hCEACAM1介导淋病奈瑟菌对COS-1细胞的黏附

目的 研究人癌胚抗原相关细胞黏附因子1(hCEACAM1)在淋病奈瑟菌黏附宿主细胞过程中的作用.方法 将hCEACAM1 cDNA置于hCD46启动子和兔β-球蛋白第2内含子之后,构建重组真核表达载体pCDPGICEA1.转染COS-1细胞,G418筛选和流式细胞术分选稳定表达hCEACAM1的基因转染细胞.细菌黏附实验检测淋病奈瑟菌对基因转染COS-1细胞的黏附.结果 在hCD46启动子和兔β-球蛋白第2内含子的作用下,hCEACAM1 cDNA在COS-1细胞获得高效表达,淋病奈瑟菌可以黏附表达hCEACAM1的COS-1细胞.结论 hCEACAM1可以介导淋病奈瑟菌对动物细胞的黏附,可能是淋病奈瑟菌特异性感染人体的一种重要受体,可用来制备淋病奈瑟菌感染的相应转基因动物模型.     

淋病奈瑟菌NspA原核表达及其抗原性研究

目的 研究淋病奈瑟菌表面蛋白A(NspA)的抗原性及其在淋病快速诊断技术中的应用价值.方法 用PCR技术克隆淋病奈瑟菌NspA基因,构建其原核表达载体,在大肠杆菌中表达重组NspA,表达产物复性后免疫小鼠,Western blot、ELISA分别检测免疫血清与淋病奈瑟菌细胞裂解液中NspA及与淋病奈瑟菌全细胞的结合能力.结果 NspA基因可在大肠杆菌中表达,纯化并复性的重组NspA(rrNspA)可在小鼠体内刺激产生高效价特异性抗体,抗rrNspA抗体可与淋病奈瑟菌完整细胞及细胞裂解液中NspA特异性结合.结论 获得的rrNspA及抗rrNspA抗体在建立淋病奈瑟菌抗体或抗原快速检测方法中具有潜在的应用价值.     

淋病奈瑟菌临床菌株外膜蛋白PIA基因序列分析及原核表达系统的构建

目的： 分析本地区淋病奈瑟菌临床菌株外膜蛋白PIA基因核苷酸及氨基酸序列，构建PIA基因的原核表达系统。方法：采用高保真PCR扩增9株淋病奈瑟菌全长PIA基因序列，T-A克隆测序后与GenBank公布的序列进行同源性比较。构建PIA原核表达系统。采用不同浓度IPTG诱导重组目的蛋白rPIA表达，10%SDS-PAGE和Bio-Rad凝胶图像分析系统检测rPIA表达情况。采用Ni-NTA亲和层析法提纯rPIA，SDS-PAGE检测提纯效果。结果： 与报道的PIA基因序列（GenBank No: L19962）比较，9株淋病奈瑟菌核苷酸和氨基酸序列相似性分别高达99.6%-100%和99.1%-100%，均属于IA6血清型。rPIA表达量可占细菌总蛋白量的50.1%，提纯后仅显示单一的目的蛋白条带。结论： IA6为本地区淋病奈瑟菌优势血清型，该基因序列相当保守。所构建的PIA基因原核表达系统能高效表达rPIA，为今后研制淋病奈瑟菌血清学检测试剂盒及疫苗研制奠定了基础。     

慢病毒载体法制备荧光素酶转基因小鼠

目的基于慢病毒介导的转基因方法制备荧光素酶（Luc）转基因小鼠。方法制备携带Luc基因的慢病毒,将其注入小鼠单细胞受精卵卵周隙以感染受精卵,然后将胚胎移植进假孕母鼠体内以获得仔鼠,应用小动物活体成像仪及PCR等在蛋白和DNA水平上筛选和鉴定Luc转基因小鼠。结果移植慢病毒隙感染后的成活胚胎63枚。将其移植至3只假孕母鼠,其中2只怀孕,共生仔鼠11只;利用小动物活体成像仪检测Luc表达,在蛋白水平证实11只F0代中,3只（命名为S1、S2、S3）表达Luc;DNA水平检测证实,3只Luc阳性小鼠的基因组中整合有外源转基因Luc。此外,Luc转基因首建鼠基因组中整合的Luc转基因可稳定遗传至下一代,并能正常表达。Luc转基因小鼠主要脏器如睾丸、肾脏、胃、肠、肺、脑、胸腺、肝脏和心脏等均可见Luc信号,但不同脏器间Luc强度有差异。结论成功制备Luc报告基因转基因小鼠。     

淋病奈瑟菌rmp基因缺失突变株的构建及其抗体杀菌作用研究

目的研究淋病奈瑟菌的外膜蛋白Rmp在免疫阻抑中的作用及其消除策略。方法PCR扩增淋病奈瑟菌rmp基因,将rmp中间200个核苷酸残基用卡那霉素抗性基因Kan取代,含rmp两侧侧翼区和Kan的DNA片段△rmp∷Kan转化淋病奈瑟菌 WHO-A菌株,PCR和Western blot 鉴定野生rmp被突变基因（△rmp∷Kan）取代并不能表达Rmp的突变株。突变株免疫小鼠,并用抗体介导的补体杀菌作用研究免疫血清的抗菌活性。结果构建了rmp基因缺失的淋病奈瑟菌突变株,突变株诱生的抗体具有更强的杀伤淋病奈瑟菌活性。结论淋病奈瑟菌rmp基因缺失突变株可能消除了野生株中Rmp的免疫阻抑作用,在新型全细胞减毒活疫苗研制中具有潜在应用价值。     

人Man2c1转基因小鼠模型的建立

目的 为在体内研究MAN2C1的生物学意义而建立转hMan2c1基因的小鼠。方法 构建pIRKS2-EGFP-hMan2c1重组表达载体，经体外转染实验鉴定转染的基因能在COS-7细胞表达后，注射人ICR小鼠受精卵，以制备转基因小鼠。用基因组PCR鉴定目的基因在宿主基因组DNA的整合。用RT-PCR和Westernblot分析hMan2c1在转基因小鼠的表达。结果 在116只原代小鼠中，有7只hMan2c1基因组PCR阳性。在所检测的20只F1代小鼠中，有9只hMan2c1基因组PCR阳性。在所检测的21只F2代小鼠中，有16只基因组PCR阳性。用鼠尾组织RT-PCR和Western blot检测hMan2c1基因表达，确定基因组PCR阳性的7个系中有4个系阳性。结论 建立了4个稳定表达hMan2c1的转基因小鼠系，为深入研究MAN2C1的生物学意义打下了基础。     

产青霉素酶淋病奈瑟菌的基因检测

目的 了解常州地区产青霉素酶淋病奈瑟菌（penicillinase-producing Neisseria gonorrhoeae,PPNG)在淋病奈瑟菌中的构成比。方法 用套式聚合酶链反应（nested polymerase chain reaction,nPCR)对分离自常州地区的81株淋病奈瑟菌进行PPNG特有耐药基因检测。结果 81株淋病奈瑟菌中有62株检出耐药基因，即PPNG已占淋病奈瑟菌总数的76.5%。结论 常州不仅存在PPNG，而且已构成流行。     

携带淀粉样前体蛋白瑞典型突变基因转基因小鼠的制备及体内表达研究

目的 建立携带人类淀粉样前体蛋白瑞典型突变（Swedish mutation of amyloid precursor protein,APPSWE)基因的转基因小鼠模型。方法 采用受精卵原核显微注射法，将人类APPSWE转基因导入C57及昆明种小鼠受精卵内，然后将注射后保持完整的受精卵移植到假孕母鼠的输卵管内，然后应用聚合酶链反应（polymerase chain reaction,PCR)、荧光原位杂交及逆转录PCR分析子代小鼠中外源基因的整合及表达情况。结果 注射后卵的存活率和幼子出现率分别为76.62%和10.38%；外源基因整合率为35.29%；共获得6只首建小鼠，已稳定传3代，PCR检测共55只阳性；提取阳性小鼠心、脑、肝、肾组织及骨骼肌以人类APPSWE基因外显子特异的引物进行逆转录PCR分析，结果发现其心、脑组织及骨骼肌中具有人类APPSWE基因的表达。结论 说明携带淀粉样前体蛋白瑞典型突变基因的转基因小鼠模型已制备成功。     

淋病奈瑟菌NspA基因疫苗诱导小鼠特异性体液免疫应答

1999年Martin等首先克隆了淋病奈瑟菌表面蛋白A（Neisseria surface protein A，NspA）基因，发现其在细胞表面持续表达，菌株之间NspA氨基酸序列的同源性高达98％，提示NspA可能是研制淋病疫苗的理想抗原。我室已成功构建NspA真核表达载体pcNspA，本文研究了pcNspA基因免疫在小鼠体内诱生特异性抗体的作用。     

SV40T胃壁细胞定位表达转基因小鼠的建立

目的 构建胃壁细胞定位表达SV40T的真核表达载体并制备转基因小鼠动物模型,为研究胃癌发病机制提供动物模型.方法 从构建的胃壁细胞特异性表达载体pcDNA3.1(-)/HKSV中酶切回收3.8kb的基因片段H -K ATPase β promoter/SV40T,通过显微注射的方法制备转基因小鼠,PCR和Southern blotting检测阳性转基因小鼠并建系繁殖,RT-PCR检测基因的表达情况.结果 将422枚注射过的受精卵移植给16只假孕雌鼠,共生出77只仔鼠,移植成功率为18.2%.在出生的77只仔鼠中,2#、4#、8#、16#、24#、51#、57#、61#、68#、73#经PCR检测为阳性首建鼠.除68#不育外,其他9个品系首建鼠共生出99只F1代鼠.8#品系23只F1代尚未发现阳性鼠,另8个品系F1代经PCR和Southern blotting检测发现31只阳性鼠,阳性率为40.8%(31/76).RT-PCR检测F1代阳性鼠均仅在胃组织中有SV40T基因的表达,而在心、肝、肾、肺、食道、肠、骨骼肌等组织中均不表达.不育首建鼠处死解剖发现胃组织有肿瘤存在.结论 建立了胃壁细胞定位表达SV40T基因的转基因小鼠动物模型.     

Hybrid transgenic immune tolerant mouse model for assessing the breaking of B cell tolerance by human interferon beta

To date, the therapeutic efficacy of recombinant human proteins is limited by their potential to break B cell tolerance in patients. The formation of neutralising antibodies (NABs) directed against recombinant human interferon beta (rhIFNβ) is associated with a decrease in the therapeutic effect of the protein. For this reason, there is a need to study factors that can cause the immunogenicity of rhIFNβ. Transgenic C57Bl/6 mice that are immune tolerant for human interferon beta (hIFNβ) have been employed in a mouse model for assessing the breaking of immune tolerance by rhIFNβ. In this study, we used the original C57Bl/6 mouse model as well as the hybrid offspring from crossings of transgenic C57Bl/6 mice with wildtype FVB/N mice to study the immunogenicity of three commercial rhIFNβ products, Rebif^®, Avonex^® and Betaferon^®. As determined by ELISA, wildtype C57Bl/6 mice failed to form binding antibodies (BABs) against Rebif^® and Avonex^® formulated with human serum albumin. Because not all interferon beta products induce antibodies in wildtype C57Bl/6 mice, the transgenic C57Bl/6 mice cannot be used to study the breaking of tolerance by these products. However, the crossing of transgenic C57Bl/6 mice with FVB/N mice resulted in wildtype hybrid offspring in which all products were immunogenic and transgenic hybrid offspring that showed immune tolerance for hIFNβ. Thus, these C57Bl/6 × FVB/N hybrid transgenic mice can be used to study the breaking of immune tolerance for all rhIFNβ products. Of the three products, only Betaferon^® was able to break immune tolerance in the transgenic hybrids. With an MxA gene expression inhibition assay, NABs were detected in Betaferon^® treated wildtype hybrid mice, but not in transgenic hybrid mice, indicating a distinct immune mechanism in wildtype and transgenic mice. A pegylated rhIFNβ-1a variant, PEG-rhIFNβ-1a, induced antibodies in wildtype hybrid mice, but did not break the immune tolerance of transgenic hybrid mice. This suggests that pegylation did not affect the potential of rhIFNβ-1a to break B cell tolerance.     

免疫磁珠法分离白血病转基因小鼠骨髓造血干细胞

本文分离转基因小鼠骨髓造血干细胞。运用针对小鼠干细胞表面特异表达的干细胞抗原 1(stemcellantigen 1,Sca 1)的单克隆抗体和包被于磁颗粒表面的第二抗体 ,采用磁吸附细胞分选方法 (MACS )分离小鼠骨髓造血干细胞 ,用流式细胞术 (FACS )检测MACS分离后骨髓细胞中干细胞 (Sca 1阳性细胞 )比例 ,监测细胞分选效果。结果显示MACS分离后骨髓细胞中Sca 1阳性细胞所占比例达 85 %以上 ,涂片观察发现细胞组成和细胞形态学特征与FACS所得结果一致。MACS可以从转基因小鼠全骨髓细胞中分离出Sca 1阳性的骨髓造血干细胞 ,细胞纯度可达 85 %以上。     

R6/2型亨廷顿病转基因小鼠胰岛β细胞功能损伤

目的：研究R6/2型亨廷顿病(HD) 转基因小鼠胰岛β细胞的功能,揭示HD转基因小鼠继发糖尿病的机制。方法：利用R6/2 型HD转基因小鼠模型,检测正常和HD小鼠空腹血糖以及血清胰岛素水平;并应用HE染色和免疫荧光技术分析正常和HD小鼠胰岛形态学差异。结果：与正常小鼠相比,R6/2 型HD小鼠空腹血糖显著增高,血清胰岛素水平明显降低,胰岛萎缩,β细胞数量减少,细胞功能指数降低,而胰岛素抵抗指数正常。结论：胰岛β细胞功能损伤是引起R6/2 HD转基因小鼠继发糖尿病发生的主要因素。     

Tg2576小鼠海马发育过程中小胶质细胞和血管的变化

目的 探讨Tg2576转基因小鼠发育过程中海马CA1区小胶质细胞增殖和血管变化的规律。方法 取不同发育时间(P0、P7、P30、P180、P360) Tg2576转基因模型鼠与同时间点野生鼠,通过应用免疫组织化学、TUNEL、墨汁灌注、RT-PCR和透射电镜等方法研究海马发育过程中小胶质细胞和血管的变化。结果 随着小鼠的生长发育,P180后转基因组海马CA1区小胶质细胞密度和血管体密度高于对照组小鼠,RT-PCR结果显示,P360时转基因组海马CA1区小胶质细胞更多处于激活状态。 结论 小胶质细胞与血管改变的共同作用加重了阿尔茨海默病。     

Analysis of the immune response induced by a single xenoantigen in vivo

Transgenic mice expressing human major histocompatibility complex (MHC) class II molecules would provide a valuable model system for studying murine anti-human MHC immune response. We have previously shown that skin from HLA-DR1 transgenic mice was rejected by control littermates and spleen cells from rejecting mice were able to proliferate to donor cells. The aim of this paper is to analyze the mechanism of recognition of this xenoantigen and the possible involvement of antibody response in anti-HLA-DR1 immune response. Control littermates were immunized with spleen cells from HLA-DR1 transgenic (TG) mice; at indicated times, xenoantigen-specific proliferation and IFNgamma production was assessed using APC obtained from HLA-DR1 TG mice. Mixed direct-indirect pathway of xenoantigen recognition was suggested by the following findings: i)T cell response to HLA-DR1 was inhibited adding in culture monoclonal antibodies directed either to donor (HLA-DR) or to recipient MHC (I-A); ii) APC from control mice pulsed with purified DR1 molecules were able to induce proliferation by FVB/N mice immunized with transgenic spleen cells. HLA-DR1 recognition permits DR peptide-specific T cell response by lymphocytes of control littermates immunized with the xenoantigen. In addition, we detected xenoreactive IgM and IgG2 antibodies. Our data suggest that HLA-DR1 xenoantigen may be recognized through direct or indirect pathway and provide additional information on mouse anti-human HLA immune response.     

四环素调控的PTA1/CD226转基因小鼠的初步建立

为研究小鼠PTA1分子在体内的功能,建立四环素调控的小鼠PTA1/CD226转基因小鼠,我们构建了pBI-5-mPTA1载体,显微注射入B6D1F1受精卵,使用PCR检测新生小鼠基因组DNA中的PTA1与荧光素酶(luciferase)基因。将mPTA1和荧光素酶双阳性小鼠耳成纤维细胞转染含rtTA的pUHD17.1质粒,用含有盐酸强力霉素(Dox)的培养基进行培养,检测细胞裂解液中荧光素酶的活性。将荧光素酶表达依赖Dox的小鼠与C57BL/6小鼠交配,采用PCR对子代鼠进行检测。最终共获得7只首建鼠,其目的基因表达高度依赖Dox,并得到了其中2只首建鼠的F1代小鼠。     

Neisseria gonorrhoeae-mediated inhibition of apoptotic signalling in polymorphonuclear leukocytes

The human pathogen Neisseria gonorrhoeae recruits and interacts extensively with polymorphonuclear leukocytes (PMNs) during infection. N. gonorrhoeae is able to survive the bactericidal activity of these innate immune cells and can actively modulate PMN functions in vitro. PMNs are short-lived cells which readily undergo apoptosis, and thus the effect of N. gonorrhoeae infection on PMN survival has implications for whether PMNs might serve as an important site of bacterial replication during infection. We developed and validated an HL-60 myeloid leukemia cell culture model for PMN infection and used both these cells and primary PMNs to show that N. gonorrhoeae infection alone does not induce apoptosis and furthermore that N. gonorrhoeae can inhibit both spontaneous apoptosis and apoptosis induced by the intrinsic and extrinsic apoptosis inducers staurosporine (STS) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), respectively. N. gonorrhoeae infection also results in the activation of NF-κB signaling in neutrophils and induces secretion of an identical profile of proinflammatory cytokines and chemokines in both HL-60 cells and primary PMNs. Our data show that the HL-60 cell line can be used to effectively model N. gonorrhoeae-PMN interactions and that N. gonorrhoeae actively inhibits apoptosis induced by multiple stimuli to prolong PMN survival and potentially facilitate bacterial survival, replication, and transmission.     

A T cell receptor transgenic model of severe, spontaneous organ-specific autoimmunity.

The development of mouse models of human organ-specific autoimmune diseases has been hampered by the need to immunize mice with autoantigens in potent adjuvants. Even autoantigen-specific T cell receptor transgenic models of autoimmunity have proven to be complex as the transgenic mice frequently fail to develop disease spontaneously. We have isolated a CD4(+) T cell clone (TxA23)that recognizes the gastric parietal cell antigen, H/K ATPase alpha-chain(630-641), from a mouse with autoimmune gastritis that developed after thymectomy on day 3 of life. The T cell receptor alpha and beta genes from this clone were used to generate A23 transgenic mice. All A23 transgenic animals spontaneously developed severe autoimmune gastritis, and evidence of disease was detected as early as day 10 of life. Gastritis could be transferred to immunocompromised mice with a limited number of transgenic thymocytes (10(3)), but as many as 10(7) induced only mild disease in wild-type animals. Due to the complete penetrance of spontaneous disease, identity of the auto-antigen, susceptibility to immunoregulation, and close relation to autoimmune gastritis in man, A23 transgenic mice represent a unique CD4(+) T cell-mediated disease model for understanding the multiple factors regulating organ-specific autoimmunity.     

Experimental Gonococcal Genital Tract Infection and Opacity Protein Expression in Estradiol-Treated Mice

The development of effective prophylactic agents against gonorrhea and the study of adaptation by Neisseria gonorrhoeae to the urogenital mucosa are hindered by the lack of a well-established animal model of gonococcal genital tract infection. Here, a murine model of long-term gonococcal genital tract infection is described. Female BALB/c mice were treated with 17-beta-estradiol and inoculated intravaginally with wild-type gonococcal strain FA1090 or MS11. N. gonorrhoeae was recovered from vaginal swabs for an average of 12 to 13 days following inoculation with 10(6) CFU of either strain. Inflammation occurred in over 80% of infected mice, and diplococci were associated with epithelial cells and neutrophils in stained vaginal smears. Ascended infection occurred in 17 to 20% of mice inoculated with strain FA1090. An outbred mouse strain (SLC:ddY) previously reported to be naturally susceptible to N. gonorrhoeae was also tested; however, as with BALB/c mice, estradiol was required for prolonged infection. Although piliation was not maintained during experimental murine infection, 46 to 100% of vaginal isolates from four of eight BALB/c mice and three of four SLC:ddY mice expressed one or more opacity (Opa) proteins within 4 days after inoculation with an Opa-negative variant of strain FA1090. The observed selection for and/or induction of gonococcal Opa protein expression during murine infection appears to parallel events that occur during experimental urethritis in volunteers.