经基磷灰石(H A )的制备方法及其研究进展

HA生物材料作为最有发展前途的生物硬组织替代材料之一。已经成为生物医用材料研究的重点内容。本文在综合了大量的国内外文献的基础上,对羟基磷灰石的不同制备方法及国内外研究现状进行了总结及评论。并提出了相应的设想与展望。     

Recent avian H5N1 viruses exhibit increased propensity for acquiring human receptor specificity

Adaptation of avian influenza viruses for replication and transmission in the human host is believed to require mutations in the hemagglutinin glycoprotein (HA) which enable binding to human α2-6 sialosides and concomitant reduction in affinity for avian α2-3 linked sialosides. Here, we show by glycan microarray analyses that the two mutations responsible for such specificity changes in 1957 H2N2 and 1968 H3N2 pandemic viruses, when inserted into recombinant HAs or intact viruses of some recent avian H5N1 isolates (clade 2.2), impart such attributes. This propensity to adapt to human receptors is primarily dependent on arginine at position 193 within the receptor-binding site, as well as loss of a vicinal glycosylation site. Widespread occurrence of these susceptible H5N1 clade 2.2 influenza strains has already occurred in Europe, the Middle East, and Africa. Thus, these avian strains should be considered high-risk, because of their significantly lower threshold for acquiring human receptor specificity and, therefore, warrant increased surveillance and further study.     

湖南湖北两省H6亚型禽流感病毒表面基因的序列分析

2008年至2009年间,在湖南和湖北两省的活禽市场中分离到了14株H6亚型禽流感病毒,为了解这14株病毒之间的分子特征和差异,我们运用PCR和测序鉴定对这14株病毒的NA基因进行了分型,并对其表面基因HA和NA进行序列测定及序列分析.14株H6亚型病毒中,H6N2亚型12株,H6N6亚型2株.序列测定和进化分析结果显示:DK/HN/284的HA基因与其它13株的HA差异性较大,差异性达到19.4%～20.2%,其余13株毒同源性在94.2%～99.9%;N2亚型NA基因的同源性在91.1%～99.9%,差异性比较大;两株N6亚型NA基因同源性为89.5%,差异明显.这些数据表明:不同毒株呈现一定的地域性差异.与我国周边其它地区的H6亚型禽流感毒株序列进行比较发现,只有DK/HN/284的HA基因与香港早期的毒株可能有着共同的来源,其余都与香港和韩国等的毒株有着较大的差异性,并且各个毒株的HA基因上潜在的糖基化位点和受体结合位点也有所不同,这些数据表明,这些毒株表现出明显的异源性.     

Letter: Filamentous bacterial viruses. X. X-ray diffraction studies of the R4-protein mutant

Eukaryotic DNA fragments that are totally denatured by alkali swiftly re-form duplex regions that are several hundred up to several thousand nucleotide pairs in length. A combination of sedimentation and electron microscopic studies demonstrate that they arise by the folding-back of a single linear chain, and not from cross-linking between the two complementary chains. Thus these “hairpin”-like structures must come from inverted repetitions of the type A B C t C′B′A′ that are located at intervals along the chromatid. Electron microscopic studies, reveal no unpaired single-chain regions in the “turn-around” t. The resistance of these hairpins to single-chain specific nucleases indicates that t must only consist of a few nucleotides. Therefore we call these regions in double-chain DNA palindromes, because, given the antiparallel arrangement of the polynucleotide chains, these sequences read the same both backwards and forwards. The thermal stability profile of these hairpins is nearly identical to that of sonicated duplex fragments of comparable length. Since these hairpins have the same average base composition as bulk DNA, the palindromes are nearly perfect. By studying the fraction of DNA retained on hydroxyapatite as a function of chain length, one may determine the distribution of palindromes along the DNA. These experiments are best explained by clusters of palindromes located at intervals of 10 to 80 /gm depending on the species. The presence of such long, well-matched palindromes suggests that the linear double helix may sometimes adopt an alternative configuration, the cruciform, in which mismatches that may occur are eliminated by excision and repair.     

Proteolytic activation of Vibrio mimicus (Vm) major outer membrane protein haemagglutinin (HA) with Vm-HA/protease: Implication for understanding bacterial adherence

Vibrio mimicus (Vm) haemagglutinins (HAs), such as an extracellular HA/protease (Vm-HA/protease) and a major outer membrane protein-HA (Vm-OMPHA), have been recognized as the putative adherence factors for the bacterium. However, the mechanism by which HAs coordinate the adherence function of the bacterium remains as yet unknown. We report herein the positive interaction between Vm-HA/protease and Vm-OMPHA resulting in significant enhancement of the haemagglutinating ability. In this interaction, no cleaved polypeptide was detected; however, limited proteolysis of Vm-OMPHA was confirmed by SDS-PAGE. The proteolytic activation of the native cell-associated Vm-OMPHA by limited proteolysis was also demonstrated in several V. mimicus strains. Proteolytic activation of OMPHA was also achieved with various proteases from bacterial and eukaryotic sources. These findings may indicate a novel coordination of V. mimicus HAs in the adherence of the bacterium.     

Stability of Hemagglutinating Activity of Extracellular and Intracellular Forms of Japanese Encephalitis Virus Exposed to Acidic pH

The hemagglutinating (HA) activity of extracellular and intracellular forms of Japanese encephalitis (JE) virus was comparatively titrated by exposure to acidic pH below 7.0. A pH-dependent irreversible loss in titer was observed with the virus grown in both C6/36 and BHK 21 (BHK) cells maintained in the pH range of 5.8 to 7.0 for 10 min at 37 C. The HA activity of intracellular virus was relatively more stable than that of extracellular virus in the pH range of 5.8 to 6.4. Virion structural components, envelope glycoprotein (E), capsid (C), and membrane (M) proteins in extracellular virus and E, C, and the precursor form of M (prM) proteins in intracellular virus were detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. A panel of monoclonal antibody (mAb) directed for nine antigenic epitopes on the JE virus E protein molecule was used for the analysis of antigenic reactivity of E protein after treatment at pH 6.0. The reaction between the extracellular virus and three HA-inhibiting (HI) mAbs was significantly reduced after acid treatment; however, the antigenic reactivity of intracellular virus was much more stable with a 100- to 1,000-fold difference. Infectivity titers of extracellular and intracellular viruses in Vero cells were reduced by 1/24,100 and 1/21,666 after acidic treatment at pH 6.0. In contrast, the infectivity of intracellular viruses was more stable, with residual infectivity of 1/182 and 1/340 for BHK and C6/36 cell-grown virus, respectively. Acidic treatment of JE virus not only resulted in the irreversible loss of its HA activity but also affected the antigenic reactivity of HI epitopes on its E protein molecule.     

贝飞达对慢性肝病患者纤维化指标及TNF-α的影响

目的探讨贝飞达在治疗慢性肝病患者时,对血清HA、NL、IV-C及TNF-α的影响。方法血清HA、LN、IV-及TNF-α检测采用放免法。结果60例肝功能减退者,经3个月贝飞达治疗与对照组比较。HA、LN、IV-C及TNF-α值下降水平平均低于对照组,经统计学处理差异有显著性。结论应用微生态调节剂贝飞达对慢性肝病患者进行抗纤维化治疗。通过调整肠道菌群失调。控制内毒素血症,可以降低血清HA、LN、IV-C及TNF-α水平,从而达到预防和控制纤维化的目的。     

密码子优化的人H5N1流感病毒HA基因在哺乳动物细胞中获得高效表达

为了提高人禽流感病毒血凝素HA的表达量,应对流感大流行疫苗的需求,按照人的偏爱密码子将H5N1(A/Anhui/1/2005)流感病毒的HA基因进行优化改造,经全基因合成后插人到真核表达载体pDC315中,构建了真核表达质粒pDC315-Mod.HA;将此质粒和含野生HA基因的真核表达质粒pDC315-Wt.HA分别转染293T细胞,比较HA蛋白的表达量.结果表明:经间接免疫荧光实验及Western blot实验比较和鉴定,密码子优化后,HA蛋白在293T细胞中的表达水平显著提高,为流感大流行疫苗的研究打下了基础.     

蛋氨酸脑啡肽（MEK）抗B型流感病毒感染作用的研究

研究MEK抗B型流感病毒感染的作用。采用MDCK细胞和9～10日龄鸡胚,按不同的顺序加入不同剂量MEK和B型流感病毒,共培养72 h后做血凝实验。所有加入B型流感病毒的MDCK细胞均培养出病毒,HA滴度为1：64。在鸡胚尿囊腔中,先注入MEK孵育24 h后,再注入B型流感病毒的鸡胚也培养出病毒,HA滴度为1：6.8,与病毒对照组比较P〈0.01,有统计学意义。实验结果未见MEK直接抗B型流感病毒感染MDCK细胞株的作用,但可见MEK抗B型流感病毒感染鸡胚的作用。     

Identification of amino acids of influenza virus HA responsible for resistance to a fusion inhibitor, Stachyflin

We have recently described a novel hemagglutinin (HA) conformational change inhibitor of human influenza virus, Stachyflin (Yoshimoto et al, Arch. Virol., 144, 1-14, 1999). Stachyflin-resistant variants of human influenza A/WSN/33 (H1N1) virus were isolated in vitro and the nucleotide sequences of their HA genes were determined. The relation of amino acid substitutions and Stachyflin resistance was analyzed with in vitro membrane fusion between HA-expressing cells and octadecylrhodamine (R18)-labelled chick erythrocytes (RBC). The amino acid substitutions, lysine to arginine at position 51 or lysine to glutamic acid at position 121 of the HA2 subunit of the HA protein was enough to confer a Stachyflin-resistant phenotype of HA protein. The molecular mechanism of anti-HA conformational change activity of Stachyflin is discussed.     

核糖体失活蛋白体内外抗病毒的实验研究

核糖体失活蛋白是一类广泛存在于高等植物细胞内的能抑制核糖体功能的植物蛋白。建立了从苦瓜籽中提纯核糖体失活蛋白的方法,蛋白质电泳结果显示提取液中含有一定量的核糖体失活蛋白。在鸡胚成纤维细胞和鸡胚上进行核糖体灭活蛋白体内外抗马立克病病毒、新城疫病毒和鸡痘病毒试验,结果均表明核糖体灭活蛋白对3种病毒有一定的抑制作用。     

广东地区1996年流感暴发的分子变异基础

1996年广东地区流感毒株发生明显的血清学抗原漂移;引起广东地区流感暴发的分子基础是流感毒株HA基因编码的A、B、C、D和E五个抗原决定簇位点变异,尤其是A、C、E位点发生氨基酸改变;而受体结合位点的氨基酸改变对此流感流行未发挥明显影响。HA基因编码氨基酸的第145号和第193号位点变异导致流行毒株的生物学特性改变,即分离毒株适应于MDCK细胞株生长,而难以适应鸡胚生长环境。     

南京市2011年乙型流感血凝素基因分子特征分析

[目的]分析2011年南京市乙型流感病毒的血凝素(HA)分子学特征.[方法]选择7株2011年南京市不同时间段有代表性的乙型流感毒株进行HA基因序列测定,通过生物信息学方法对HA分子学特征进行分析.[结果]7株乙型流感毒株分为两个系,4株为Victoria,3株为Yamagata;与2011年度疫苗株相比,Victoria和Yamagata系毒株分别在抗原位点146、197和116、198发生了氨基酸替换;其中197和198位点分别是Victoria和Yamagata毒株的受体结合位点,由于上述位点的替换使得Victoria系/Yamagata系毒株分别在197/196位增加了一个潜在的糖基化位点.[结论]2011年南京市乙型流感Victoria 系和Yamagata系病毒同时存在,Victoria/Yamagata毒株197/198位点的氨基酸替换,值得做进一步的探讨.     

我国H3N8马流感病毒血凝素基因分子进化树的分析

根据已发表的马流感病毒血凝素基因序列,设计并合成一对特异性引物,经反转录聚合酶链反应(RTPCR)成功扩增出了我国马流感病毒青海株(AEquineQinghai194)、吉林株(AEquineJilin189)及黑龙江株(AEquineHeilongjiang189)血凝素基因,将片段分别连接到PGEMTeasy载体并转化DH5α,提取阳性菌落的质粒经EcRo1酶切和PCR鉴定其大小均为17kb左右,对其测序并构建HA基因进化树。经过比较分析,青海株与AEquineKentucky286、AEquineMiami163等关系较近,核苷酸同源率为973%～847%,而与我国马流感吉林株和黑龙江株关系较远,同源率仅为552%。经过比较,青海株与我国香港1992年马流感病毒关系密切 。     

单向放射免疫扩散法测定流感灭活疫苗血凝素含量的研究

为改进流感灭活疫苗血凝素含量的测定方法 ,将待测样品经去污剂处理后 ,用WHO标准品作对照进行免疫扩散测定 10个流感灭活疫苗样品血凝素含量。在血凝效价相同的情况下 ,疫苗中三价 (A1,A3,B)血凝素含量存在不同程度的差异。可见 ,单向放射免疫扩散法较血球凝集法更适用于流感灭活疫苗血凝素含量的测定。     

Antigenic Drift of the Hemagglutinin from an Influenza A (H1N1) pdm09 Clinical Isolate Increases its Pathogenicity In Vitro

Virologica Sinica - The influenza A (H1N1) pdm09 virus emerged in 2009 and has been continuously circulating in humans for over ten years. Here, we analyzed a clinical influenza A (H1N1)...     

AIV血凝素亚型同步检测基因芯片技术研究

对流感病毒14个血凝素亚型的基因芯片检测技术进行了初步研究。通过RT-PCR克隆禽流感病毒血凝素基因片段,获得重组质粒。从重组质粒扩增大约500bp的DNA片段,浓缩后点到氨基化玻璃载体上,制成芯片。待检病毒样品用TRIzolLS提取RNA,反转录过程中用Cy5标记样品cDNAs。将标记样品与芯片杂交,扫描芯片上待检样品与芯片上捕捉探针的结合位点,杂交信号与预期设想一致。结果显示,DNA芯片技术可以提供一种有效的AIV血凝素亚型鉴别诊断方法。     

Existence of a Novel Hemagglutinin Having No Protease Activity in Vibrio mimicus

The protease elaborated by Vibrio mimicus is known to possess hemagglutinating ability to chicken erythrocytes, the well-known HA/protease. A non-protease hemagglutinin (HA) with strong agglutinating ability towards rabbit erythrocytes was obtained from 32 hr culture supernatant of a pathogenic environmental strain of V. mimicus. This HA (V. mimicus HA: VMHA) appeared stable at relatively higher temperature and agglutinated the erythrocytes from rabbit, guinea pig and mouse but not the erythrocytes from chicken, bovine, horse and sheep. Simple sugars, metal ions and chelating agents failed to inhibit the activity of VMHA. The activity of VMHA was found to be sensitive to digestion by proteolytic enzymes including HA/protease. These results provide evidence for the existence of novel HA other than HA/protease in V. mimicus.     

马链球菌透明质酸合成酶基因的分子克隆及表达

根据Streptococcus equisimilis、Streptococcus pyogenes、Streptococcus uberis三种链球菌透明质酸合成酶(seHAS、spHAS、suHAS)基因的高度保守区,设计一对简并引物,用两次PCR从Streptococcus equi的总DNA中扩增出sqHAS基因。构建表达质粒pSE-sqHAS并转化大肠杆菌DH5α,诱导培养后在细胞膜中检测到sqHAS蛋白及活性。利用携带该酶的细胞膜以UDP-GlcA和UDP—GlcNAc为底物在体外合成了分子量为3.6×10~6Da的HA,分别是发酵法生产和提取法生产的HA的分子量的2.5倍和5倍左右。马链球菌透明质酸合成酶基因的克隆及表达,国内外文献尚未见报道。本研究为体外酶法生产透明质酸做了初步探索。