; 2+, 26-50% cells; 3+, 51-75%; and 4+, >75% cells. The 3+ and 4+ staining patterns were considered homogeneous patterns of potential clinical significance and were scored positive for statistical analysis. In low-grade tumors, the most immunoreactivity was seen with mAb M3H67 but little labeling was observed with the other monoclonal antibodies. In high-grade tumors, monoclonal antibodies M3H67 (25%), 57B (23%), and CT7-33 (20%) showed the highest reactivity, while ES121 showed the lowest immunoreactivity (6%). The staining pattern was mostly heterogeneous. Statistical significance was found solely for the correlation of monoclonal antibody 57B staining and p53 expression. No correlation was found for any anti-CT monoclonal antibody staining and clinical stage or for anti-CT staining and WT1 expression. CT antigens CT7, MAGE-A3 and MAGE-A4, but not NY-ESO-1, are expressed in high-grade endometrial carcinomas, and expression of MAGE-A4 is correlated with the presence of overexpressed p53.     

Growth of Trichinella Spiralis Larvae in Rats Receiving Folic-Acid-Deficient Diet

A study of the effect of growth of Trichinella spiralis in rats fed a folic-acid-deficient diet is described. Trichinella spiralis larvae encysted in the diaphragms of two groups—rats fed a folic acid-deficient diet and rats fed a complete (normal) diet—were examined. In rats fed a folic-acid-deficient diet, the number of the encysted larvae was larger than that in the controls. However, the encysted larvae were substantially longer in the control group of rats fed complete (normal) diets.     

Minor interspecies variations in the sequence of the gp53 TSL-1 antigen of Trichinella define species-specific immunodominant epitopes

Among the Trichinella TSL-1 antigens, whose antigenicity is generally due mainly to tyvelose-containing epitopes, gp53 is unusual in that its antigenicity is due mainly to protein epitopes. In the present study we mapped two of these epitopes, recognized by monoclonal antibodies (mAbs) that specifically recognize gp53 from all encysting Trichinella species (mAb US9), or gp53 from Trichinella spiralis alone (mAb US5). Based on previously published sequences of this glycoprotein [Mol. Biochem. Parasitol. 72 (1995) 253], in this study, we cloned the full gp53 cDNA from a new strain, Trichinella britovi (ISS 11; AN: ), and from another T. spiralis isolate (ISS 115; AN: ). The gp53 sequence comprised an ORF of 1239bp, coding for 412 amino acids, with 61 nucleotide differences (resulting in 38 residue changes) between the two species. Mapping of US5- and US9-recognized epitopes was undertaken through the construction and expression in the pGEX4T vector of truncated gp53 peptides, and by the construction of peptides derived from the antigenic regions. The epitope recognized by mAb US9 was a linear peptide of 8 residues, 33Met- 40Ser, located in the amino-terminal region, while the corresponding epitope recognized by mAb US5 was a 47-amino acid sequence containing two alpha-helix regions flanked by random coils, 290Thr- 336Lys. Molecular modeling of these peptides seems to indicate that recognition of the US9 epitope depends on the presence of two available hydroxyl groups provided by one methionine and one serine on T. spiralis gp53 (not present on Trichinella pseudospiralis gp53). Additionally, the stability of the US5 epitope seems to depend on correct folding of the 47-amino acid sequence (only present in T. spiralis). The relevance of these findings for understanding the antigenic recognition of Trichinella TSL-1 antigens, and for further studies to investigate possible function(s) of gp53 in Trichinella, is discussed.