乙醇诱导肝癌细胞凋亡及其单克隆抗体的制备

目的：探讨原发性肝细胞癌（简称肝癌）细胞凋亡过程中相关分子的表达情况,制备抗肝癌凋亡细胞相关抗原的单克隆抗体（mAb)。方法：用含6％乙醇的培养液培养肝癌细胞系HCC－9204细胞6h诱导其凋亡,透射电镜观察细胞形态变化,TUNEL法检测细胞DNA片段化发生情况,流式细胞仪分析细胞DNA含量变化。环磷酰胺消减免疫法免疫小鼠。杂交瘤法制备抗凋亡细胞的mAb。ELISA法筛选在凋亡细胞高表达,而在非凋亡细胞低表达的mAb。有限稀释法连续克隆化。用免疫细胞化学ABC法进一步筛选和分析,并进行杂交瘤细胞染色体和免疫球蛋白（Ig)类型分析。结果：6％乙醇作用HCC－9204细胞6h后,可见明显的凋亡形态学变化,大部分细胞为TUNEL染色阳性,并且流式细胞仪检测可见亚二倍体凋亡峰。经连续克隆化筛选出1株在乙醇诱导凋亡的HCC－9204细胞的胞核高表达,而在未经诱导凋亡的HCC9－9204细胞低表达的mAb。染色体分析结果符合mAb杂交瘤细胞特征,其Ig类型为IgG1。结论：低浓度乙醇诱导凋亡的HCC－9204肝癌细胞中有某些相关分子的表达升高。初步获得了1株可能是抗乙醇诱导的肝癌细胞凋亡相关抗原的mAb。     

凋亡对肿瘤细胞表面抗原活性的影响

通过以常规免疫法和削减法分别制备抗кＢ凋亡细胞相关抗原的抗体Ａｂ１和Ａｂ１ａ，以ＥＬＩＳＡ法检测抗原抗体反应，观察肿瘤细胞在凋亡过程中表面相关抗原活性的变化及新抗原的表达。结果显示Ａｂ１与кＢ凋亡及非凋亡细胞的表面相关抗原均有较强反应，而Ａｂ１ａ，只与凋亡细胞表面抗原反应较强。     

新的人突触相关蛋白(FRG4)抗原表位分析及抗体制备

目的：分析一个新的人突触相关蛋白(FRCA)抗原表位并制备其多克隆抗体。方法：从人胎肝文库PCR扩增获得FRCA基因全长cDNA序列；通过生物信息学分析，预测FRG4编码氨基酸序列的二级结构、抗原决定簇、功能结构域，并进行了多序列比对；采用固相多肽合成法合成了FRCA抗原多肽，并免疫家兔；用免疫组化检测蛋白在人肝癌细胞HepG2细胞中的表达。结果：通过生物信息学分析选取抗原13肽PKLVKEEVFWRNY，制备兔抗人FRCA多克隆抗体。高效液相色谱检测显示抗体纯度达82．79％，抗体滴度为1：16000，Western blot证实该抗体具有较好的反应性和特异性，免疫组化证实其主要在HepG2细胞胞浆中表达。结论：成功制备了新的人突触相关蛋白(FRCA)多克隆抗体。     

甲胎蛋白对树突状细胞免疫表型影响的体外研究

目的：研究甲胎蛋白（Alpha Fetoprotein,AFP)对树突状细胞（DendriticCell,DC)免疫表型的影响。方法：用流式细胞仪分析体外培养扩增的小鼠骨髓DC表面H-2K^b、I-A^b、B-7-1和B7-2等免疫分子的表达。结果：与对照组相比,AFP可明显上调DC的B7-1和B7-2分子水平、下调H-2K^b和I-A^b的表达、但抑制I-A^b的程度较轻;AFP抗原抗体复合物极度抑制H-2K^b分子表达、对I-A^b的下调程度高于AFP组,而对B7-1和B7-2分子几乎无影响：AFP抗体和人血清白蛋白（HSA）对DC免疫分子活性的影响均较弱,结论：AFP可作为肝癌相关抗原,致敏DC,部分上调DC免疫活性分子的表达。     

抗汉坦病毒NP抗原细胞内抗体的瞬时表达及活性检测

目的 :在真核细胞中表达抗汉坦病毒NP抗原鼠源性单克隆抗体 (mAb) 1A8的scFv ,并检测表达产物的活性。方法 :将真核表达载体 1A8 scFv Cκ/pCI neo用脂质体转染COS 7细胞 ,用间接免疫荧光和免疫共沉淀法检测瞬时表达产物的免疫学活性。结果 :间接免疫荧光的结果表明 ,约 1%细胞胞质中出现弥散荧光 ,在免疫共沉淀 /免疫印记中 ,表达的抗体片段可与汉坦病毒的NP抗原特异性结合。结论 :细胞内表达的1A8 scFv Cκ可与NP抗原特异性结合 ,为进一步研究细胞内抗体的抗病毒功能奠定基础     

免疫学

猪FasL基因在猪软骨细胞上的表达和鉴定；重组蓖麻毒紊A链的融合表达、纯化及活性研究；凋亡抑制蛋白survivin在活化T细胞中的表达及其意义；人CD59基因的突变和表达及其活性研究；预先形成的重链二硫键有助于HLA-A2-抗原肽复合物体外折叠；人源噬菌体抗体库的构建及抗人NH-LBP抗体的筛选与鉴定；抗禽流感病毒轻链抗体库的构建；CTL识别的HLA-A2限制性人卵巢癌相关抗原OVA66表位的鉴定；含多抗原表位的嵌合SARS-CoV基因疫苗的构建和免疫原性研究；TM-TNF-α介导的反向信号协同增强sTNF-α，对U937细胞的激活作用；NK92细胞的胞吐效应与SNAP-23蛋白转位相关；CXCL16抗体可阻断BCG和LPS诱导的小鼠免疫性肝损伤；GM-CSF对抗原化抗体基因免疫TH细胞应答的调节作用；趋化性细胞因子受体在Tc1和Tc2亚群的差异表达；在单个细胞水平上分析抗原特异性Th1／Th2细胞因子产生的关联性；人OX40-Fc分子的构建、真核表达及活性研究；人免疫缺陷病毒Ⅱ型核心蛋白DNA疫苗的实验免疫研究；PD-L1 mAb的制备、鉴定及其特性的研究；噬菌体呈现的多聚多肽3A8诱导Jurkat细胞的凋亡效应；T细胞受体Dβ-Jβ sjTRECs连接区多样性；α-Dystroglycan在胸腺T细胞的表达及其对胸腺发育的作用；Bcl-2家族蛋白参与调节CD3ε和5-氟尿嘧啶介导的T淋巴细胞凋亡；人白细胞介素21在真核细胞中表达及其体外促T细胞增殖作用；小干扰RNA特异性抑制淋巴细胞共刺激分子CD28基因表达的研究；猪-猕猴延迟性异种移植排斥反应的发生机制；天花粉蛋白在不同小鼠品系中区分性下调树突状细胞IL-12分泌和CD80表达；复合HA-2氨基多肽可增强chitosan-DNA疫苗的免疫保护效果；CD4^＋CD25^＋调节性T细胞抑制小鼠自身免疫性甲状腺炎的发生；人β2-微球蛋白在pET-22b（＋）表达载体中的克隆、表达与纯化；B7-1／B7-H1相对比例变化对Hsp70-肽复合物抗肿瘤免疫效应的影响。     

抗人凋亡诱导因子单克隆抗体的制备与鉴定

背景：凋亡诱导因子不仅具有氧化还原和电子传递功能,还具有促细胞凋亡功能,从而在维持细胞正常生理活动中具有重要作用。 目的：制备抗人凋亡诱导因子单克隆抗体并进行生物学特性鉴定。 方法：利用Ensembl数据库及DNAstar软件包对凋亡诱导因子氨基酸序列进行分析,获得优势表位多肽,采用碳化二亚胺法将多肽与载体蛋白偶联制备完全抗原免疫动物,采用杂交瘤技术制备凋亡诱导因子单克隆抗体并纯化。 结果与结论：间接ELISA法检测显示,免疫鼠腹水中抗人凋亡诱导因子单克隆抗体效价达到1∶252 400。Western blot结果显示,存在与抗原带一致的相对分子质量67 000的特异条带。免疫组化检测结果显示,在结肠癌组织细胞中有棕色阳性颗粒表达,说明此抗体也可用于免疫组识化学染色。提示实验获得了高活性、高纯度、高特异性抗人凋亡诱导因子单克隆抗体。     

基于可重复利用免疫磁珠的抗体检测方法的建立

目的基于可重复利用的免疫磁珠,建立一种简捷快速的特异性抗体检测方法。方法设计人类巨细胞病毒（human cytomegaloviruses,HCMV）PP150蛋白的抗原表位,并合成8分枝多聚抗原肽PP150-8MAPs。将PP150-8MAPs以共价结合形式包被于Dynabeads M-450 Tosylactivated磁珠表面,制备特异性免疫磁珠。用PP150-8MAPs免疫Balb／c小鼠制备抗此抗原表位的标准抗血清。应用免疫磁珠检测标准抗血清中的抗体,优化检测条件。在抗体检测反应结束后,洗脱抗体-二抗复合物,再生后的免疫磁珠重复用于标准抗血清样品的检测分析,并分析免疫磁珠可重复利用的次数。结果制备免疫磁珠时,PP150-8MAPs的最佳包被量为100μg／mL,包被效率为79％。用PP150-8MAPs免疫小鼠后,得到的抗血清滴度可达到1：12800。用免疫磁珠法检测小鼠标准抗血清,免疫磁珠可重复进行20次以上的检测分析。结论基于酶联免疫检测方法,包被抗原的免疫磁珠可重复应用于血清样品中特异性抗体的检测分析。此实验为建立一种新的可重复检测的高效清样品中特异性抗体的检测分析。此实验为建立一种新的可重复检测的高效免疫磁珠抗体分析方法奠定了基础,在基础研究与临床检测中具有广泛的应用前景。     

抗人FKBP52分子单克隆抗体的制备和鉴定

目的；研制抗人FKBP52单克隆抗体（mAb)，并分析其免疫生物学特性。方法：采用纯化的人FKBP52蛋白免疫BALB／c小鼠。用ELISA法鉴定mAb的特异性；用Western blot鉴定制备的9株mAb所识别的抗原相对分子质量（Mr)及结合人FKBP52分子的功能区。结果：共获得9株分泌抗人FKBP52 mAb的杂交瘤细胞。抗人FKBP52 mAb可识别相对Mr为52000在原核系统中表达的人FKBP52蛋白，也可识别Jurkat细胞浆中Mr为52000的天然FKBP52蛋白。两株mAb均可识别人FKBP52分子中的第2功能区。其余7株mAb可识别人FKBP52分子中的第3功能区。结论：成功地制备了抗人FKBP52分子mAb。     

肝癌凋亡细胞相关蛋白的表达分析

目的；分析肝癌凋亡细胞与肝癌细胞所表达的蛋白质在分子量分布上的差异。方法：ＳＤＳ＿ＰＡＧＥ电泳后薄层扫描。结果：肝癌凋亡细胞所表达蛋白质的分子量普遍较大。在分子量大于６．７×１０＾４ｕ时，凋亡的肝癌细胞有７个较为明显的蛋白扫描峰，与未凋亡的肝癌细胞具有明显的区别。部分大分子量的凋亡相关蛋白在肝癌凋亡细胞表达量很高，其蛋白质扫描峰的面积可占总面积的８％。结论：肝癌凋亡细胞有较大分子量的蛋白质表达，可     

Identification of epididymis-specific antigens using neonatal tolerization

PROBLEM: Conventional immunization using whole sperm containing multiple antigens as the immunogen followed by hybridoma technology usually gives antibodies to antigens invariably of testicular origin, probably because of the strong immunogenic nature of these antigens. Therefore, an alternate approach of neonatal tolerization or subtractive immunization has been utilized to raise antibodies specific to epididymis by suppressing immune response to testicular antigens. METHOD OF STUDY: Neonatal mice were tolerized with testicular sperm proteins on days 0 and 5. These animals were then immunized with epididymal sperm proteins on day 21, followed by two boosters at biweekly intervals. Sera from these mice were used to localize epididymis-specific antigens. RESULTS: Sera from mice that were tolerized to testicular sperm proteins and later immunized with epididymal sperm proteins reacted only with epididymal proteins. CONCLUSION: The results of this study demonstrate that neonatal tolerization with testicular sperm proteins, followed by immunization with epididymal sperm proteins, enhances the production of antibodies to proteins exclusively of epididymal origin.     

Mucosal immunity in the genital tract: prospects for vaccines against sexually transmitted diseases--a review.

PROBLEM: Consistent with the absence of protective immunity resulting from previous infection with Neisseria gonorrhoeae, the genital mucosal immune response in human gonorrhea is weak: only low levels of immunoglobulin G (IgG) and immunoglobulin A (IgA) antibodies are detectable against gonococci, and inflammatory cytokine responses are poor. METHOD OF STUDY: Mucosal immunization strategies designed to induce persisting genital antibody responses might afford protection against infection, if appropriate conserved antigens can also be identified. RESULTS: Intragastric or intranasal immunization with bacterial antigens expressed as recombinant chimeric proteins with cholera toxin A2/B subunits induced persisting IgA antibodies in genital and other secretions, and circulating IgG antibodies. CONCLUSION: Although gonococci may avoid inducing or even suppress immune responses during natural infection, alternative approaches to vaccine development may be successful. However, inadequate understanding of the origins of antibodies in the genital tract, and their effector mechanisms, will need to be rectified to make this possible.     

DNA immunization followed by a single boost with cells: a protein-free immunization protocol for production of monoclonal antibodies against the native form of membrane proteins

Recent advancements in antibody-based therapies require the development of an efficient method for generation of monoclonal antibodies (MAbs) against the native form of membrane proteins. We examined DNA immunization followed by a single boost with cells as a protein-free immunization protocol for production of MAbs. Mice immunized with plasmid cDNAs encoding human CD30 or Ret tyrosine kinase were given a single boost with cells expressing the corresponding antigen prior to cell fusion. A total of nine cell fusion experiments revealed that the cell boost is necessary for efficient generation of hybridomas and the DNA-cell boost method gave good yields of specific MAbs (5–59 MAbs from one mouse). All IgG isotypes except IgG3 were generated, although IgG2a was the dominant isotype. All the MAbs reacted with native antigens expressed on cells in a fluorescence-activated cell sorter (FACS) analysis as well as with recombinant CD30 or Ret protein genetically fused with human Fc in an enzyme-linked immunosorbent assay (ELISA). The affinities of the anti-CD30 MAbs to CD30-Fc protein ranged from 0.9 to 12.4 nM K_ds, which were comparable to existing MAbs to these proteins, which range from 3.0 to 13.0 nM. Western blot analysis and topographical epitope mapping experiments based on the mutual competition of pairs of the anti-CD30 MAbs revealed that about 40% of the epitopes were linear epitopes and that each epitope was topographically classified into one of six groups. The large number of MAbs that react with high affinities to a variety of epitopes on the native form of antigens indicates that the method presented in this paper could be generally useful for generating MAbs to other membrane proteins.     

Antigenic analysis of the Epstein-Barr virus major membrane antigen (gp350/220) expressed in yeast and mammalian cells: implications for the development of a subunit vaccine

The Epstein-Barr virus (EBV) major surface membrane antigen, gp350/220, was expressed in recombinant yeast cells and in several recombinant mammalian cell lines. Each of the expressed proteins was analyzed for its ability to bind to a panel of anti-gp350/220 monoclonal antibodies and to a series of anti-EBV positive human sera. The antigens also were used as immunogens for the immunization of rabbits. Each expressed protein was found to be unique both in its pattern of reactivity to the various antibodies and in the spectrum of antibody induced following animal immunization. These results suggest that cell-specific post-translational modifications critically influence the antigenic presentation of the expressed proteins. Nonetheless, all of the mammalian cell-derived versions of the membrane antigen were found capable of inducing EBV-specific neutralizing antibodies.     

A rapid and simple approach to preparation of monoclonal antibody based on DNA immunization

Inoculation with purified specific protein is usually the first step for preparation of monoclonal antibody (mAb).But it is quite difficult to obtain pure proteins especially with natural structures.Here we attempt to replace theprotein inoculation with DNA immunization in the preparation of mAb.The eukaryotic expression vectorspcDNA3-PreS2/S and pVAX-PreS2/S encoding the HBV M protein were constructed and prepared for DNAimmunization.Female BALB/c mice developed a well antibody response to the target antigen after muscleinjection with corresponding plasmids.The mice with effective antibodies induced were used for preparation ofmAb.We found the mice immunized with three administrations of pcDNA3-PreS2/S and boosted byintrasplenic injection with the same plasmid could be exploited for preparation of mAb.And positivehybridoma cell 2D3 that can secrete specific mAb was cloned and analyzed.Our studies demonstrate that geneimmunization may provide a convenient and efficient way to prepare mAbs.Cellular & Molecular Immunology.2004;1(4):295-299.     

不同免疫方案制备抗HAb18G/CD147胞外区多克隆抗血清的比较

目的: 制备针对肝癌相关抗原HAb18G/CD147胞外区不同表位的抗血清, 比较不同免疫方案的免疫效果。方法: 以原核表达的GST -HAb18GEF融合蛋白、重组真核表达质粒pcDNA3 /HAb18G及HCC细胞为免疫原, 分别采用蛋白常规免疫、DNA肌肉免疫及pcDNA3 /HAb18G -HCCbooster(DNA -cellbooster)免疫接种BALB/c小鼠。采用间接ELISA和细胞ELISA, 同时测定免疫血清中抗变性和天然HAb18GEFIgG抗体的滴度和Ig亚类。用Westernblot检测各免疫方案制备的抗血清, 与变性HAb18GEF抗原结合的特异性。用免疫荧光法验证DNA- cellbooster免疫接种法制备的抗血清, 与细胞膜上天然HAb18G抗原的结合特异性。结果:以GST- HAb18GEF常规免疫后, 可诱导高滴度的IgG1抗体产生, 但针对的多为HAb18GEF的变性或线性表位; 以pcD -NA3 /HAb18G肌肉免疫后, 可诱导针对其天然表位的IgG2a抗体产生, 但滴度较低; 以DNA -cellbooster免疫后, 可诱导中等滴度、针对其天然表位的IgG2a和IgG1抗体产生。结论: 不同免疫方案可诱导针对HAb18GEF不同表位、不同滴度的多克隆抗血清, 为淘筛针对其不同表位的多样性抗体奠定了基础。     

蛋白表达的亚细胞定位影响基因免疫诱导的体液免疫应答水平的研究

目的研究表达产物的不同亚细胞定位对基因免疫诱导的体液免疫应答水平的影响,为基因疫苗的设计提供参考。方法利用分子克隆技术,分别构建能表达不同细胞定位的EGFP-HPVl6L1融合蛋白和截短型EGFP-HPV16L1△NLS融合蛋白的重组pcDNA-EGFP-HPV16L1和pcDNA-EGFPHPV16L1△NLS真核表达载体;通过转染CHO细胞,并在倒置荧光显微镜下观察表达产物的细胞定位;用重组pcDNA载体免疫BALB/c小鼠;EGFP作为抗原,ELISA法检测血清抗体吸光度。结果①重组pcDNA-EGFP-HPV16L1转染的CHO细胞核内可见到绿色荧光,pcDNA-EGFP-HPV16L1△NLS真核表达载体转染的CHO细胞质内可见到绿色荧光:②两种不同的重组pcDNA载体均可诱导BALB/c小鼠体液免疫反应,但重组pcDNA-EGFP-HPV16L1△NLS真核表达载体免疫组小鼠IgG抗体A_(450)值显著高于重组pcDNAEGFP-HPV16L1真核表达载体免疫组小鼠IgG抗体A_(450)值(P0.001)。结论表达产物的不同细胞定位可影响基因免疫诱发的体液免疫应答水平,定位于细胞质内的蛋白分子较定位于细胞核的蛋白分子能诱导机体更强的体液免疫反应,这为以增强体液免疫反应为目的的基因疫苗的设计提供了参考。     

Effects of immunization and checkpoint inhibition on amodiaquine-induced liver injury

If idiosyncratic drug-induced liver injury (IDILI) is immune-mediated, it is possible that an individual’s prior exposure to antigens may affect their susceptibility to IDILI. An individual’s repertoire of memory immune cells is shaped by every past exposure to antigens. Subsequent drug-induced adverse drug reactions may therefore involve an immune cell’s cross reactivity between a prior antigen and resulting drug-modified proteins. Therefore in this experiment, mice were immunized with amodiaquine (AQ)-modified hepatic proteins to mimic a previous exposure; treated with a RIBI adjuvant and anti-CD40 antibodies to stimulate an immune response; and, treated with anti-PD1 and anti-CTLA-4 antibodies prior to AQ treatment in order to overcome immune tolerance. This treatment led to greater liver injury than treatment with AQ alone. However, the mice did not develop serious liver injury. PD1^?/? mice were then immunized and treated with AQ and anti-CTLA-4 antibodies so that immune tolerance would be impaired, both during immunization and also during AQ treatment. However, even this did not result in liver failure, and the liver injury was not significantly increased relative to un-immunized PD1^?/? mice treated with anti-CTLA-4 and AQ. From these results we conclude that, although previous antigen exposure may affect the risk of IDILI, it appears that a very strong stimulus is required, and impairing immune tolerance remains the most effective method for producing an animal model of IDILI.