SED超抗原MHCⅡ类分子结合位点的研究

目的：运用定点突变技术确定SED超抗原的MHCⅡ结合位点。方法：首先检测突变体的促T淋巴细胞增殖活性；用FTTC标记SED，对增殖活性降低的突变体，进一步用竞争结合实验检测其MHCⅡ结合活性。结果：突变体SEDN23A、SEDN23A/H26R和SEDF45A的促增殖活性均显著降低，初步推测23、26和45位氨基酸可能参与了SED与MHC的相互作用。竞争结合实验证明F45位氨基酸是SED与MHCⅡ类分子结合的关键位点。结论：F45位氨基酸是SED与MHCⅡ类分子结合的关键位点；23和26位氨基酸可能参与了SED与T细胞受体(TCR)Vβ的识别，值得进一步研究。     

猪Ia相关恒定链定位基元突变对其定位功能的影响

目的研究猪Ia相关恒定链(Ii)胞质区2个亮氨酸基元(Leu7/Ile8和Met16/Leu17)周围氨基酸对基元内膜系统定位功能的影响。方法通过大引物PCR定点突变法,将2个亮氨酸基元及其周围氨基酸分别突变并导入pEGFP-C1真核表达质粒,获得21个突变Ii重组质粒。经LipofectamineTM2000将突变Ii重组质粒分别转染COS-7细胞,荧光显微镜检测突变Ii在细胞内的定位。结果 Leu 7/Ile 8和Met 16/Leu 17独立调控Ii分子的细胞内化。当保留其中一个亮氨酸基元,突变另一个亮氨酸基元周围氨基酸时,GFP-Ii融合蛋白或定位于内膜系统或分布于整个细胞。结论猪Ii链含有2个亮氨酸定位基元,亮氨酸基元的定位功能需要周围特定位置的氨基酸的支持。     

新城疫病毒HN糖蛋白促细胞融合区域内保守氨基酸基因突变分析

目的 确定新城疫病毒(NDV)血凝素神经氨酸酶(HN)糖蛋白促细胞融合区域内的保守氨基酸功能,探讨HN糖蛋白的促细胞融合机制.方法 采用PCR定点突变与体内同源重组相结合的方法将HN糖蛋白促细胞融合区域内6个保守氨基酸突变为丙氨酸(A),在BHK21细胞内表达后,流式细胞仪定量分析蛋白的表达效率,并分别检测其促细胞融合活性、血细胞吸附能力(也称为受体识别活性)和神经氨酸酶活性.结果 L74A蛋白表达效率降低为72.7％,各突变株蛋白表达效率与野毒株相比差别无统计学意义(P＜0.05).各突变株蛋白促细胞融合活性都有不同程度的下降,其中I103A下降幅度最大,为野毒株的9.1％,各突变株蛋白血细胞吸附能力也都出现不同程度的降低,其中I1iA的下降最明显,为28.2％,各突变株蛋白的神经氨酸酶活性变化程度不同,其中174A略有升高,为118.6％,L110A下降幅度最大,为5.2％,I103A仅次于L1 10A,为5.7％.结论 新城疫病毒HN糖蛋白促细胞融合区域内的保守氨基酸在细胞融合中发挥着重要作用,第103位异亮氨酸(Ⅰ)是此区域内的关键氨基酸.     

一种基于分子动力学模拟来识别GPIbα与vWF-A1结合面上重要残基的新方法

目的 发展一种识别受体与配体相互作用中关键氨基酸残基的计算机新方法。方法 GPIb?/vWF-A1的晶体结构取自PDB数据库;利用自由分子动力学模拟,观察GPIb?/vWF-A1复合物结合面上的盐桥和氢键的形成和演化;利用分析计算得到的这些盐桥和氢键的生存率的高低,作为度量相互作用残基对之重要性的判据。结果 在GPIb?/vWF-A1的结合面上,GPIb?的21个残基和vWF-A1的21个残基显著参与了GPIb?和vWF-A1间的相互作用,这些残基中的20个已得到突变实验的证实。结论 该方法能较好地预报和识别受体-配体相互作用中的关键残基,并可为传统的氨基酸残基突变实验和抗凝血栓药物设计提供指导。     

修饰后中国山东地方株HPV16 E6E7基因的转化活性检测及免疫原性研究

目的 利用突变修饰后消除转化活性并保留抗原性的中国山东地方株人乳头瘤病毒16型（HPV16）E6E7基因，研制HPV16 DNA疫苗。方法 定点突变E6的终止密码，并保证E7读码框架不定；定点突变E7蛋白的Rb结合区中对其转化活性维持起关键作用的第24位氨基酸。突变修饰后的基因命名为fmE6E7。PCR扩增fmE6E7，重组人pLNCX载体，脂质体法转染3T3细胞，免疫荧光组织化学及Western blot检测转染细胞蛋白的表达。经软琼脂集落培养法和BALB／c裸鼠皮下接种法检测fmE6E7的转化活性。然后PCR扩增fmE6E7，构建pVR1012-fmE6E7真核表达质粒，于C57BL／6小鼠肌肉内直接进行裸DNA免疫，^51Cr释放法体外分析免疫鼠的细胞毒性T淋巴细胞活性，间接ELISA法检测免疫鼠血清中E7特异性抗体。结果 测序证实获得了预期的突变结果。pLNCX-fmE6E7转染细胞体外软琼脂培养3周未见集落形成；裸鼠皮下接种2月后未见移植瘤形成（0／3）。免疫鼠获得了较好的E7特异性的抗体E和抗原特异性的CTL。结论 修饰后E6E7基因可融合表达，转化活性消除的同时还可诱发特异的细胞免疫和体液免疫，表明中国山东地方株的E6E7基因可作为HPV16治疗性DNA疫苗的靶基因。     

调节破骨细胞分化的RANK特异性基序的再确认

目的： 进一步确认核转录因子NF-κB受体激动剂（RANK）蛋白中调节破骨细胞(OC)分化的特异性基序(motif),为阐明OC分化机制提供理论依据。方法: 分别突变RANK蛋白膜内部分第533-540之间的8个氨基酸（突变前氨基酸序列为DIIVVYVS,突变后氨基酸序列为ELLAAFAA）,用定点突变方法构建8个由肿瘤坏死因子受体1(TNFR1)和RANK跨膜部分和膜内部分共同组成的TNFR1/RANK突变嵌合体(TNFR1/RANK-533-TNFR1/RANK-540),每1个突变体在其RANK膜内部分含1个突变氨基酸。用包装细胞plat E分别将各突变体包装成逆转录病毒,通过逆转录病毒感染分别将这些突变体转染到TNFR1/TNFR2基因敲除小鼠的骨髓巨噬细胞(BMM)中。用TNF-ɑ和单核细胞集落刺激因子（M-CSF）刺激、观察转染哪种突变体的BMM不能诱导OC形成,不能诱导OC形成的突变体所包含的突变前氨基酸就是RANK调节OC分化的关键氨基酸,多个关键氨基酸组成的片段就是RANK特异性motif。结果: 转染TNFR1/RANK-533、TNFR1/RANK-539和TNFR1/RANK-540的BMM均能分化成OC,表明氨基酸D533、V539、S540突变后对OC分化无影响;转染TNFR1/RANK-534的BMM有极少数能分化成OC,表明氨基酸I534突变后对OC分化有部分影响;而转染TNFR1/RANK-535、TNFR1/RANK-536、TNFR1/RANK-537和TNFR1/RANK-538的BMM均不能分化成OC,表明氨基酸I535、V536 、V537和Y538突变后对OC分化起关键影响。结论: I534、I535、V536 、V537和Y538这5个氨基酸组成的片段（534-IIVVY-538）可能就是RANK调节OC分化的特异性motif。     

表达Cys548雌激素受体突变型cDNA的真核载体构建及其促转录活性分析

目的 考察一个548位点C→T单核苷酸突变的雌激素受体(ER)在体外条件下,对乳腺癌细胞株(MCF-7)信号通路的影响,以探讨ER单核苷酸突变引起非雌激素依赖性性早熟的可能机制.方法 用重叠延伸PCR定点诱变技术对548位点的碱基进行定点突变.构建定点突变表达载体pSG5-MuER.构建含雌激素受体反应元件(ERE)的萤光素酶报告基因载体pGL3-ERE-Luc.将野生和突变ER质粒分别和pGL3-ERE-Luc共转染MCF-7细胞,观察萤光素酶的变化,以检测突变ER反应活性的变化.结果 成功构建ER突变质粒psG5-MuER和ER报告质粒pGL3-ERE-Luc,psG5-MuER较pSG5-ER能够增加萤光素酶的产生.结论 该突变雌激素受体在体外具有高促转录活性特征,构建的载体可用于进行进一步的相关研究.     

蒙药材瑞香狼毒体外细胞毒性的研究

目的研究蒙药瑞香狼毒炮制前后的体外细胞毒性和抗肿瘤活性。方法利用石油醚、醋酸乙酯和正丁醇分别萃取获得瑞香狼毒药材生品、奶制品、煎膏制品的各溶剂提取物,再通过萃取、分离纯化方法分别获得瑞香狼毒的9个单体化合物、总蛋白质和6个相对分子质量不同的蛋白质组分(Pr.1~Pr.6)。将各溶剂提取物及单体化合物制成浓度为2.0mg/ml的溶液,总蛋白质制成浓度分别为0.5、1.0、2.0、4.0、6.0、8.0mg/ml的系列溶液,各蛋白质组分制备成浓度均为10mg/ml的溶液,分别作为样品溶液,采用MTT法分别测定各样品对人肝癌SMMC-7721细胞、狗肾MDCK细胞的生长抑制作用。结果瑞香狼毒总蛋白质抗肿瘤活性和细胞毒性作用随其浓度增加而增强,其中4个蛋白质组分呈现较强的抗肿瘤活性,其活性由强至弱依次为Pr.4≥Pr.6≥Pr.3=Pr.2,3个蛋白质组分具有细胞毒性,其毒性由强至弱依次为Pr.4≥Pr.3≥Pr.1。瑞香狼毒单体化合物中,西瑞香素、狼毒色原酮、异狼毒素呈现不同程度的抗肿瘤活性,而狼毒色原酮、异狼毒素同时具有较弱的细胞毒性。除石油醚和多糖提取物外,瑞香狼毒药材生品各溶剂提取物均呈现不同程度的抗肿瘤活性和细胞毒性,并呈药物剂量依赖性;而炮制后,在保留一定程度抗肿瘤活性的基础上,其相应各溶剂提取物的细胞毒性均降低。结论瑞香狼毒的细胞毒性主要源自多成分的协同作用,其蛋白质可能是瑞香狼毒的主要毒性位点之一,而炮制方法可降低瑞香狼毒药材的毒性且保留其生物活性。     

催化甾体羟基化的P450氧化酶BM3的蛋白质工程的研究进展

<正>甾体关键位点的羟基化在药用甾体的合成中发挥重要作用。为探究细胞色素P450(CYP450)氧化酶BM3在甾体羟基化合成中的潜在应用,基于细胞色素P450 BM3的蛋白质工程逐渐发展起来。在取得的若干P450 BM3突变酶的基础上,通过新一代测序技术和生物信息学分析等方法,筛选出催化甾体羟基化的相关CYP450。根据易错PCR等定向进化技术获得了突变位点信息,进一步采用(饱和)定点突变等进化技术对活性氨基酸位点进行分析,再经过筛     

HIV-1B′亚型毒株抵抗VRC01抗体中和作用的机制研究

目的:探讨具有广谱中和活性的患者DRVI01血浆来源的HIV-1B′亚型毒株抵抗VRC01抗体中和的机制。方法:比较同期感染相同亚型且对VRC01抗体敏感的病毒序列,结合文献报道筛选可能影响VRC01中和作用的关键氨基酸,通过定点突变、不同来源膜蛋白序列交换,验证这些位点氨基酸对VRC01抗体中和作用的影响。结果:位于...     

Small deletion in v-src SH3 domain of a transformation defective mutant of Rous sarcoma virus restores wild type transforming properties.

RSV mutant virus PA101T was obtained while assaying the tumorigenicity of parental PA101 virus in chickens. PA101 is a transformation defective mutant of RSV which has a low src kinase activity. However, PA101 retained a temperature-sensitive ability to induce sustained proliferation of neuroretina cells. PA101T appeared as a wild-type phenotype revertant of PA101. Molecular cloning and sequencing of PA101T showed that this reversion is due to additional mutations in PA101 src gene. These mutations are a deletion eliminating three amino acids in the N-terminal region of SH3 domain and mutation of Ala 426 to Val. Analysis of the properties of chimeric src genes associating either half of PA101T with the complementary regions of PA101 or wild-type virus showed that the N-terminal moiety of PA101T src, which contains the deletion, confers wild-type transforming properties, whereas its C-terminal moiety, which contains single amino acid mutation, confers a partially temperature-sensitive phenotype. These results are consistent with other reports showing that mutations or deletions in this region of SH3 activate the transforming potential of c-src. They support the hypothesis that the N-terminal region of SH3 interacts with a cellular negative regulator of src activity.     

The influence of amino acids on mitogen-activated proliferation of human lymphocytes in vitro

Recurrent infections are common features in patients affected by various aminoacidopathies. Since these disorders are biochemically characterized by tissue accumulation of amino acids, it is possible that these compounds may act as immunosuppressants. We therefore investigated the influence of 21 amino acids on in vitro cellular growth of lymphocytes stimulated with phytohaemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM), a recognized test of cellular immunocompetence. Human peripheral lymphocytes were cultured in flat-bottomed 96-well microplates at 37°C for 96 (PHA and Con A) or 144 h (PWM) in the presence of one mitogen at different concentrations and of one amino acid added at doses of 2, 4 or 8 mM. Cell reactivity was measured by the incorporation of tritiated thymidine into cellular DNA and compared to that of identical cultures with no amino acids added (controls). We found that among the 21 amino acids tested, cysteine stimulated lymphocyte growth, whereas glutamate, tryptophan, phenylalanine and glutamine caused significant inhibition. These results may reflect an immunomodulatory role for some amino acids.     

Identification of amino acids in the V3 region of gp120 critical for virus neutralization by human HIV-1-specific antibodies.

The importance of the dependence on single amino acids in the V3 region of HIV-1 gp120 was evaluated for virus neutralization and antibody-dependent cellular cytotoxicity (ADCC). Synthetic overlapping 15-mer peptides and a set of omission peptides covering amino acids 301-317 were used. Sera from 29 HIV-1-infected individuals at different stages of disease were tested for neutralization, ADCC and specific IgG reactivity. Six HIV-1 neutralizing monoclonal antibodies (mAb) acted as controls. All mAb reacted with a region (amino acids 304-318) of gp120, previously shown to induce neutralizing antibodies. The amino acids essential for reactivity were identified to be within the sequence GPGR (amino acids 312-315). The importance of this region for occurrence of neutralizing antibodies in infected humans was investigated using the same set of peptides. Out of 29 individuals, 21 were found to have neutralizing antibodies in titres between 100 and 1000. Among the neutralization-positive sera, 17/21 (81%) reacted with amino acids 304-318, compared with only one of eight sera (13%) negative in neutralization. When any of the four amino acids G, P, G or R were deleted, the seroreactivity decreased considerably. The conserved sequence GPGR was therefore considered to be the most important for neutralization in this region in human sera as well. Thus, the conserved sequence GPGR in the V3 region of gp120 is critical for virus neutralization by human HIV-1-specific antibodies.     

Biological activity of mutants of human tumour necrosis factor-alpha.

Point mutations in different regions of the tumour necrosis factor-alpha (TNF-alpha) molecule influence anti-tumour cytotoxic/cytostatic activities as well as haemorrhagic tumour necrosis, tumour regression and lethal toxicity in mice. Mutations in the C-terminal region in positions 150 and 155 markedly decrease cytotoxicity for murine L929 fibroblasts and human MCF7 mammary carcinoma cells. Competitive binding experiments with 125I-labelled TNF-alpha revealed that the loss of cytotoxicity is caused by a loss of target cell binding. In contrast to the reduced activity against L929 and MCF7 cells, neither binding to nor cytostatic activity against the human myeloid leukaemia cell lines HL60 and U937 are affected. This target cell type-dependent behaviour is probably due to the fact that L929 and MCF7 cells express different types of TNF receptor compared with myeloid leukaemia cells. While a mutation in position 127 decreases the overall activity of TNF-alpha, a deletion of four N-terminal amino acids does not reduce biological activity. In vivo the TNF mutants differed in their anti-tumour effects and lethal toxicity, but a segregation of anti-tumour activity and toxicity was not observed.     

Nucleotide sequences of the PA and PB1 genes of B/Ann Arbor/1/66 virus: comparison with genes of B/Lee/40 and type A influenza viruses

The complete sequences of the PA and PB1 genome RNA segments of B/Ann Arbor/1/66 virus have been determined. The PA vRNA is 2308 bases long. Its complementary RNA has a single open reading frame of 2187 bases, capable of encoding a PA protein of 726 amino acids with a molecular weight of 83,175 Da. The predicted PA polypeptide has an overall net charge of -7.5 at pH 7.0. The PB1 vRNA is 2369 bases long. Its complementary RNA has a single open reading frame of 2277 bases, capable of encoding a PB1 protein of 752 amino acids with a molecular weight of 84,332 Da. The predicted PB1 polypeptide has an overall net charge of +18.5 at pH 7.0. Sequence homology comparisons of the PA and PB1 polypeptides from B/Ann Arbor/1/66 virus to the PA and PB1 polypeptides of type A influenza virus reveal respective homologies of approximately 38 and 60%. This high cross-type homology (61%) was previously reported for the PB1 protein of B/Lee/40 virus (Kemdirim et al., 1986). The cross-type homology for the PA protein is similar to that of other non-polymerase proteins, but is substantially lower than that seen for the PB1 protein. Thus, the high cross-type homology that exists for the PB1 gene does not appear to be a characteristic of all polymerase genes.     

The effect of surface charge on in vivo biodistribution of PEG-oligocholic acid based micellar nanoparticles

To systematically elucidate the effect of surface charge on the cellular uptake and in vivo fate of PEG-oligocholic acid based micellar nanoparticles (NPs), the distal PEG termini of monomeric PEG-oligocholic acid dendrimers (telodendrimers) are each derivatized with different number (n = 0, 1, 3 and 6) of anionic aspartic acids (negative charge) or cationic lysines (positive charge). Under aqueous condition, these telodendrimers self-assemble to form a series of micellar NPs with various surface charges, but with similar particle sizes. NPs with high surface charge, either positive or negative, were taken up more efficiently by RAW 264.7 murine macrophages after opsonization in fresh mouse serum. Mechanistic studies of cellular uptake of NPs indicated that several distinct endocytic pathways (e.g., clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis) were involved in the cellular uptake process. After their cellular uptake, the majority of NPs were found to localize in the lysosome. Positively charged NPs exhibited dose-dependent hemolytic activities and cytotoxicities against RAW 264.7 cells proportional to the positive surface charge densities; whereas negatively charged NPs did not show obvious hemolytic and cytotoxic properties. In vivo biodistribution studies demonstrated that undesirable liver uptake was very high for highly positively or negatively charged NPs, which is likely due to active phagocytosis by macrophages (Kupffer cells) in the liver. In contrast, liver uptake was very low but tumor uptake was very high when the surface charge of NPs was slightly negative. Based on these studies, we can conclude that slightly negative charge may be introduced to the NPs surface to reduce the undesirable clearance by the reticuloendothelial system (RES) such as liver, improve the blood compatibility, thus deliver the anti-cancer drugs more efficiently to the tumor sites.     

Evolutionary pathways of the PA genes of influenza A viruses

Nucleotide sequences of the PA genes of influenza A viruses, isolated from a variety of host species, were analyzed to determine the evolutionary pathways of these genes and the host specificity of the genes. Results of maximum parsimony analysis of the nucleotide sequences indicate at least five lineages for the PA genes. Those from human strains represent a single lineage, whereas the avian genes appear to have evolved as two lineages--one comprising genes from many kinds of birds (e.g., chickens, turkeys, shorebirds, and ducks) and the other comprising only genes from gulls. H3N2 swine influenza virus PA genes are closely related to the currently circulating duck virus PA gene. By contrast, the H1N1 swine and equine virus PA genes appear to have evolved along independent lineages. Comparison of predicted amino acid sequences disclosed 10 amino acid substitutions in the PA proteins of all avian and H3N2 swine viruses that distinguished them from human viruses. The H1N1 swine viruses seem to be chimeras between human and avian viruses and they contain 8 amino acids not shared by other viruses. The equine viruses also appear to show their own amino acid substitutions. These findings indicate that the PA genes of influenza A viruses have evolved in different pathways defined by apparently unique amino acid substitutions and host specificities. They also indicate that influenza A viruses have been transmitted from avian to mammalian species.     

Subcutaneous release of amino acid loads on food and water intakes in the rat

A cellophane sac containing a mixture of amino acids was implanted under the skin of the freely fed and watered rat. After its dissolution in body fluid, the load diffused out of the sac according to an exponential function. Water intake increased maximally during the period of most rapid release of amino acids into the body. Food intake was depressed most effectively somewhat later. The anorexigenic effect of a mixture consisting solely of essential amino acids was greater than that of a mixture containing a proportion of dispensable amino acids. Effects of amino acids which facilitate the urea cycle indicated that ammonia toxicity contributed to the suppression of intake by the highest dose of essential amino acids (90 mmoles/kg). The moderate suppression of feeding by a mixture containing both essential and dispensable amino acids was potentiated by inclusion of glucose. This suggested that metabolic effects of the amino acids, other than ammonia production, contributed to the observed inhibition of feeding.     

异形矿物粉尘巨噬细胞毒性研究

为研究粉尘样品对兔肺泡巨噬细胞（AM）产生的不同毒性。探讨巨噬细胞受损的机制，采用体外细胞培养技术，观测兔肺泡巨噬细胞死亡率，丙二醛（MDA）、乳酸脱氢酶（LDH）及超氧化物歧化酶（SOD）的活性及变化。结果表明．沸石、硅灰石无细胞毒性，而其它的纤维状及颗粒状矿物粉尘则表现出不同程度的细胞毒性。纤维状矿物尘对AM毒性大于颗粒状矿物，毒性程度与粉尘中的活性OH^-含量正相关，但并不一定与SiO2含量相关。粉尘所形成的高pH值不利于细胞的生存，低生物持久性的粉尘对人体是安全的。粉尘中变价元素的含量可能影响其毒性。表面电位是粉尘毒性的非稳定因素。矿物尘的微形态是影响其毒性的因素之一．而矿物尘的毒性主要依赖于其特性。     

Epitope mapping of the influenza A virus RNA polymerase PA using monoclonal antibodies

Summary.  To obtain reagents to functionally map the PA protein, we produced monoclonal antibodies specific to this protein. Twenty-two monoclonal antibodies reacting with PA protein in ELISA were divided into 10 groups on the basis of competitive binding patterns to this protein. Of these, seventeen monoclonal antibodies bound to PA polypeptide spanning amino acids 101–400 and three bound to that of amino acids 518–600, while the other two did not react with any PA polypeptides tested with the exception of full-length PA. Among these monoclonal antibodies, only five reacted with PA in A/PR/8/34 virus-infected cells in indirect immunofluorescence assay. Thus, we obtained monoclonal antibodies that recognize at least 10 distinct regions of the PA molecule. These monoclonal antibodies should be useful in dissecting functions of the PA protein. Received September 6, 1999/Accepted January 5, 2000