B组轮状病毒ADRV与RDRV主要基因的克隆及其相关？…

对２株ＰＡＧＥ图型相似的Ｂ组轮状病毒－成人腹泻轮状病毒（ａｄｕｌｔｄａｒｒｈｅａｒｏｔａｖｉｒｕｓ,ＡＤＲＶ）和大白鼠服腹泻轮状病毒（ｒａｔｄｉａｒｒｈｅａｒｏｔａｖｉｒｕｓ,ＲＤＲＶ）主要基因的相关性进行研究。方法ＲＴ－ＰＣＲ扩增ＡＤＲＶ第４、５、９三个基因的ｃＤＮＡ全长拷贝,并获其ｃＤＮＡ克隆。用ＡＤＲＶ第９基因末端引物ＲＴ－ＰＣＲ成功扩增了ＲＤＲＶｄｓＲＮＡ,获得相对分子质量与ＡＤＲＶ第９基     

应用痘苗病毒体表达猴轮状病毒VP4抗原基因

把编码猴轮状病毒Ｖｐ４抗原的第４基因片段插入到痘苗病毒表达载体ｐＪＳＡ１１７５的Ｐ７．５启动子下游，构建成在痘苗病毒Ｐ７．５启动子调控下表达猴轮状病毒Ｖｐ４抗原基因的重组质粒ｐＪＳＡ１１７５－Ｖｐ４。应用磷酸钙沉淀技术将ｐＪＳＡ１１７５－Ｖｐ４ＤＮＡ转入ＴＫ－１４３细胞，在ＢＵＤＲ和Ｘ－ｇａｌ 存在下筛选蓝色蚀斑。     

食管癌低表达cDNA片段C6-2A的克隆及表达分析

目的　克隆人食管癌发生相关的基因。方法　用ｍ Ｒ Ｎ Ａ 差异显示技术分离、克隆食管癌组织中不表达或低表达的ｃ Ｄ Ｎ Ａ 片段,再通过 Ｎｏｒｔｈｅｒｎ ｂｌｏｔ 、ｄｏｔ ｂｌｏｔ 和 Ｒ Ｔ Ｐ Ｃ Ｒ 证实。结果　获得２８０ｂｐ 的ｃ Ｄ Ｎ Ａ 片段,命名为 Ｃ６２ Ａ。与 Ｇｅｎ Ｂａｎｋ 基因数据库比较,未发现 Ｃ６２ Ａ 与任何已知基因有同源性。查询 Ｅ Ｓ Ｔ 数据库,发现 Ｃ６２ Ａ 与ｎｅ２７ｂ０３ ,ｓ１ Ｎ Ｃ Ｉ Ｃ Ｇ Ａ Ｐ Ｃ０３ 人ｃ Ｄ Ｎ Ａ 克隆８９８５４１ 及人卵巢肿瘤ｃ Ｄ Ｎ Ａ 克隆７５５１９６ 等高度同源。 Ｎｏｒｔｈｅｒｎ ｂｌｏｔ 结果显示６／６ 例食管癌组织表达丧失或低表达,ｄｏｔ ｂｌｏｔ 分析表明７／８例食管癌组织低表达, Ｒ Ｔ Ｐ Ｃ Ｒ 显示食管癌细胞系 Ｅ Ｃ１０９ 、 Ｅ Ｃ８７１２ 、 Ｅ Ｃ９７０６ 和肺腺癌细胞系 Ｇ Ｌ Ｃ８２ 极弱表达,１７／２０ 例食管癌组织表达丧失或低表达,胎儿食管、皮肤、大脑、胎盘较高表达,胎儿胃、肝较弱表达,胎儿心、小肠、肾不表达。结论　在食管癌细胞系和食管癌组织高频率的不表达或低表达提示, Ｃ６２ Ａ 很可能与食管癌的发生、发展有关。     

先天性尿道下裂与SRD5A2及SRY基因突变关系研究

目的　探讨中国人先天性尿道下裂的发生与５α还原酶２（ Ｓ Ｒ Ｄ５ Ａ２） 基因突变及 Ｓ Ｒ Ｙ 基因突变的关系。方法　收集先天性尿道下裂患儿静脉血２３ 例,抽提 Ｄ Ｎ Ａ。经 Ｐ Ｃ Ｒ 分别扩增 Ｓ Ｒ Ｄ５ Ａ２ 基因的第１ 至第５ 外显子及 Ｓ Ｒ Ｙ 基因的 Ｄ Ｎ Ａ 片段。对各扩增片段采用单链构象多态性诊断法（ Ｐ Ｃ Ｒ Ｓ Ｓ Ｃ Ｐ） ,筛选基因突变标本,有电泳异常的标本进行测序分析。结果　经 Ｐ Ｃ Ｒ Ｓ Ｓ Ｃ Ｐ 发现３ 例 Ｓ Ｒ Ｄ５ Ａ２ 基因的第４ 外显子 Ｄ Ｎ Ａ 扩增片段电泳带位置异常,经 Ｄ Ｎ Ａ 序列分析证实２ 例为纯合子型第２２７ 位密码子由 Ｃ Ａ Ａ 替代 Ｃ Ｇ Ａ（ Ａｒｇ２２７ Ｇｌｎ） ;１ 例为双重杂合子型突变,第２２７ 位密码子由 Ｃ Ａ Ａ 替代 Ｃ Ｇ Ａ,同时第１８６ 位密码子由 Ｔ Ｔ Ｇ 替代 Ｔ Ｔ Ｔ（ Ｐｈｅ１８６ Ｌｅｕ） 。 Ｐ Ｃ Ｒ Ｓ Ｓ Ｃ Ｐ 检测 Ｓ Ｒ Ｙ 基因片段未发现异常电泳带。结论　 Ｓ Ｒ Ｄ５ Ａ２ 基因突变,可能是先天性尿道下裂的病因之一, Ｓ Ｒ Ｄ５ Ａ２ 基因的第２２７ 位密码子可能为中国人该基因的突变热点,而尿道下裂中 Ｓ Ｒ Ｙ 基因的突变少见。     

轮状病毒1,2血清型VP7蛋白基因的扩增及其克隆

用聚合酶链反应扩增了轮状病毒Ｗａ株（第１血清型）和ＤＳ－１株（第２血清型）的全长ＶＰ７蛋白基因，利用两引物５＇末端的多接头位点，以ｐＵＣ１８质粒为载体构建了含两株ＲＶＶＰ７基因ｃＤＮＡ拷贝的重组体。经ＰＣＲ扩增制备得１～４血清型ＲＶＶＰ７基因高变区（５１－３９２核苷酸）￣（３２）Ｐ标记核酸探针，对重组质粒型别进行了Ｓｏｕｔｈｅｒｎ和斑点杂交鉴定，结果Ｗａ株ＶＰ７基因重组体只与１型探针呈强杂交信号，ＤＳ－１株重组体只与２型探针呈强杂交信号。     

应用痘苗病毒载体表达猴轮状病毒VP4抗原基因

把编码猴轮状病毒（Ｒｈｅｓｕｓｒｏｔａｖｉｒｕｓ，ＲＲＶ）Ｖｐ４抗原的第４基因片段插入到痘苗病毒表达载体ｐＪＳＡ１１７５的Ｐ７．５启动子下游，构建成在痘苗病毒Ｐ７．５启动子调控下表达猴轮状病毒Ｖｐ４抗原基因的重组质粒ＰＪＳＡ１１７５－ＶＰ４。应用磷酸钙沉淀技术将ＰＪＳＡ１１７５－ＶＰ４ＤＮＡ转入ＴＫ－１４３细胞，在ＢＵＤＲ和Ｘ－ｇａｌ存在下筛选蓝色蚀斑。经３代以上纯化和病毒增殖，获重组病毒Ｒ－ＶＪＳＡ１１７５－Ｖｐ４。蚀斑滴定其满度达到１５×１０１１ＰＦＵ／Ｌ。经核酸杂交试验证明所获得的重组痘苗病毒带有猴轮状病毒Ｖｐ４抗原基因。用重组病毒感染ＴＫ－１４３细胞（或Ｖｅｒｏ细胞），在感染后４８ｈ，用酶免疫法（ＥＩＡ）检测受染细胞上清液和细胞裂解液中表达的猴轮状病毒Ｖｐ４抗原基因均呈阳性反应。本试验为本研究室轮状病毒基因工程疫苗的一部分，为深入了解轮状病毒基因结构及其功能在方法学上奠定了必要的基础。     

T细胞识别HLA-DRB1*0901分子显示TCR BV基因取用的高度局限性

目的分析ＤＲ９纯合细胞刺激下ＤＲ９阴性正常人ＴＣＲＢＶ基因片段的取用格局,确定和分离出ＤＲ９关联性疾病中的自身反应性Ｔ细胞克隆。方法常规方法获取ＰＢＭＣ经ＰＣＲ－ＳＳＯ和ＰＣＲ－ＲＦＬＰＤＮＡ分型,确定ＤＲ、ＤＱ、ＤＰ等位基因后,进行单向混合淋巴细胞培养,抽提总ＲＮＡ并合成第一链ｃＤＮＡ,定量ＰＣＲ检测２２种ＴＣＲＢＶ基因片段的取用格局。结果Ｔ细胞对ＤＲ９分子的识别和扩增具有寡克隆性,２例都有ＢＶ７和ＢＶ１６片段的优势取用,而片段ＢＶ１９和ＢＶ１０则在２例中分别表达。结论实验结果表明,共有ＢＶ７和ＢＶ１６片段的优势取用是ＤＲ９抗原等分子经直接途径激活了带有相应ＴＣＲＢＶ细胞的结果;而片段ＢＶ１９和ＢＶ１０在２例中分别表达可能是不同ＨＬＡ背景的ＡＰＣ间接递呈ＤＲ９抗原的结果。     

汉坦病毒A9株M基因片段全长cDNA克隆及其在痘苗病毒中的瞬时表达

采取反转录一聚合酶链反应（ＰＣＲ），分３个片段扩增Ｉ型汉坦病毒（野鼠型）中国毒株Ａ９株Ｍ基因片段，分别克隆入ＰＧＥＭ－Ｔ载体中。选择３个正向插入的ＴＡ９ＩＢ、ＴＡｇＣＤ、ＴＡｇＥＡ克隆，选用ＣｌａＩ、ＥｃｏＲＶ进行酶切、连接，获得了１个在ＰＧＥＭ－Ｔ载体中插入３．６ｋｂ的Ａｇ株Ｍ基因片段ｃＤＮＡ克隆，酶切图谱分析证实播入片段正确。将Ａ９Ｍ片段在痘苗病毒／Ｔ７噬菌体ＲＮＡ聚合酶瞬时（Ｔｒａｎｓｉｅｎｔ）表达系统中进行表达，用抗汉坦病毒糖蛋白单克隆抗体进行免疫荧光检测，观察到很强的特异性荧光，证明ＡｇＭ基因ｃＤＮＡ能进行表达。     

多重PCR方法检测多发性骨髓瘤患者重排基因的研究

目的　探讨分子生物学方法在多发性骨髓瘤诊断中的意义。方法　采用多重 Ｐ Ｃ Ｒ 联合 Ｓｏｕｔｈｅｒｎ 杂交方法检测２９ 例多发性骨髓瘤患者骨髓及外周血标本 Ｉｇ Ｈ Ｃ Ｄ ＲⅢ及 Ｔ Ｃ Ｒ ＶγⅠ Ｊγ重排基因。结果　８９ ．７ ％ （２６／２９） 和６５ ．５ ％ （１９／２９） 的患者骨髓标本存在 Ｉｇ Ｈ 及 Ｔ Ｃ Ｒ ＶγⅠ Ｊγ重排基因,而在外周血标本,仅５５ ．２ ％ （ １６／２９） 和３７ ．９ ％ （１１／２９） 存在 Ｉｇ Ｈ 及 Ｔ Ｃ Ｒ 重排基因。联合检测 Ｉｇ Ｈ Ｃ Ｄ ＲⅢ和 Ｔ Ｃ Ｒ ＶγⅠ Ｊγ重排基因,９３ ．１ ％ （２７／２９） 骨髓标本及５８ ．６ ％ （１７／２９） 外周血标本存在 Ｉｇ Ｈ 或／ 和 Ｔ Ｃ Ｒ 重排基因,外周血标本Ⅱ期和Ⅲ期患者重排基因阳性率（７１ ．４ ％ ） ,显著高于Ⅰ期患者（２５ ％ ）（ Ｐ＜ ０ ．０５） 。结论　 Ｐ Ｃ Ｒ 技术检测多发性骨髓瘤患者重排基因对诊断是有帮助的, 尤其是骨髓标本。多重 Ｐ Ｃ Ｒ 方法可在一个 Ｐ Ｃ Ｒ 循环中同时扩增两个重排基因,更简便、经济,更适宜临床实际应用。     

庚型肝炎病毒NS5区部分基因的表达及其在EIA中的 …

目的 在大肠杆蓖中表达ＨＧＶ ＮＳ５抗原,以建立ＨＧＶ抗体血清学检测方法。方法 利用扫转录ＰＣＲ（ＲＴ－ＰＣＲ）法从人血浆中分离庚型肝炎病毒ＮＳ５部分基因,克隆到ｐＲＳＥＴ Ａ载体,经酶切初步鉴定后进行ＤＮＡ序列分析,结果表明克隆序列正确。以ｐＰＲＯＥＸ－１为表达载体,转化ＤＫ５α,诱导表达后进行ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ ｂｌｏｔ分析。结果 克隆的基因片段在大肠杆菌中表达出相对分子质量（Ｍ     

Temporal distribution of human rotavirus serotypes 1,2,3, and 4 in Venezuelan children with gastroenteritis during 1979-1989.

The temporal distribution and clinical severity of rotavirus VP7 serotypes 1, 2, 3, and 4 recovered from 427 Venezuelan children with acute gastroenteritis over a period of 11 years were studied. Rotavirus VP7 serotype was established by ELISA serotyping in 298 (69.78%) of the specimens while the serotype of the remaining 129 (30.21%) samples could not be determined. Of the specimens typed, 85 (19.90% of the total) were serotype 1, 43 (10.07%) were serotype 2, 105 (24.59%) were serotype 3, and 65 (15.22%) were serotype 4. Yearly changes in the frequency of individual serotypes were observed. The predominance of a single serotype with minor contribution from others was noted every year. In this study, serotype 1 appears to induce a less severe illness in comparison with serotypes 2, 3, and 4. No apparent association between the proportion of each serotype and the children's age were found.     

Severity and age of rotavirus diarrhea,but not socioeconomic conditions,are associated with rotavirus seasonality in Venezuela

Rotavirus (RV) epidemiology presents differences between developing and developed countries among which are seasonality, age at first infection, variability of strain in circulation and severity of disease. Since, in Venezuela, we have distinct seasonal patterns of RV occurrence, we examined the epidemiological profile of RV disease associated to these differences by analyzing data from previous studies conducted in Venezuela. Data were collected from children <5 years of age with diarrhea seen in six hospitals located in five cities. Socio‐demographic and clinical characteristics of RV illness were analyzed according to RV identification by ELISA assay and the seasonal patterns of RV circulation (marked versus minimal seasonality). A total of 6,742 episodes of diarrhea (1,820 rotavirus positive and 4,922 rotavirus negative) were evaluated: 1,951 in Caracas, Cumaná and Pto. Ordaz (minimal seasonality) and 4,791 in Mérida and Valencia (marked seasonality). Mean age (months) of children with RV was 8.14 ± 6.74 and 12.21 ± 9.62 (P < 0.0001) in areas with minimal and marked seasonality, respectively. RV disease was more frequent (33% vs. 24%; OR = 1.536; 95% CI: 1.36–1.73), more severe (dehydration: 64% vs. 29%; OR = 4.436; 95% CI: 3.61–5.44) and more common in infants than in older children (79% vs. 60%; OR = 2.521; 95% CI: 2.01–3.14) in cities with minimal seasonality than in those with marked seasonality. Socioeconomic conditions were not associated with seasonality. In environments with minimal seasonality, children are infected with RV at younger ages and the disease is more severe regardless of malnutrition and poverty. J. Med. Virol. 81:562–567, 2009. © 2009 Wiley‐Liss, Inc.     

两株Wa株人源轮状病毒NSP4蛋白的表达及其致ICR小鼠腹泻的毒力比较

目的研究两株Wa株人源轮状病毒NSP4蛋白139位氨基酸位点的变异对毒力的影响。方法从2002～2004年昆明地区婴幼儿轮状病毒感染者大便样品中,通过RT-PCR,PCR扩增了22株轮状病毒株的NSP4全长基因、cDNA序列,并对cDNA序列测序,发现这些病毒株的NSP4氨基酸序列高度保守,仅在79位、139位氨基酸位点存在差异。选取139位氨基酸不同(V/I)的两株病毒株,在E.coli BL21中,用pGEX5X-1载体融合表达了两株病毒的86～175位氨基酸(GST-NSP4_(86-175);并酶切融合头得到两种NSP4_(86-175)蛋白,在ICR乳鼠中比较这4种不同蛋白致小鼠腹泻的差异。结果发现GST-NSP4_(86-175)蛋白毒性比NSP4_(86-175)蛋白强,而139位氨基酸不同(V/I)的两株病毒株的NSP4_(86-175)蛋白毒性没有明显的差异。结论139位氨基酸位点的变异并不影响NSP4蛋白的毒力改变。     

Human rotavirus in an adult population with travelers' diarrhea and its relationship to the location of food consumption.

The role of human rotavirus in adult diarrhea was evaluated in 164 newly arrived US students attending summer school at an urban Mexican university. Rotavirus was identified in stool samples by electron microscopy. Rotavirus was found in 26 of 109 students with diarrhea (24%) and in 8 of 55 asymptomatic control students (15%). Although bacterial pathogens were recovered from virus positive students with diarrhea, viral shedding also occurred independently of other agents. Clinical disease in students excreting only rotavirus tended to be mild and was accompanied by a low density of viral shedding. Food consumption in the home and at public eating establishments was examined the week before illness. While the location of food consumption was found to be important in the acquisition of diarrhea, there was no apparent relationship of the site where meals were eaten and the acquisition of rotavirus by students newly arrived in Mexico. These data support our previous study in a US student population residing in a rural setting in Mexico and implicate rotavirus as a cause of diarrhea among students traveling to Mexico from the United States. The present study offers additional evidence that rotavirus infection in this population might be spread by a nonfood vehicle of transmission which differs from spread of enterotoxigenic E coli, Shigella, or Salmonella strains in the same population.     

A组轮状病毒单克隆抗体特性鉴定及临床初步应用

本文报道了A组RV McAb的特性鉴定及临床初步应用。用从河南省腹泻犊牛粪便中分离的HN-7毒株(RV)抗原免疫BALB/c小鼠,用杂交瘤技术获得2株分泌A组RV特异性McAb的杂交瘤细胞系。经鉴定,所获2个McAb(2C7.3C9)与A组RV第1血清型Wa株、第3血清型SA-11株,第6血清型NCDV株以及从国内猪、牛粪便中分离的4株未分型毒株的间接ELISA效价达10~(-5)～10~(-6),但与这7R-V毒株的血凝抑制滴度≤1:8,中和效价≤1:100,表明,这2个McAb是针对A组RV共同抗原的。琼脂糖双向扩散结果,2C7为小鼠IgG1亚类,3C9待鉴定。用RVMcAb建立的单夹心ELISA技术测定162例人,牛腹泻粪样,阴阳性总符合率与常规 ELISA相比较,为98.8％(160/162)。     

2005—2007年广州地区腹泻儿童感染轮状病毒情况分析

目的了解近3年来广州地区儿童肠道轮状病毒感染情况。方法收集2005年1月至2007年12月收治的包括门诊和住院的急性腹泻患儿的大便标本,采用免疫胶体金技术对标本进行轮状病毒抗原检测。结果轮状病毒2005、2006和2007年平均感染率分别为44．70％、38．22％和39．99％;与2006、2007年相比,2005年轮状病毒感染高峰和波谷均向后推延1个月。抗原检测阳性例数在6～24个月婴幼儿最高,全年感染率在性别上差异无统计学意义。结论轮状病毒是引起婴幼儿病毒感染性腹泻的主要病原体,气候可能对广州地区轮状病毒感染有较大影响。     

Molecular epidemiology of group A rotavirus in Buenos Aires,Argentina 2004–2007: Reemergence of G2P[4] and emergence of G9P[8] strains

Detection and characterization of group A rotavirus in Buenos Aires, Argentina, was conducted on 710 fecal samples from children 0–15 years old collected between 2004 and 2007. Rotavirus was detected in 140 (19.7%) samples with G9P[8] (30.0%) and G2P[4] (21.4%) as the most common genotypes. Mixed (G and/or P) infections accounted for 17.9% of the samples and the emerging G12 strain was detected during 2004 (3.5%) and 2007 (2.5%). Genotype G2 was the most prevalent during 2004 (43.9%) and 2007 (57.5%) and G9 during 2005 (58.0%) and 2006 (61.5%). Analysis of genotype prevalences from studies performed since 1996 in the same area showed striking natural fluctuations in G and P genotype frequencies. In particular, G2P[4] strains disappeared after 1999 and reemerged in 2004 to become the predominant strain by 2007 with a concomitant major decrease in G1P[8] prevalence. The VP7 genes from Argentinian G9 and G2 strains were sequenced and phylogenetic analysis was conducted in order to compare with sequences from strains isolated in regional countries reported previously. Several changes in the deduced amino acid sequence in antigenic regions of the VP7 protein from Argentinian and Brazilian strains were identified compared to vaccine strains. Overall, this study revealed relationships in the circulation of rotavirus strains in South American countries and major replacements in dominant genotypes, including the virtual disappearance of G1P[8] strains in a non‐vaccinated population. High numbers of mixed infections speeding up evolution, circulation of rare serotypes, and antigenic drift could, eventually, become challenges for new vaccines. J. Med. Virol. 82:1083–1093, 2010. © 2010 Wiley‐Liss, Inc.     

Homotypic immune response to primary infection with rotavirus serotype G1

Some aspects of rotavirus humoral immunity were assessed on the basis of distinguishing se-rotype-specific specificities (VP4/VP7) by using rotavirus reassortants, human and animal strains in neutralization assays in serum samples obtained during the acute phase, and 1, 6 and 12 months after primary natural infection. In this study, all the infecting virus strains were characterized as G type and some also as P type. Primary natural infection induces a significantly greater homotypic neutralization response than heterotypic response. In addition, there was no significant difference in the number of homotypic or heterotypic responses following reinfection. Transplacentally acquired homotypic antibodies were associated with protection against dehydration during rotavirus gastroenteritis. © Wiley-Liss, Inc.