河南地区HIV-1毒株Gag基因抗原表位变异特征及准种特点研究

目的 探讨中国河南地区人免疫缺陷病毒Ⅰ型(HIV-1)毒株GAG蛋白抗原表位变异特征,并对其准种特点加以分析.方法 套式聚合酶链反应(Nested-PCR)扩增确认HIV阳性样本gagp17～p24基因区段并测序,PCR产物纯化后克隆,挑选克隆株鉴定为阳性后测序,以MEGA(version 3.0)等软件进行分析.结果 河南HIV毒株为B'亚型;gag基因p17区段抗原表位突变有E62G(55.80%),Y79F(48.90%),T84V(48.90%),144V(44.20%),gag基因p24区段抗原表位未见明显变异.结论 HIV-1 B'亚型毒株gag基因p17区段的4个抗原表位,存在较大变异,p24区段较为保守,适合抗原表位疫苗的研制.     

中国HIV-1型B、C亚型主要流行株外膜蛋白基因V3-V4区序列的变异分析

目的　研究我国人类免疫缺陷病毒 1型 (HIV 1)B、C亚型主要流行株在感染过程中基因变异的特点及其与选择压力的关系。方法　应用巢式聚合酶链反应 (nested PCR)对 2 5 8例HIV 1感染者血样中的HIV 1外膜蛋白 (env)基因进行扩增 ,并使用ABI 377型测序仪对扩增产物测序后 ,选择其中 37份B亚型和 35份C亚型HIV 1毒株env基因包括V3～V4区的序列进行比较分析 ,并计算和分析氨基酸同义替换与非同义替换的比值 (Ks Ka)。结果　B亚型毒株V3～V4区的基因离散率高于C亚型毒株。无论B亚型 ,还是C亚型毒株 ,其V4区基因序列较V3区变异更大。在C亚型毒株中 ,V3区基因序列变异甚至比V3上游区和C3区小。B和C亚型毒株整个V3～V4基因区的Ks Ka比值均 <1,差异有非常显著性 (P <0 0 0 1) ,其中B亚型毒株以V3区的Ks Ka比值最小 ,而C亚型毒株则以V4区的Ks Ka比值最小。结论　B和C亚型毒株env基因的变异主要发生在V4区而不是V3区。C亚型毒株V3区较V3上游区和C3区还要保守 ,是本研究的特殊发现。这两种亚型在我国快速流行中发生的变异是在选择压力下发生的 ,而不是随机进化的结果 ,而且选择压力对这两种亚型毒株V3、V4区的作用程度也不一样。这将为我国艾滋病防治策略的制定和疫苗研究提供科学的依据。     

早期HIV-1感染env基因的动态变异研究

目的 追踪观察HIV-1新发感染者体内CRF07_BC重组毒株膜蛋白基因的变异性.方法 从HIV-1感染者血浆中提取总RNA,通过逆转录多聚酶链反应(RT-PCR)获得HIV-1 gp120全长及C2-C5区段基因.纯化后装入T载体,转化至Top10大肠埃希菌内增殖,通过蓝白斑筛选获得阳性克隆,运用PCR方法进行鉴定,最后对所获得的目的克隆测序.结果从两个感染者血浆样品中获得感染后半年到2年半间多个时间点的gp120基因克隆共135个及gp120基因C2-C5区段克隆15个,分析显示这些克隆均为HIV-1 CRF07_BC亚型.随感染时间的延长,HIV-1 env基因的离散率与多样性均有增加的趋势.env基因同义突变与非同义突变比较结果显示,C1、C3与V4区段非同义突变比率比gp120基因的其他区域都高.基因多态性分析的结果显示,同一时间点内不同毒株基因在不同区段的变异是不同的,基因多样性介于0与0.066±0.028之间.结论 随着患者感染时间的推移,HIV-1膜蛋白基因的变异逐渐增大.C1、C3和V4区的高突变率提示,该基因区可能是HIV-1病毒生存与宿主免疫压力相互作用的主要位点.     

HIV/AIDS患者特异性细胞毒性T细胞功能的研究

目的 了解中国HIV／AIDS患者HIV特异性细胞毒性T细胞(CTL)功能。方法 将覆盖HIV-1 P15、P17和P24 Gag全长的94个重叠多肽作为抗原，用IFN-γ ELISPOT方法检测HIV／AIDS患者HIV-1特异性CTL功能。结果 HIV-1抗原多肽P17-15、P17-16、P24-7、P17-8，P24-28最易被HIV／AIDS患者特异性CTL识别。HIV感染者识别HIV-1多肽的数量和强度均高于AIDS患者。结论 我国HTV／AIDS患者体内存在识别不同HIV-1 Gag多肽的特异性CTL，且HIV特异性CTL功能与疾病进展相关。     

黑龙江省17例HIV/AIDS患者的病毒亚型及基因序列特征分析

目的了解黑龙江省内部分人免疫缺陷病毒1型（HIV-1）的亚型及基因序列特征。方法用巢式聚合酶链反应（nested-PCR），对黑龙江省内17份HIV-1感染者外周血单个核细胞（PBMC）中前病毒脱氧核糖核酸的膜蛋白（env）基因进行扩增，并对C2-V3的核苷酸序列进行测定和分析。结果系统树分析显示，17份样本中病毒与HIV泰国B（B’）亚型聚在一起，基因离散率为（6．94±1．01）％，与欧美B亚型基因离散率为（12．94±2．19）％，与其他亚型的离散率大于20％。对于其V3环四肽序列的分析表明，具有GPGQ的8例，占47．06％；具有GPGR的7例，占41．18％；1例为GQGR；1例为GPGH。通过序列分析预测，大部分利用CCR5辅助受体。结论所检测的黑龙江省17例HIV-1均为B’亚型，提示黑龙江省的HIV-1流行株可能以B’亚型为主，其V3环顶端四肽序列特征以GPGQ和CP（承为主。     

人类免疫缺陷病毒-1包膜糖蛋白进化对抗原表位及病毒亲嗜性的影响

目的调查同一供体来源的人类免疫缺陷病毒（HIV）-1感染不同个体后病毒包膜糖蛋白的变异、病毒侵入靶细胞能力以及包膜抗原主要中和表位的变化,为了解病毒感染规律及机体抗病毒免疫奠定基础。方法对病毒包膜糖蛋白基因序列进行基因离散分析;用包膜蛋白表达质粒与HIV-1骨架质粒共转染293T细胞构建包膜包膜假病毒,用假病毒感染HIV-1靶细胞U87．CD4．CCR5或U87．CD4．CXCR4细胞检测假病毒侵入靶细胞的能力及病毒亲嗜性;对包膜糖蛋白中已知的广谱中和抗体识别表位进行分析。结果24个有完整开放读码框的env基因克隆与河南省HIV-1毒株CNHN24的基因离散率为（7．91±0．78）％,与云南省分离毒株RIA2的基因离散率为（6．904-0．79）％。各可变区基因离散率呈现严重不均衡性,其中,VI／V2区的离散率最高,V4区的离散率次之,V3区离散率最小。包膜假病毒中既有CCR5亲嗜性和CXCR4亲嗜性的,也有双亲嗜性的包膜。而且上述包膜中主要中和表位IgGlbl2、2F5和4E10抗体识别表位保守,但447—52D抗体识别表位变异较大。结论同一来源的HIV包膜糖蛋自在4～7年间的不同受者体内发生了较大变异并影响了病毒侵入靶细胞的能力;主要广谱中和抗体的识别表位部分保守。     

不同疾病进展阶段的HIV-1 B亚型毒株感染者对Gag、Nef肽段库的特异性CTL应答比例的比较和分析

目的 对不同疾病进展阶段的HIV-1B亚型毒株感染者Gag、Nef特异性细胞毒性T淋巴细胞（cytotoxic Tlymphocyte，CTL）应答进行研究，比较和分析不同患者群对不同肽段库应答比例的异同，探讨针对不同肽段库的特异性CTL应答在延缓病程进展中所起的作用。方法 选取56例未经抗病毒治疗的中国HIV-1B亚型毒株感染者。其中包括长期无进展者（long-term nonprogressors，LTNP）、HIV感染早中期患者和AIDS患者3组不同疾病进展阶段患者。以覆盖HIV-1 Gag全长和部分Nef的14个肽段库为刺激原，应用IFN-γ ELISPOT法测定3组患者的特异性CTL应答，并比较3组患者对不同肽段库的应答比例。结果 3组不同进展阶段患者对14个肽段库总体的特异性CTL应答的平均反应宽度和强度间差异均无统计学意义。3组患者对14个肽段库的识别模式可分为两种类型：（1）对Gag-p24-1、Gag-p24-5、Gag-p 2/7/1/6-1以及Gag-p2／7／1／6-3这4个肽段库的识别比例高低与病情进展情况相平行。3组患者对4个肽段库整体的识别比例间差异有统计学意义（P=0．041）；（2）对其他10个肽段库的识别与病情进展不平行，在HTV感染早中期患者中比例高，而在LTNP中低。结论 针对不同肽段库的CTL应答可能在控制病毒复制过程中发挥不同的作用，对疾病进展的控制可能需要针对多个抗原表位的有效CTL应答。     

HIV DNA疫苗与重组腺病毒伴随病毒联合免疫效果的研究

目的 构建含HIV-1B亚型中国株gagV3基因的DNA疫苗及重组腺病毒伴随病毒(rAAV)疫苗，并研究DNA疫苗和rAAV联合免疫的免疫效果。方法 将HIV-1B亚型中国株gagV3基因克隆入真核表达载体pCI-neo上，构建了含HIV-1 gagV3基因的DNA疫苗pCI-gagV3。采用电击法将pCI-gagV3质粒转染p815细胞，用G418压力筛选，得到转入重组质粒的细胞系p815-gagV3，用免疫酶法检测细胞系中HIV-1基因的表达。用该DNA疫苗进行小鼠免疫实验，检测免疫效果；用该DNA疫苗初次免疫，含同样gagV3基因的重组腺病毒伴随病毒rAAV-gagV3加强免疫，采用免疫酶法检测免疫小鼠血清中HIV-1特异性的抗体水平，用乳酸脱氢酶法检测免疫小鼠的HIV-1特异性CTL水平。结果 pCI-gagV3可以在p815细胞中表达HIV-1的基因，免疫BALB／c小鼠后可以在小鼠体内诱发HIV-1特异性的细胞和体液免疫反应。HIV-1特异性抗体滴度为1：20；效靶比为50：1时，CTL平均杀伤率为41．7％。pCI-gagV3与rAAV-gagV3联合免疫并不能明显提高抗体水平，但可以提高CTL反应，效靶比为50：1时，CTL平均杀伤率为61.3％，高于单独用DNA疫苗或重组AAV疫苗免疫后产生的CTL活性。结论 DNA疫苗与重组腺病毒伴随病毒联合免疫可以提高免疫小鼠产生的HIV-1特异性CTL反应。     

2006年中国流感病毒抗原性及基因特性研究

目的分析2006年中国季节性流感的流行状况，以及病毒的抗原性和基因变异情况。方法对来自流感监测网络的毒株进行单向血凝抑制试验，在此基础上选择不同时间、地点分离的毒株进行血凝素基因的序列测定，然后分析其基因特性。结果2006年我国同时流行A型（H1N1亚型、H3N2亚型）和B型流感病毒。H1N1亚型毒株和B型Victoria系流感病毒为优势毒株。对H1N1亚型毒株的HA1区序列比较发现，2006年分离的毒株与A，湖北洪山／53／2005（H1N1）比较，在192、193、196、198位发生氨基酸替换的毒株．这些位点位于抗原决定簇的B区。H3N2亚型毒株与A，云南，1145／2005（H3N2）比较，在142、144位发生氨基酸替换。我国流行的B型流感毒株无论是Victoria系和Yamagata系毒株的抗原性均没有发生变异，与2005--2006年我国的流行株B／shenzhen／155／2005、B／tianjin／144／2005类似。结论2006年中国流行的H1N1亚型和H3N2亚型流感病毒的抗原性及基因特性已经发生改变；B型流感病毒的抗原性和基因特性没有改变。     

中国人群HIV-1B亚型Nef蛋白特异性细胞毒性T细胞反应的研究

目的探讨中国人群HIV-1B亚型Nef蛋白特异性细胞毒性T细胞(CTL)反应特征及其与病毒载量以及CD4细胞数量间的关系。方法选取33例HIV-1B亚型感染者。用合成的HIV-1B亚型Nef全基因序列肽库作为抗原,ELISPOT方法检测HIV-1B亚型Nef蛋白特异性CTL反应,同时测定病毒载量及CD4细胞数量。结果70％的感染者对Nef产生特异性CTL反应,单一肽段能够被识别的频率不超过40％,应答强度为(1102±2136)SFC(斑点形成细胞数)／106 PBMC。HIV-1B亚型Nef特异性CTL应答的强度和频率之间没有显著的相关性。HIV-1 Neff特异性CTL反应强度与病毒载量间存在显著负相关,与CD4细胞数量间存在显著正相关。结论初步确定了Nef蛋白CTL应答的优势区域。这些区域主要集中在一些高度保守的氨基酸序列。提示HIV-1B亚型Nef特异性CTL应答在疾病进展中对机体具有保护性作用。     

Characterization of SE‐P3, P16, P37, and P47 bacteriophages infecting Salmonella Enteritidis

The aim of this study was to identify and characterize the SE‐P3, P16, P37, and P47 phages infecting Salmonella Enteritidis. Transmission electron microscopy analysis showed that the SE phages belonged to the Myoviridae or Siphoviridae family and had plaque sizes between 0.622 ± 0.027 and 1.630 ± 0.036 mm in diameter. sefC, pefA, spvC, sopE, and gipA virulent gene regions were absent in their genome and their calculated genome sizes were between 35.9 and 37.8 kbp. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the protein profiles of each phage were different. The SE phages had a short latent period (10–20 min), large burst size (76–356 PFU/cell), and a short burst time (25–35 min). The multiplicity of infection values and mutant frequency of the phages were 0.01–0.0001 and 10⁻⁷, respectively. They were very infective against their host bacteria when applied at 20°C, 30°C, or 37°C and adsorbed to their host cells by 96.20–97.65% in the first 5 min of incubation, and also Ca²⁺ ions did not have a significant effect on their adsorption. The SE phages were resistant to wide pH ranges and high temperatures. These results indicate that the SE phages are good candidates as therapeutic and biocontrol agents against foodborne pathogenic S. Enteritidis.     

Wavelet Analysis of P3a and P3b

Target/standard discrimination difficulty and the degree of stimulus "novelty" were manipulated systematically in a three-stimulus oddball task to assess how these variables affect target and non-target P300 scalp distributions for visual stimuli. Wavelet transformation (WT) analyses were performed on the non-target (P3a) and target (P3b) ERPs to assay how the underlying electroencephalographic (EEG) activity was affected by both the difficulty and novelty factors. When target/standard discrimination was easy, P300 amplitude was higher for the target than the non-target across all electrode sites, and both demonstrated parietal maximums. In contrast, when target/standard discrimination was difficult, non-target amplitude (P3a) was higher and earlier over the frontal/central electrode sites for both levels of novelty, whereas target amplitude (P3b) was greater parietally and occurred later than the non-target components and was generally unaffected by non-target novelty level. The WT analyses indicated that appreciable theta activity was related to the more novel non-target stimuli; primarily target component delta coefficients were affected by the discrimination difficulty variable. The findings suggest that target/standard discrimination difficulty, rather than stimulus novelty, determines P3a generation for visual stimuli but that the underlying theta oscillations are differentially affected by stimulus novelty. WT analysis methods are discussed along with the theoretical and neurophysiological implications of the findings.     

P300 and sleep-related positive waveforms (P220, P450, and P900) have different determinants

Stimulus factors known to influence the amplitude of the well known endogenous event-related potential (ERP) component P300 were manipulated to determine whether they have the same, or a different, influence on the amplitude of positivities of the sleep ERPs identified as P220, P450, and P900. Behavioral responsiveness and ERPs were recorded as subjects moved from wakefulness to sleep while performing an oddball task. The task consisted of sequential presentation of target and non-target tone stimuli with instructions to respond to targets with a finger--lift response. The probability of the target and non-target stimuli was varied (0.2/0.8, 0.5/.05 and 0.8/0.2) across three test conditions. While subjects were awake, P300 was maximal parietally with amplitude inversely related to the relative probability of the evoking stimulus and directly related to its task relevance. Positive waveforms (P220, P450, P900) recorded in sleep were largest at frontal and central recording sites. P220 and P900 amplitudes were inversely related to stimulus probability. P220 was smaller following target relative to non-target stimuli. Processes underlying P220, P450, and P900 sleep-related waveforms are different from those underlying the P300 component seen in alert wakefulness. The sleep positivities may be state-related waveforms subject to modulation by psychological processes.     

The formation of P particle increased immunogenicity of norovirus P protein

As a commentary on a recently published paper in Immunology, this article summaries the principle of norovirus P particle as a promising vaccine against noroviruses. It emphasizes the importance of P particle formation in the immune enhancement of the vaccine and methods for production/verification of high quality P particles which may be easily neglected by researchers.     

Functional Differences Between Members of the P300 Complex: P3e and P3b

The main findings of this study bear upon differences in the functional roles of P3b and a shorter latency, more centrally distributed endogenous positive component denoted as P3e. At the present writing, we have observed P3e only in conjunction with P3b. As in the case of P3b, P3e is fully endogenous in that it can be- elicited by omission of a stimulus if stimulus omission conveys relevant information to the subject. It was found that P3e and P3b relate differently to information delivery. Information delivery was manipulated by varying event probabilities and the discriminability of the events. The well known properties of P3b, namely that its amplitude is large when elicited by low probability (high information content) events and is reduced by perceptual difficulty (information loss-equivocation), were replicated in the current study. In contrast to P3b, variation of event probability had no effect upon P3e amplitude, but increased perceptual difficulty markedly reduced P3e amplitude. In addition, two CNV-type negativities were observed in the epochs prior to presentation of the informative signal event: 1) A negativity that was maximal over central scalp related to the subject's prediction that a rare or frequent event would be presented; 2) A negativity that was maximal over occipital scalp related to a stimulus that informed the subject whether the subsequent discrimination of the signal would be easy or difficult. Finally, there was a serendipitous Hading of an apparently new short duration component, tentatively labeled Px, which is elicited by presentation of the signal that informs the subject whether the subsequent discrimination will be easy or difficult.     

The Development of Visual P3a and P3b

The relationship of visual P3a and P3b to age and neuropsychological performance was investigated in 26 healthy children (6.8–15.8 years) and 129 adult volunteers (20.0–88.8 years). Within the sample of children, an effect of age on midline topography was observed, with higher frontal amplitudes in the youngest compared to the oldest children. Increasing age was associated with lower P3a and P3b amplitude and shorter P3b latency at Fz. Performance on neuropsychological tests (matrix reasoning from WASI, digit span from WAIS, word order and hand movement from Kaufman) was only weakly associated with measures of P3a and P3b. The analyses were then repeated with the full life-span sample (n = 155). It was found that for P3a, amplitude decreased and latency increased with age. For P3b, the pattern was more complex, with a nonlinear amplitude reduction and no latency change with age. It appears that the development of P3a in children represents the start of processes that later continue in the adult life-span, but that the automatic processes indexed by P3a seems to mature earlier than the controlled processes reflected by P3b. Finally, it was demonstrated that the relationships between neuropsychological test scores (matrix reasoning, digit span) and P3 parameters were complex, following a mix of linear and nonlinear patterns. It is suggested that the neuropsychological significance of the different P3a and P3b parameters may change from childhood to the adult life-span.     

The development of visual P3a and P3b

The relationship of visual P3a and P3b to age and neuropsychological performance was investigated in 26 healthy children (6.8-15.8 years) and 129 adult volunteers (20.0-88.8 years). Within the sample of children, an effect of age on midline topography was observed, with higher frontal amplitudes in the youngest compared to the oldest children. Increasing age was associated with lower P3a and P3b amplitude and shorter P3b latency at Fz. Performance on neuropsychological tests (matrix reasoning from WASI, digit span from WAIS, word order and hand movement from Kaufman) was only weakly associated with measures of P3a and P3b. The analyses were then repeated with the full life-span sample (n = 155). It was found that for P3a, amplitude decreased and latency increased with age. For P3b, the pattern was more complex, with a nonlinear amplitude reduction and no latency change with age. It appears that the development of P3a in children represents the start of processes that later continue in the adult life-span, but that the automatic processes indexed by P3a seems to mature earlier than the controlled processes reflected by P3b. Finally, it was demonstrated that the relationships between neuropsychological test scores (matrix reasoning, digit span) and P3 parameters were complex, following a mix of linear and nonlinear patterns. It is suggested that the neuropsychological significance of the different P3a and P3b parameters may change from childhood to the adult life-span.