嵌合抗CD20抗体片段F(ab'')2的表达及活性

目的 :为了简化生产步骤 ,提高抗体蛋白的生物活性 ,探索在工程菌体内直接进行抗CD2 0F(ab’) 2 的高效率分泌性表达。方法 :采用单因素考察法优化培养条件 ,使蛋白G柱和S2 0 0 HR分子筛柱分离纯化目的蛋白 ,用MTT法检测抗CD2 0F(ab’) 2 抑制Daudi细胞体外生长的活性。结果 :发现用论文所选定的最适培养条件 ,抗CD2 0F(ab’) 2 的产量有了明显提高 ,从1 9～ 2 .2mg L达到了 3 7～ 4 3mg L ,F(ab’) 2 在表达产物中所占的比例也从 9 7%～ 13 2 %提高到了 38 1%～ 4 6 8% ;使用S2 0 0 HR分子筛柱对蛋白G柱亲和纯化后的产物进一步分离纯化 ,可以使F(ab’) 2 的纯度达到 85 %以上 ;MTT法检测结果证明F(ab’) 2 抑制Daudi细胞生长的IC50 值为 14 6 μg ml,而Fab’为 39 5 μg ml。 结论 :实现了抗CD2 0F(ab’) 2 在工程菌体内的高效率分泌性表达 ,而且所表达的抗CD2 0F(ab’) 2 比抗CD2 0Fab’具有更强的抑制Daudi细胞体外生长的能力     

Single-cycle immunodeficiency viruses provide strategies for uncoupling in vivo expression levels from viral replicative capacity and for mimicking live-attenuated SIV vaccines

To reduce the risks associated with live-attenuated immunodeficiency virus vaccines, single-cycle immunodeficiency viruses (SCIVs) were developed by primer complementation and production of the vaccine in the absence of vif in a vif-independent cell line. After a single intravenous injection of SCIVs into rhesus monkeys, peak viral RNA levels of 10(3) to 10(4) copies/ml plasma were observed, indicating efficient expression of SCIV in the vaccinee. After booster immunizations with SCIVs, SIV-specific humoral and cellular immune responses were observed. Although the vaccine doses used in this pilot study could not protect vaccinees from subsequent intravenous challenge with pathogenic SIVmac239, our results demonstrate that the novel SCIV approach allows us to uncouple in vivo expression levels from the viral replicative capacity facilitating the analysis of the relationship between viral expression levels or viral genes and immune responses induced by SIV.     

MECP2 duplication syndrome in a patient from Cameroon

MECP2 duplication syndrome (MDS; OMIM 300260) is an X‐linked neurodevelopmental disorder caused by nonrecurrent duplications of the Xq28 region involving the gene methyl‐CpG‐binding protein 2 (MECP2; OMIM 300005). The core phenotype of affected individuals includes infantile hypotonia, severe intellectual disability, very poor‐to‐absent speech, progressive spasticity, seizures, and recurrent infections. The condition is 100% penetrant in males, with observed variability in phenotypic expression within and between families. Features of MDS in individuals of African descent are not well known. Here, we describe a male patient from Cameroon, with MDS caused by an inherited 610 kb microduplication of Xq28 encompassing the genes MECP2, IRAK1, L1CAM, and SLC6A8. This report supplements the public data on MDS and contributes by highlighting the phenotype of this condition in affected individuals of African descent.     

人外周血淋巴细胞褪黑素受体的基因与蛋白表达的研究

目的:研究人外周血淋巴细胞褪黑素受体(MR)的基因与蛋白表达的特点。方法:分离健康成年人外周血淋巴细胞,采用异硫氢酸胍-苯酚-氯仿一步法抽提总RNA,分别采用Northern印迹和RT-PCR方法检测MR亚型mt₁和MT₂的mRNA,并将RT-PCR的阳性产物通过自动测序仪测序;同时应用免疫组化染色方法检测mt₁和MT₂受体亚型蛋白在淋巴细胞内的分布特点。结果:Northern印迹方法检测出长约1.1 kb mt₁ mRNA,但未检出长约1.1 kb MT₂ 的mRNA;RT-PCR方法则得到了368 bp mt₁ cDNA片段,无321 bp MT₂ cDNA片段,同时将mt₁阳性条带通过胶回收、测序,结果显示扩增产物与人mt₁受体亚型的基因序列相吻合;免疫组化结果显示:人外周血淋巴细胞存在mt₁受体蛋白,主要分布于细胞膜和细胞浆,呈棕褐色阳性颗粒,而细胞核上未见阳性颗粒,MT₂受体蛋白则未检出。结论:证实正常人外周血淋巴细胞存在mt₁的基因与蛋白的表达,而无MT₂的基因与蛋白的表达,提示外周血淋巴细胞为褪黑素(Mel)作用的外周靶器官,为研究生理及病理情况下Mel对免疫系统的调节奠定了基础。     

肿瘤转移抑制基因nm23—H1 mRNA在乳腺癌中的转录表达

目的：探讨nm23—H1基因mRNA在乳腺患组织中的表达及其与临床的关系．方法:采用半定量RT—PCR方法，检测30例乳腺患组织nm23—H1的表达．结果:①淋巴结阳性的原发灶组织nm23—H1 mRNAA表达明显低于淋巴结阴性的原发灶组织，Ⅲ期乳腺患组织nm23—H1 mRNA水平较Ⅰ、Ⅱ期的明显低．②多因素分析发现淋巴结转移与nm23—H1 mRNA的表达有显著性相关．结论nm23—H1基因mRNA表达强度与淋巴结转移呈负相关．在乳胰患转移过程中，nm23—H1 mRNA起重要的作用。     

IL—6基因转染的瘤苗体内诱导的抗肿瘤免疫功能与效果

经丝裂霉素C体外处理后,将IL-6基因转染的、高分泌的IL-6的B16黑色素瘤细胞制成瘤苗。结果发现,体内注射IL-6基因转染的瘤苗后,小鼠脾脏CTL活性、NK活性及IL-2诱导的LAK活性显著升高。经IL-6基因转染瘤苗体内治疗后,荷瘤小鼠的皮下肿瘤生长显著减慢、肺转移结节数显著降低、存活期显著延长,若同时合用低剂量IL-2,则上述治疗效果更好。可见IL-6基因转染的瘤苗能有效地通过诱导机体抗肿瘤免疫功能而发挥抗肿瘤作用,与低剂量IL-2合用后,IL-6基因转染的瘤苗的抗肿瘤效果更佳。     

利用寡核苷酸芯片检测乙型肝炎病毒基因突变

目的　利用寡核苷酸芯片平行分析乙肝病毒表面抗原区S、前核心抗原pre C区、X区、聚合酶P区 12个已知的突变位点。方法　针对S、pre C、X、P区的 12个突变位点 ,设计位于乙肝病毒反义链的 2 4条寡核苷酸探针和两对PCR引物 ,探针长度为 14～ 18bp ,其 5′端连有氨基己烷和T15间隔子 ,合成后经点样仪点到醛基化玻片上。两对PCR引物分别用于扩增S与P区内的 5个突变位点和X、前C区内的 7个突变位点 ,其中上游引物含有荧光标记。不对称PCR扩增所得到的荧光标记的单链DNA与寡核苷酸阵列杂交、清洗后经扫描分析实验结果。结果　在对 12个乙肝阳性样品前核心抗原C和X区的突变检测中 ,存在 176 2A→T ,176 4G→A联合突变的有 2例、1896G→A突变的 3例、同时存在 176 2A→T、176 4G→A联合突变和 1896G→A突变的 1例、未存在任何突变的样品 6例。在 12个乙肝阳性样品的表面抗原S的突变检测中 ,未发现有突变出现。部分样品的随机测序结果与寡核苷酸阵列杂交结果一致。结论　寡核苷酸芯片适于快速、平行、大量检测突变     

Isolation of a DNA fragment which complements glutamine synthetase deficient strains ofS. pombe

Summary From a gene bank ofS. pombe DNA, a 5.6 kb clone was isolated which complemented mutants defective in glutamine synthetase (GS) activity. Sub-cloning fragments of this 5.6 kb clone showed that the complementing activity was localised in a 1.6 kb HindIII-Aval fragment and a partial DNA sequence revealed an open reading frame preceded by TATA sequences and a TGACTA sequence. Plasmid constructs carrying up to 3.4 kb of DNA used to transformgln ⁻ strains gave transformants which showed a wide range of GS activity, in some cases 100 times the wild-type level. These constructs identify DNA sequences lying downstream from the putative coding sequence which have effects on the total amount of enzyme activity, but do not affect the control imposed by the nitrogen source on which the cells are grown.     

应用微阵列初步分析髓母细胞瘤的基因表达谱

目的　应用微阵列技术研究髓母细胞瘤的分子发病机理。方法　收集新鲜髓母细胞瘤 4例及正常脑组织 1份的组织标本 ,提取总 RNA,逆转录成 32 P标记的 c DNA探针 ,与 Atlas人肿瘤芯片杂交 ,通过放射自显影获得基因谱 ,应用 Atlas Image TM1.0 1a分析。结果　与正常脑组织相比 ,髓母细胞瘤下调基因 6个 ,上调基因 35个 ;逆转录 -聚合酶链反应技术验证结果与芯片检测结果相符。除少数基因外 ,大部分基因的表达趋势与肿瘤生物学特性相符。结论　髓母细胞瘤是与星形细胞起源胶质瘤具有不同分子发病机理的多基因病变 ,不同基因之间可能存在复杂的相互作用和联系 ,值得进一步研究。     

Non-redundancy of cytidine deaminases in class switch recombination

Class switch recombination (CSR), somatic hypermutation, and gene conversion are immunoglobulin diversification mechanisms that are strictly dependent on the activity of the activation-induced cytidine deaminase (AID). The precise role and substrate(s) of AID in these processes remain to be well defined. The closest homologue of AID is APOBEC-1, a bona fide mRNA-editing enzyme, which shares with AID the ability to deaminate cytidines within single-stranded DNA in vitro and in prokaryotic cells. To determine whether APOBEC-1 can therefore substitute for AID in activated B cells, we expressed human AID, a catalytic mutant thereof, and rat APOBEC-1 in AID-deficient murine B cells. Whereas AID rescued CSR, neither the inactive mutant nor APOBEC-1 could complement AID deficiency. This indicates that cytidine deaminase activity is necessary but not sufficient to initiate CSR, and suggests that AID is specifically targeted to its cognate substrate, the immunoglobulin genes or a distinct mRNA, by an as-yet-unknown mechanism.