酸性成纤维细胞生长因子的分离纯化及活性鉴定

目的：从牛脑中分离纯化酸性成纤维细胞生长因子（aFGF)并鉴定生物活性，方法：根据DenisGOSPODAROW-ICZ的方法，新鲜牛脑匀浆，三步硫酸铵盐析。最后沉淀物溶解透析后予离子交换层析及琼脂糖胶亲和层析，促3T3细胞DNA合成率鉴定生物活性。结果：用1.0mol/LNaCl的缓冲液洗脱得到分子量为13600的多肽，得率为610μg/kg牛脑，氨基酸组成分析与已知的aFGF相同，促3T3细胞DNA合成的最低浓度为0.5ng/ml，最大效应浓度50ng/ml，ED50值为8ng/ml。结论：aFGF具有强烈的促细胞增殖作用。     

多粘菌素B琼脂糖层析柱清除体液中内毒素

用多粘菌素B琼脂糖亲和层析法清除内毒素,结果表明:5ml多粘菌素B层析柱的总吸附内毒素能力为450 μg,用此法可完全清除体液或各种液体中的内毒素,对血清及腹水中的内毒素也有明显的吸附作用,而其他各种主要成分(除内毒素外)经过处理后无明显改变。去氧胆酸是一种强有力的去污剂,可使已饱和的柱子复活,复活率达85％左右。该方法有简便,可靠,吸附能力大,柱子的复活率高等优点。本方法的建立为内毒素血症的治疗展示了新的前景。     

凝集素受体在膀胱癌中的分布

目的：探讨膀胱癌中凝集素受体分布与其分化程度和浸润深度的关系。方法：应用生物素标记的花生凝集素（ＰＮＡ）、麦胚凝集素（ＷＧＡ）及刀豆凝集素（ＣｏｎＡ）等３种凝集素对５２例人体膀胱癌、１０例正常人体膀胱粘膜，进行亲合组织化学法研究。结果：发现正常膀胱粘膜ＰＮＡ、ＷＧＡ受体阴性，ＰＮＡ受体阳性率随膀胱癌病理分级的上升而递增，差异具有显著性意义（Ｐ＜０．０５）。ＰＮＡ、ＷＧＡ受体阳性率在浸润性肿瘤中明显高于浅表性肿瘤（Ｐ＜０．０５）。结论：提示ＰＮＡ、ＷＧＡ受体阳性率与膀胱癌分化程度和浸润深度有关。     

Response of V beta 8.1+ T cell clones to self Mls-1a: implications for the origin of autoreactive T cells.

Clonal deletion and anergy are two major mechanisms of self-tolerance. However, the molecular mechanisms underlying clonal deletion and anergy, as well as the threshold of TCR affinity/avidity required for these processes, are not known. Expression of the V beta 8.1 TCR correlates with the reactivity of the T cells to the minor lymphocyte stimulating locus-1a (Mls-1a) and T cells expressing this TCR are deleted in the thymus of Mls-1a mice. Similarly, in TCR V beta 8.1 transgenic mice, the number of CD4+CD8-T cells is reduced in Mls-1a mice. However, small numbers of CD4+CD8-T cells remain in the periphery of adult Mls-1a transgenic mice. We have generated T cell clones from TCR V beta 8.1 transgenic mice by stimulation of lymph node T cells with C57BL/6 alloantigens. Interestingly, CD4+CD8-V beta 8.1+ clones isolated from the transgenic mice of Mls-1a background responded to the self-antigen Mls-1a, to which they did not respond in primary assay. Reactive patterns of the clones were compared with clones derived from Mls-1b mice. Proliferation and cytokine production of the clones from Mls-1a mice to the self-antigen Mls-1a were generally reduced when compared with clones from Mls-1b mice. More importantly, T cell clones from Mls-1a mice required more Mls-1a antigen for their activation, and were more susceptible to the inhibitory effects of anti-CD4 antibody on the proliferative responses to Mls-1a than those from Mls-1b mice. These results suggest that the T cell receptor on clones derived from Mls-1a mice have functional but reduced affinity/avidity for self-antigen Mls-1a.     

兔乳酸脱氢酶C4的分离纯化及动力学研究

建立兔乳酸脱氢酶Ｃ４（ＬＤＨ－Ｃ４）分离纯化的新方法，研究该酶的动力学特性。采用Ｂｌｕｅ Ｄｅｘｔｅｒａｎ和Ｓｅｐｈａｒｏｓｅ ４Ｂ交联制成Ｂｌｕｅ Ｄｅｘｔｒａｎ－Ｓｅｐｈａｑｒｏｓｅ ４Ｂ染料配体亲和层析柱，结合ＱＡＥ－Ｓｅｐｈａｄｅｘ Ａ４５０离子交换层析分离兔ＬＤＨ－Ｃ４，以作图法测定该酶动力学数据。     

胎盘生长因子串联scFv质粒构建表达及亲和力测定

目的:构建并表达胎盘生长因子串联scFv,提升对胎盘生长因子的亲和力。方法:设计不同长度的柔性连接子连接的串联scFv,构建串联scFv质粒,并分别将串联scFv与亲本scFv于原核表达系统进行表达。通过间接ELISA分别测定串联scFv及对应亲本scFv亲和力,分别对数据进行单因素ANOVA方差分析,并使用Dunnett-t检验进行组间比较。结果:柔性连接子长度的不同,会对串联scFv亲和力造成差别。单因素ANOVA方差分析结果表明,各组亲和常数间存在明显的统计学差异,Dunnett-t检验组间比较结果可知,当串联scFv间柔性连接子（GGGGS）n长度n=4和n=5时,亲和力较亲本提升不明显（P＞0.05）;但亲和力在n=6（t335=33.90,t7A10=32.00,均P<0.000 1）及n=7（t335=91.68,t7A10=90.71,均P<0.000 1）时较两亲本均有明显提升。结论:串联scFv可以明显提高单体scFv亲和力。     

Qualitative difference between the cytotoxic T lymphocyte responses to melanocyte antigens in melanoma and vitiligo

Vitiligo is a skin disorder characterized by depigmented macules secondary to melanocyte loss. An unusual facet is its relation to melanoma: cytotoxic T lymphocytes directed to melanocyte antigens are found in both conditions and imply a breakdown of tolerance, yet the resulting immune reaction is the opposite. The mechanisms at the basis of these opposite effects are not known. Here, we performed a direct comparison of whole melanocyte-specific T cell populations in the two diseases. We demonstrate that neither precursor frequencies of Melan-A/MART-1-specific T lymphocytes nor their status of activation differ significantly. However, by using a tetramer-based T cell receptor down-regulation assay, we documented a higher affinity of vitiligo T cells. We calculated that the peptide concentration required for 50% of maximal receptor down-regulation differed by 6.5-fold between the two diseases. Moreover, only vitiligo T cells were capable of efficient receptor down-regulation and IFN-gamma production in response to HLA-matched melanoma cells, suggesting that this difference in receptor affinity is physiologically relevant. The differences in receptor affinity and tumor reactivity were confirmed by analyzing Melan-A/MART-1-specific clones established from the two diseases. Our results suggest that the quality, and not the quantity, of the melanocyte-specific cytotoxic responses differs between the two pathologies.     

生物工程技术制备人源抗-HBs Fab 片段

目的：用生物工程技术制备人源性抗-HBsFab。方法：将从抗体文库中筛选出的人源抗-HBsFab基因克隆入pBAD/gⅢA载体，进而转化Tpo10大肠杆菌，对重组质粒菌发酵表达后，利用Ni-NTA-Agarose螯合层析柱纯化周质腔可溶性Fab蛋白。对所得包涵体依次变性，溶解，纯化后，利用透析进行复性，用Western blot检测Fab蛋白的特异性，Dot blot测定其生物学活性。结果：经Ni-NTA-Agarose柱纯化的周质腔可溶性Fab蛋白，有较好的生物学活性，并且总量达到80mg/L。对所获包涵体进行透析复性后，也可得到少量有活性的蛋白，但比例很小。结论；用pBAD/gⅢA-Top10表达系统表达人源抗-HBsFab片段，发酵培养后，经有效纯化可得到生物学活性较好的可溶性蛋白。为人源抗-HBsFab片段的大量制备提供了有效手段。     

抗SARS病毒N蛋白单克隆抗体的制备和初步应用

目的 制备抗SARS病毒N蛋白的单克隆抗体并研究其初步应用。方法 用基因重组N蛋白免疫小鼠，取免疫后的鼠脾细胞与骨髓瘤细胞融合，筛选分泌抗SAPS病毒N蛋白单克隆抗体细胞株。将阳性细胞株接种小鼠腹腔制备单克隆抗体腹水并对抗体进行纯化，分析纯化抗体的相对亲和力。选择亲和力较高的抗体制备检测SARS病毒抗原的酶联免疫诊断试剂，并对其敏感性和特异性进行分析。结果 共获得11株单克隆抗体细胞株，其中3株单抗与N蛋白具有较高的亲和力，4株纯化单抗与N蛋白反应很弱，其余4株单抗介于两者之间。用亲和力较高的单抗制备检测SARS病毒抗原的诊断试剂，其敏感性可达31PFU／ml，而且与其他呼吸道病毒无交叉反应。结论 该试剂特异性较好，可用于SARS病毒抗原的检测，其敏感性仍需用临床急性期样品进行评价。