应用噬菌体随机肽库技术筛选幽门螺杆菌中性粒细胞激活蛋白的抗原表位

目的 筛选和鉴定幽门螺杆菌(Helicobacter pylori,Hp)中性粒细胞激活蛋白(neutrophil-activating protein,NAP)的有效抗原表位,为Hp疫苗的研制提供基础.方法 以抗NAP的单克隆抗体作为固相筛选分子,经3轮吸附-洗脱-扩增免疫,筛选噬菌体随机7肽库,随机挑选噬菌体克隆,经噬菌体酶联免疫吸附试验(ELISA)、交叉反应试验及竞争抑制试验鉴定阳性克隆,测定阳性克隆所携带DNA序列并进行计算机辅助分析.以制备的阳性噬菌体克隆短肽液免疫小鼠,免疫血清与NAP经Western blot分析,以验证NAP的模拟表位.结果 经3轮免疫筛选后挑选到45个阳性克隆,经ELISA鉴定有12个阳性克隆,测序结果显示5种表位,其中P17噬菌体展示肽FAHLATQ与NAP氨基酸序列(137～143)高度同源,位于NAP高抗原区域(118～140),免疫血清可识别NAP.结论 用噬菌体随机7肽库成功筛选到了NAP的模拟表位,为基于NAP的诊断和疫苗的研制提供了基础.     

鸡柔嫩艾美耳球虫cDNA文库构建与表达序列标签分析

为系统分析鸡球虫敏感虫株和抗药虫株不同发育阶段的基因表达情况,利用柔嫩艾美耳球虫敏感株和抗马杜霉素株(由敏感株诱导)的未孢子化卵囊、孢子化卵囊、子孢子和裂殖子为材料构建了一个混合cDNA文库,并用该文库获得了2806条3'端高质量的表达序列标签(ESTs)。通过生物信息学分析:EST序列拼接出1424个假定独立转录本(TUTs),冗余度为49.3%。从cDNA文库中筛选出大量的低丰度表达基因,约占TUT总数的91.5%,且功能未知基因约占TUT总数的83.6%。在功能注释基因中,编码MIC2蛋白、BT1家族蛋白和核糖体蛋白等入侵和发育相关的基因高丰度表达。     

人源抗人免疫缺陷病毒1型噬菌体抗体库的构建及筛选

目的构建人免疫缺陷病毒１型（ＨＩＶ－１）特异性噬菌体抗体库，制备人源抗ＨＩＶ－１ｇｐ１２０单克隆抗体。方法以半巢式聚合酶链反应（ＰＣＲ）从ＨＩＶ－１感染者外周血单个核淋巴细胞中扩增抗体重链Ｆｄ和轻链（ｋ）基因，与噬菌体载体ｐＣｏｍｂ３连接，构建噬菌体抗体Ｆａｂ组合文库。对抗体库进行３轮吸附－洗脱－扩增的亲和选择后，以ＥＬＩＳＡ法筛选抗ＨＩＶ－１ｇｐ１２０噬菌体抗体，并进行ＤＮＡ序列分析和Ｆａｂ的可溶性表达。结果半巢式ＰＣＲ有效地扩增出Ｆｄ和ｋ基因，以此构建成容量为１９５×１０７的噬菌体抗体库。３轮亲和选择使特异性抗体得到高度富集，抗ＨＩＶ－１ｇｐ１２０噬菌体抗体阳性克隆占３２％。对一阳性克隆抗体基因ＣＨ１和ＣＬ部分ＤＮＡ序列进行了测定，并在大肠杆菌表达出可溶性Ｆａｂ。结论抗ＨＩＶ－１特异性噬菌体抗体库的构建和人源抗ＨＩＶ－１ｇｐ１２０单克隆抗体的制备为今后筛选抗ＨＩＶ中和抗体奠定了基础，具有重要的应用价值。     

斯氏肺吸虫成虫半胱氨酸蛋白酶cDNA片段的克隆和序列分析

目的　克隆斯氏肺吸虫成虫半胱氨酸蛋白酶cDNA片段 ,并对其进行测序和序列分析。方法　利用简并引物 ,进行RT PCR ,扩增斯氏肺吸虫成虫半胱氨酸蛋白酶cDNA片段。TA克隆装入pUCm T载体 ,进行鉴定、测序 ;利用DNASIS程序推导其所编码的氨基酸序列 ,并与相关虫种半胱氨酸蛋白酶进行氨基酸序列的同源性分析。结果　RT PCR扩增出了一约5 0 0bp的cDNA片段 ,对阳性克隆测序后获得其核酸序列 ,长 4 95bp。将推导的氨基酸序列作同源性分析显示 ,该序列与相关虫种半胱氨酸蛋白酶存在着较高的同源性 ,组成半胱氨酸催化三联体的半胱氨酸、组胺酸和天冬酰胺残基高度保守。结论　克隆获得了斯氏肺吸虫成虫半胱氨酸蛋白酶cDNA片段 ,该片段包含了与半胱氨酸蛋白酶活性和空间结构相关的重要基因位点。     

伤寒减毒活疫苗Ty21a免疫前后小鼠肠细胞差异表达的基因

以基因表达谱芯片对Ty2 1a免疫前后小鼠肠细胞 (包括肠粘膜上皮细胞和肠上皮间淋巴细胞 )基因表达的差异性进行研究比较。将 490条经抑制消减杂交法筛选出的与小鼠Ty2 1a免疫相关的cDNA制备成表达谱芯片 ;利用免疫前后小鼠肠细胞的mRNA通过逆转录方法 ,将Cy3和Cy5两种荧光分别标记到两种组织的cDNA上 ,制备成cDNA探针 ,并与表达谱芯片进行杂交及扫描 ,单点重复 2次实验 ,通过计算机数据处理判定基因是否在上述两种细胞群中有表达差异。筛选出差异表达基因共 98条 ,其中 92条为表达上调基因 ,6条为表达降低基因。提示 ,基因表达谱芯片技术是高通量进行基因表达模式研究的方法 ,可同时定量研究大量的基因表达水平 ,从而鉴定可能参与免疫的基因。     

HIV-1gp41核心结构模拟位的筛选与鉴定

目的：筛选并鉴定HIV-1 gp4l核心表位。方法：用识别HIV-1 gp41的构象特异性单克隆抗体NC-1筛选噬菌体12肽库，通过夹心ELISA、NC-1特异性阻断实验、竞争抑制实验鉴定阳性噬菌体克隆，DNA序列分析阳性克隆。结果：经3轮筛选，随机挑取24个噬菌体克隆，ELISA鉴定表明有lO个克隆可与NC-1结合，DNA序列分析并推导氨基酸序列，共5种序列：HDVHHRWVYLLS、ITVNEWLYTSEQ、HGRSHGMFKPKR、MGPIARPHWHLN、DMYRSPRPKPDT。其中gp41N肽和C肽所形成的复合物可特异性阻断表达HDVHHRWVYLLS，VNEWLYTSEQ和MGPIARPHWHLN的克隆与NC-1的结合。结论：所得序列HDVHHRWVYLLS，VNEWLYTSEQ及MGPIARPHWHLN模拟HIV-1 gp41六螺旋束核心表位。     

A second group of hepatitis C viruses

cDNA clone 11–7 was isolated by immunoscreening a cDNA library that was prepared from a pooled plasma of non-A non-B hepatitis (NANBH) patients using expression vector gt 11. This cDNA corresponds to known nucleotide positions 3983–4745 of the genome of hepatitis C virus (HCV). This clone was used as a probe for screening the HCV-related cDNAs in a cDNA library similarly prepared by using gt 10. As a result, six more cDNA clones were isolated and analyzed for their nucleotide sequences. The results strongly suggested that there are at least two groups of HCV, group I and group II. According to our classification, the prototype HCV and clone 11–7 belong to group I HCV, and their nucleotide and deduced amino acid sequences were diverged from those of group II HCV. Genetic variation observed in the nucleotide and the amino acid sequences between the two groups resembles that in the NS3 region of the genome between Japanese encephalitis virus and West Nile fever virus. Polypeptides produced inEscherichia coli carrying a clone 11–7 or a group II cDNA clone E reacted with antibodies in the blood of 12 or 4 out of 14 individual chronic NANBH patients, respectively. Our data clearly indicate the existence of a second group of HCV.     

Characterization of a cosmid library from flow-sorted chromosomes 11

A cosmid library specific for human chromosome 11 has been constructed from flow-sorted chromosomes. The flow-purified chromosomes were prepared from the hamster/human hybrid line J1 which contains chromosome 11 as the only human chromosome. Individual clones were sampled in 187 microtitre plates, resulting in a total of 17 952 colonies. Hybridization analysis revealed that 83.7% of these clones were of human and 10.4% of hamster origin. The average insert size was estimated at 33.6 kb, and only 2.4% of insert fragments appear to be rearranged. This should result in 494 487 kb of cloned human DNA representing 3.5 chromosome 11 equivalents. We have prepared high-density nylon membranes of the arrayed library containing 1 536 single colonies per filter. We have demonstrated the usefulness of the library in the molecular genetic analysis of human chromosome 11 by testing for the presence of possibly polymorphic simple repeat motifs, by identifying cosmids that contain inserts from the telomeric ends of chromosome 11 and by assessing the potential of the library for rapid chromosome walking.     

从卵巢癌cDNA文库中筛选新的肿瘤标志物

采用SEREX法筛选了自行构建的中国人卵巢癌cDNA表达文库 ,得到 2 7个阳性克隆 ,其中 3个为全长cDNA。序列分析和同源性比对结果表明所获得的 2 7个克隆分属于分化抗原、细胞结构蛋白及功能未知三类。选择其中一个全长cDNAMY OVA 13(该基因编码的蛋白是MAD2 ) ,利用基因工程方法克隆、表达和纯化得到目的蛋白 ,采用点杂交方法检测了MY OVA 13目的蛋白与正常人和肿瘤患者中的血清学反应。MY OVA 13抗体只在肿瘤组中检测到 (5 / 74 ) ,在正常组中均为阴性 ;间接ELISA测定结果显示 ,MY OVA 13抗体的水平在肿瘤组中均高于正常组 ,其中肝癌和前列腺癌组与正常组相比 ,在统计学上有显著差异 ,P值分别 <0 0 1和 <0 0 5。我们的初步结果表明MY OVA 13及其抗体具有一定的肿瘤特异性 ,有可能作为肿瘤诊断的标志物