目的 探讨大肠杆菌O127∶H6 (Escherichia coli O127∶H6)感染BALB/c小鼠建立肠炎模型及其受激活调节正常T细胞表达和分泌活性因子(RANTES)与炎症反应的相关性.方法 通过不同剂量大肠杆菌O127∶H6灌胃的方式,建立肠炎小鼠模型(实验组),灌胃生理盐水为对照组.取小鼠空肠标本,石蜡包埋,HE染色,观察病理变化;采用荧光定量PCR的方法,测定不同剂量组感染小鼠空肠组织的RANTES表达情况.结果 成功建立了肠炎性小鼠模型,实验组与对照组比较,空肠有明显的炎性病理变化;荧光定量PCR结果表明实验组RANTES表达量明显高于对照组.结论 通过灌胃大肠杆菌O127∶H6的方式可以建立肠炎小鼠模型,其空肠部位具有明显的病理变化;RANTES表达量的变化可以作为评价小鼠肠炎的指标之一.

Objective To obtain capsid p27 of SIV for detecting.Methods The p27 gene of SIV was synthesized and inserted into pMAL-p5x vector to construct pMAL-p5x-p27 recombinant plasmid.And then the recombinant plasmid was transformed into E.coli BL21 to express protein and the express product was purified by Amylose Resin maltose-binding protein affinity column.Results SDS-PAGE analysis indicated that pMAL-p5x-p27 can be expressed in E.coli BL21 and the protein molecular weight was about 70×103;The final p27 protein purification could reach 90%.Conclusion p27 of SIV was successfully expressed in E.coli,which lays a foudation for developing the methods of detecting SIV antibody.

Objective Atfu-B* sv1 with α2 domain deletion is a splice variant of major histocompatibility complex class I B in Ateles fusciceps.The aim of this study was to obtain the basic information of expression and intracellular localization of Atfu-B* sv1,and to compared them with that of the full-length Atfu-B gene.Methods Eukaryotic expression vectors of Atfu-B* sv1,Atfu B* 02:03 and Atfu-B* 03:01 were constructed and transfected into 293T cells,respectively.Expression of the three genes in the cells was detected by Western blotting,and the intracellular localization of the three genes was analyzed by immunofluorescence and laser confocal microscopy.Results Atfu-B* sv1,Atfu-B* 02:03 and AtfuB* 03:01 were all expressed on the plasma membrane of 293T cells and were glycosylated.Conclusions The expression and intracellular localization of Atfu-B* sv1 is not significantly different from those of full length Atfu-B genes.Further studies on the functions of Atfu-B* sv1 are needed to be done.

Objective To examine immunohistochemically the expression of estrogen receptor-α(ERα) and-β(ERβ) in different types of cells in the ovary and uterus of Beagle dogs.Methods Six multiparous female Beagle dogs were used in this study.S-P immunohistochemistry was used to detect the expression of ERα and ERβ in the dog uterus and ovary with DAB,BCIP/NBT and AEC staining.Results ERα was mainly expressed in the nuclei of granulosa cells,ovarian interstitial glandular epithelial cells and endometrial glandular epithelial cells(along with weaker staining in the cytosol).It was also sporadically expressed in the nuclei of the theca cells,peri-glandular stromal cells,arteriolar endothelial cells,artery smooth muscle cells and small vein endothelial cells.On the contrary,ERβ was mainly expressed in the cytoplasm of all of the above cells(along with sporadic staining in the cell nuclei).In addition,in the corpus luteum at different physiological periods,ERα was immunohistochemically positive in the nuclei of theca cells,and also in the nuclei and cytoplasm of luteinizing granulosa cells,while ERβ was still positive expressed in the cytoplasm of luteal cells.Coexpression of ERα and ERβ in the dog uterus was not detected with BCIP/NBT and AEC double staining.Conclusions The results of our study show that the expressions of ERα and ERβ in the uterus and ovary of Beagle dogs are different.ERα mainly located in the nuclei of different cell types(along with weaker staining in the cytosol),whereas ERβ is mainly localized in the cytoplasm of different cell types(along with sporadic staining in the nuclei).

Objective Mamu-B* 007: 03-sv1 is a splice variant of major histocompatibility complex class I in Chinese rhesus macaque devoid of α3 domain. The aim of this study was to get the basic information on expression and cellular localization of Mamu-B * 007: 03-sv1 and to compare with the full-length Mamu-B * 007: 03 gene. Methods Eukaryotic expression vectors of Mamu-B * 007: 03-sv1-myc-pEGFPN3and Mamu-B * 007: 03-myc-pEGFPN3were constructed and transfected into human renal epithelial 293T cells. The expression of the two genes in cells was detected by Western blotting,and the cellular localization of the two genes was analyzed by immunofluorescence and laser scanning confocal microscopy. Results Both Mamu-B* 007: 03-sv1 and Mamu-B* 007: 03 were expressed in 293T cells and both were glycosylated. While Mamu-B* 007: 03 gene was expressed on the cell membrane,Mamu-B* 007: 03-sv1 gene was expressed intracellularly and on cell membrane. Conclusions The expression and cellular localization of Mamu-B* 007:03-sv1 deviod of α3 is significantly different from that of the full length Mamu-B* 007: 03 gene. The functions of Mamu-B* 007: 03-sv1 remain to be further studied.

目的 建立BABL/c小鼠感染大肠杆菌O127的模型及TGF-β1的荧光定量PCR方法检测方法.方法 通过灌胃的方式建立肠炎性小鼠模型,并利用嵌合荧光定量的方法对其结肠TGF-β1定量分析实验.结果 成功建立了肠炎性小鼠模型,荧光定量PCR试验结果表明实验组TGF-p1表达量明显高于对照组.结论 利用经口灌胃的方法可以成功的建立肠炎性小鼠动物模型,TGF-β1定量方法可以作为模型建立的评价标准.

目的 研究比格犬雌激素受体β(Estrogen receptor β,ERβ)及其剪接异构体在生殖调控中的功能,构建比格犬ERβ的pMAL-p5x/ERβ 480 DE3 E.coli重组菌株,并进行纯化和鉴定.方法 Trizol法提取发情期比格犬下丘脑总RNA,RT-PCR获得cDNA,以NCBI网站公布的比格犬ERβ基因CDS序列设计特异性引物,扩增ERβ保守区基因编码序列(16-496 bp),连接原核表达载体pMAL-p5x,筛选、鉴定阳性克隆并测序.将重组质粒转化至大肠杆菌DH5α,再转化大肠杆菌BL21(DE3),用IPTG诱导表达,表达产物经经麦芽糖亲和树脂(Amylom Resin)亲和层析分离纯化,并对纯化的融合蛋白进行鉴定.结果 构建了pMAL-p5x/ER(a)480重组质粒,在DE3大肠杆菌中诱导表达出MBP-ERβ融合蛋白,SDS-PAGE显示分子量约为60 000,与预期结果一致；优化了MBP-ERβ表达体系的表达条件:分别在0.2％葡萄糖,100 μg/ml Ampcillin,0.1mmol/L IPTG 37℃培养5h或在0.2％葡萄糖,50 μg/ml Ampcillin,0.2mmol/L IPTG 37℃培养3h,融合蛋白表达效果比较好.结论 构建了pMAL-p5x/ERβ480原核表达重组质粒,获得了比格犬MBP-ERβ融合蛋白,为犬种属特异性ERβ多克隆抗体的制备及功能分析奠定了基础.

Objective To compare the uterine and ovarian morphologies between diestrus and estrus Beagles for the further study of their reproductive inefficiency.Methods Detected the serum sexual hormone of 23 Beagle bitches,which have been produced once or twice,using chemiluminescence and selected 2 estrus and 4 diestrus Beagles by serological identification for ovarian and uterine specimens,4% neutral paraformaldehyde-fixed,paraffin-embedded,routine HE stained,photographed.Results The uterus and endometrium of diestrous dogs are thin,interstitial fibrosis,corpus luteum are occupied by primary follicles,1-2 visible secondary follicles,no mature follicles.Follicular and luteal cells have more fiber,less vascularized.While the uterus and endometrium of estrus Canines are more thicken,endometrial cavity is larger,part of the gland is branched bend,glandular cells are enlarged,cytoplasm pale dyed,with a small number of visible vacuoles under the nuclear.Ovarian follicles are in large numbers,and are mainly primary,secondary and 1-2 mature follicles.There are numeric luteal cells which arranged regulatory,state clearly,loose interstitial fibrosis,vascular,and no vacuolar degeneration.Diestrus and estrus dogs' ovarian have no obvious white body.Conclusion Diestrous Beagle sex hormone is maintained at a relatively low level,in proestrus estrogen levels increased rapidly,and after ovulation progesterone levels were higher.Morphological changes of ovary and uterus are adapted to the estrous cycle of bitches.

Rhesus macaques are an animal model for the study of a variety of human diseases. The Chinese rhesus macaques have been widely used in biomedical research in recent years. However, the polymorphism of major histocompatibility complex (MHC) class I A region among different local populations of Chinese rhesus macaques has never been investigated. In this study, we identified 46 Mamu-A alleles by cDNA cloning and sequencing on a cohort of 53 Chinese rhesus monkeys including Zhiming, Chuanxi, and Fujian populations, of which 5 were first reported in rhesus monkeys. The frequencies of alleles were identified for each population. The result suggests that the repertoire of allelic variants of MHC class I A region found in different populations of Chinese macaques is largely non-overlapping. The frequencies of alleles and the popular allele are also different for different populations. PCR-SSP experiment further confirms the different frequencies of two alleles, Mamu-A*026:01 and Mamu-A*022:01, in additional 99 Zhiming monkeys and 191 Chuanxi monkeys. Our findings have important practical implications in that the origin of the individuals and the genetic polymorphism of the monkeys need to be considered at the level of local populations for Chinese rhesus monkeys in biomedical research. Further immunogenetic work is needed to investigate the MHC polymorphism among different populations of Chinese rhesus macaques and to reveal the functional implication of such polymorphism and disease outcome correlations.

Objective To synthesize the gene including the CDV-N protein antigen epitopes,and construct the baculovirus expression vector.Methods The N protein antigen epitopes was predicted by bioinformatics software and synthesized tandem.The fragment was cloned into the baculovirus expression vector pFastBac-Hta.Results The gene including the CDV-N protein antigen epitopes was obtained and successfully constructed the pFastBac-Nsynthesis expression vector.Conclusion The experiment laid the foundation for the CDV-N protein antigen epitopes expression in baculovirus.