Studies on renin-2 gene in transgenic rats

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Experimental oral and nasal transmission of rabies virus in mice.

Weanling female white Swiss mice were exposed to challenge virus standard rabies virus and street virus isolates from various domestic and wild animals. Virus was given free choice as suspension or as infected mouse brain by stomach tube, by single injection of suspension into the oral cavity of unanesthetized mice, by repeated injection into the oral cavity of anesthetized mice and by single application to the external nares of anesthetized mice. Challenge virus standard virus in mouse brain suspension and a suspension of skunk salivary glands infected with street virus (titers greater than or equal to 10(6)MICLD50/0.03 ml) consistently produced high rates of infection in mice exposed intranasally, low to high rates of infection in mice exposed by forced feeding and other artificial methods of oral exposure and very low rates of infection when given free choice. Street virus isolates passaged intracerebrally in mice had titers less than or equal to 10(4.5) MICLD50/0.03 ml and rarely caused rabies in mice exposed orally or nasally by any method. The results indicate that with the isolates used, virus of high titer (greater than or equal to 10(6)MICLD50/0.03 ml) is required to consistently produce infection in mice by the nasal route and that the mucosa of the nasal cavity probably is the chief route of infection even after oral administration.     

携带信号肽NT4-GFP-穿膜肽Ant融合基因的重组腺相关病毒载体的构建及意义

目的：构建携带具有穿膜功能的融合报告基因NT4-GFP-Ant基因的重组腺相关病毒载体。方法：应用PCR技术和T载体克隆法克隆绿色荧光蛋白（GFP）基因;用T4 DNA连接酶将测序正确的GFP与已构建成功的Ant基因片段及PBV220/NT4线性质粒载体定向连接,构建PBV220/NT4-GFP-Ant融合载体,酶切获取融合基因,并将其连入腺相关病毒载体PSSHG中,构建PSSHG/NT4-GFP-Ant重组腺相关载体并进行酶切鉴定。结果: T-easy/GFP经EcoRⅠ酶切后,可得到730 bp左右的片段,GFP基因经DNA测序证实与GenBank序列一致;pBV220/NT4-GFP-Ant经BamHI/EcoRⅠ联合双酶切后,得到约为 1 000 bp的基因片段; PSSHG /NT4-GFP-Ant经 BamHI/EcoRⅠ联合双酶切后,得到大小约为1 000 bp长度的基因片段。结论：成功克隆GFP基因,成功构建NT4信号肽-GFP-Ant穿膜肽融合基因和PSSHG/NT4-GFP-Ant重组腺相关病毒载体。     

大鼠基质金属蛋白酶抑制因子-1 pRNAT-U6.2 小干扰RNA表达载体的构建

目的:构建大鼠基质金属蛋白酶抑制因子-1(TIMP-1)小干扰RNA的真核表达质粒pRNAT-U6.2 siRNA.方法: 依据siRNA设计原则确定以序列447～465 nt、522～540 nt、142～160 nt为TIMP-1 siRNA靶序列, 体外分别合成两端含BamH Ⅰ和Xho Ⅰ酶切位点的编码短发夹RNA序列的DNA单链,退火后克隆于pGEM-T载体, T7和SP6为引物进行PCR鉴定;双酶切pGEM-T将其定向克隆到siRNA表达载体pRNAT-U6.2,用pRNAT-U6.2的插入鉴定引物进行PCR鉴定,阳性重组质粒测序.结果:DNA测序证实合成的并被克隆入真核表达载体pRNAT-U6.2的siRNA插入序列与设计完全符合.结论:TIMP-1 siRNA表达载体构建成功, 为后期研究TIMP-1 pRNAT-U6.2 siRNA作用、意义及效果奠定了实验基础.     

Molecular fingerprinting of multidrug-resistant Mycobacterium tuberculosis strains in Beirut reveals genetic diversity and father to daughter transmission

The typing of six consecutive multidrug-resistant Mycobacterium tuberculosis strains isolated from patients with tuberculosis (TB) at the American University of Beirut Medical Center, was performed by touchdown double-repetitive-element (DRE)-PCR. The isolates exhibited four distinct patterns in DRE-PCR with three isolates exhibiting unique patterns and three isolates yielded similar DNA fragment patterns (cluster pattern). Only two of the three cluster isolates exhibited identical patterns as revealed by restriction fragment length polymorphism (RFLP) targeting specific mutations in the rpoB and katG genes that confer resistance to rifampin and isoniazid, respectively. A direct epidemiological linkage for the two isolates exhibiting genotypic relatedness was also established as the isolates were recovered from a 33-year-old man and his 8-year-old daughter. The data show that transmission of multidrug-resistant M. tuberculosis strains is contributing to the emergence of drug-resistant TB in Beirut. Combining DRE-PCR with RFLP at the rpoB and katG genes could provide a powerful means for investigating the spread of multidrug-resistant M. tuberculosis strains in Lebanon.     

Experimental rabies in skunks: oral, nasal, tracheal and intestinal exposure.

Striped skunks (Mephitis mephitis) were exposed to challenge virus standard rabies virus by feeding infected mouse brain in suspension or as intact brain free choice, by forced feeding of suspension, and by intranasal, intratracheal and intraintestinal instillation of suspension. All of five skunks exposed intranasally, two of five exposed intratracheally and two of ten exposed by forced feeding developed rabies. None of the skunks exposed to challenge virus standard virus, by other methods, became rabid. Most of the survivors, when challenged intramuscularly with street rabies virus at six months, developed rabies. The results indicate that the skunk is much more susceptible to challenge virus standard rabies virus given intranasally than by the other methods used. When disease occurs following oral administration, infection may be associated with prolonged contact with buccal mucosa or accidental contact with nasal mucosa. Survivors had little or no protection when challenged intramuscularly with street rabies virus.     

增城市2002～2007年狂犬病流行特征分析及防制措施

目的分析增城市2002～2007年狂犬病的流行特征,提出有效防制措施,控制狂犬病发生,达到消除狂犬病。方法收集增城市2002～2007年狂犬病病例资料进行分析及防制措施效果评价。结果2002～2006年增城市狂犬病发病呈逐年上升,由2002年的1例上升到2006年的10例,狂犬病疫情较为严峻;2007年采用健康教育促进防制狂犬病措施,狂犬病病例明显下降,2007年发病7例,狂犬病疫情得到有效控制。结论我市狂犬病疫情比较严重,推广应用《增城市健康教育促进防制狂犬病效果研究》项目后,提高了整体人群预防狂犬病的知晓率和自我保护意识,短时间内有效控制了狂犬病疫情。     

东莞市厚街镇2005年被动物伤害人群的流行病学调查

目的分析东莞市厚街镇2005年被动物伤害人群流行病特点及狂犬疫苗接种情况。方法根据2005年广东省狂犬病暴露人群门诊登记资料,采用描述性研究的方法对东莞市厚街镇2005年被动物伤害人群的流行病学特征进行统计分析。结果东莞市厚街镇2005年被动物咬伤数共2797例,年致伤率736/10万,无狂犬病病例发生;伤人动物以家犬为主,致伤者中男∶女性别比是1.61∶1,15～44岁的中青年为最高(60.14%),下肢是最易被咬伤的部位,7～10月咬伤率最高,绝大部分人能及时全程足量注射完狂犬疫苗。结论加强犬、猫等动物管理,宣传狂犬病防治知识。     

变形链球菌表面蛋白V区、P区及C末端遗传多态性与龋病发生关系的研究

目的了解变形链球菌表面蛋白V区、P区及C末端遗传多态性与其粘附性能的关系。方法实验菌株选自本实验室前期工作所获得的粘附力较强和较弱的血清C型变形链球菌临床分离株，提取全菌DNA，经PCR分别扩增表面蛋白V区、P区编码基因spaP—pv(2060～3157bp)、C末端编码基因spaP-c(4003～4851bp)后，用限制性内切酶Alu I进行限制性片段长度多态性分析。结果两组不同粘附力的变形链球菌(血清C型)临床分离株全菌DNA扩增产物spaP—pv经Alu I酶切后，出现了两种基因型a、b。两种基因型在不同粘附力菌株的分布不同(P＜0．05)，a型在低粘附力菌株中的比例高于高粘附力菌株，而b型在高粘附力菌株中的比例高于低粘附力菌株。spaP—C经Alu I酶切后，共呈现两种基因型C、d。d型在高、低粘附力的菌株中各占1例，其分布无统计学差异。结论spaP—pv基因出现变异可能是变链菌临床分离株粘附功能出现差异的原因之一。     

平远县2001-2010年狂犬病流行病学分析

目的探讨平远县近10年来狂犬病流行病学特征及流行因素,为制订防控措施提供科学依据。方法收集2001-2010年平远县传染病疫情资料、狂犬病个案资料和狂犬病疫点调查处理资料,采用描述性流行病学方法进行分析。结果 2001-2010年平远县共报告狂犬病16例,年平均发病率为0.64/10万;死亡16例,病死率为100%;常年散发,秋季发病较多(占50%);发病以农村为主,农民和农村学生为主要发病人群;男性多于女性(性别比为5:3),40～70岁年龄组发病较多(占62.5%);头面部咬伤的潜伏期短,下肢咬伤的潜伏期较长;87.5%的病例未进行规范伤口处理,87.5%的病例未进行免疫接种。结论预防狂犬病须采取综合措施,加强犬类管理,加大健康教育力度,将农民和学生作为预防狂犬病的重点人群,规范暴露后伤口处理和提高狂犬疫苗接种率,才能有效降低发病率。     

桂林市1999-2008年狂犬病流行特征分析

目的分析1999-2008年桂林市狂犬病流行特征,探讨桂林市狂犬病流行相关因素,为狂犬病预防控制策略和措施的制定提供依据。方法收集1999-2008年桂林市狂犬病疫情资料、个案调查资料和专题调查资料,采用Excel 2003统计软件用描述性流行病学方法进行统计分析。结果10年间,桂林市共报告狂犬病400例,年平均发病率为0.82/10万。97.25%的病例发生在农村地区,0~9岁和50~59岁组分别是发病的两个年龄高峰。85.89%的伤人动物为家养动物,93.17%的伤人动物处死后未被深埋或焚烧。99.55%的病例暴露后未接受正确处置。相比没有接种史的病例,接种过疫苗仍发病的病例潜伏期更短,暴露程度更为凶险。结论桂林市狂犬病形势依然严峻,应提高全社会对犬患的足够认识,实施政府综合干预措施,坚决落实犬只捕杀办法,促进人群接受暴露后正确处置。     

重组小鼠白介素-2基因在真核细胞中的转染表达

目的：在构建重组真核表达载体pIRESneo2／mIL-2的基础上,建立能够持续稳定表达mIL-2的哺乳类工程细胞。方法：运用分子克隆技术,将由RT-PCR获得的mIL-2cDNA片断插入真核表达质粒pIRESneo2构建成mIL-2重组表达载体pIRESneo2／mIL-2。通过脂质体转染法将pIRESneo2／mIL-2导入C2C12细胞。转染后第30天,用Western blots检测mIL-2表达情况。结果：经DNA测序证明mIL-2cDNA片断插入方向和碱基组成顺序均准确无误,Western blots检测转染真核重组表达载体pIRESneo2／mIL-2的C2C12细胞系表达mIL-2。结论：利用pIRESneo2／mIL-2构建的真核表达载体在C2C12细胞系中能够持续稳定表达mIL-2。     

An Investigation of the First Case of Human Rabies Caused by a Fox in China in May 2016

This study assesses the causes and prevention measures of rabies through epidemiological investigation and analysis. A field epidemiological survey was conducted to investigate a case of rabies by fox bite. The onset of symptoms began 50 days after the bite. The patient did not receive standard treatment, rabies vaccination, or rabies immunoglobulin injection. The fox was killed on the spot. Saliva and pre-death blood samples were collected at different periods, and only blood RT-PCR tests yielded positive results. Wild fox bite is a major risk factor of rabies infection in Xinjiang Province, China.     

人端粒酶催化亚单位基因正反义腺病毒表达载体的构建及鉴定

目的: 构建hTERT正义和反义腺病毒表达载体. 方法: 用EcoRⅠ从pGRN145质粒上切下约3.5 kb的人端粒酶全长cDNA片段,然后连入pDC315质粒的EcoRⅠ酶切位点上,经BamHⅠ酶切鉴定出正义和反义表达载体,并对正反义重组质粒进一步测序鉴定其方向. 结果: 经BamHⅠ酶切后,正义质粒形成1.0 6.4 kb两条带. 反义重组质粒为1.0 2.5 3.9 kb三条带,与理论计算值完全一致,测序结果进一步确认了方向的正确性. 结论: 成功构建了hTERT的正、反义腺病毒表达载体.     

单纯疱疹病毒Ⅰ型糖蛋白D核酸疫苗的构建及初步研究

目的：构建、制备Ⅰ型单纯疱疹病毒糖蛋白D重组质粒DNA疫苗,初步检测其诱导机体产生体液免疫应答的效果,为HSV-1新型疫苗奠定基础。方法：用PCR的方法从HSV－1病毒基因组中扩增糖蛋白D（glycoprotein D,gD)基因,利用基因重组技术构建重组质粒;将重组质粒体外转染真核细胞COS－7,Western blotting检测表达产物;于BALB／C小鼠后腿胫前肌注射免疫,0、2周名免疫1次,100μg/次。初次免疫后0,2,4,6周眼眶采血,ELISA间接法检测抗体。结果：重组质粒酶切出相应大小片段,经测序证实为HSV－1gD序列。Western blotting证实能够在体外真核细胞中表达。免疫小鼠后,产生特异性抗体,抗体滴度1：2000。结论：HSV-1gD重组质粒DNA有可能作为HSV－1的DNA疫苗,用于防治HSV－1感染及其相关疾病。     

Epidemiological assessment of intestinal parasitic infections in dogs at animal shelter in Veracruz,Mexico

Objective:To determine the prevalence of infection with intestinal parasites in 101 dogs in an animal shelter in Veracruz.Mexico,and investigate whether any general characteristics of the dogs were associated with infections.Methods:Parasitologiesl examination of fecal samples from the dogs was performed by means of centrifuge-flotation using Sheather's sucrose and zinc sulfate flotation media.In addition,hematocrit was determined in each canine blood sample.Results:Intestinal parasites were found in 99(98.0%) of the 101 dogs studied.About five different intestinal parasites were identified:Ancylostoma caninum in 89 dogs(88.1%).Giardia canis in 46(45.5%).Unciiuiria stenocephalia in 43(42.6%).Trichuris vulpis in 19(18.8%)and Strongyloides canis in 16(15.8%).Multivariate analysis showed:I) Giardia infection was associated with young age and mixed breed;2) Ancylostoma was associated with young age and no rabies vaccination:and 3) Strongyloides was associated with no rabies vaccination.Unciiuiria and Trichuris infections were not associated with the variables assessed.Conclusions:A high prevalence of intestinal parasites was found in the dogs studied.This suggests that the environment is highly contaminated with intestinal parasites.Preventive and therapeutic measures should be taken against infection with intestinal parasites in dogs in this region.     

Bat rabies in Canada 1963-1967

Six hundred and twenty-eight insectivorous bats originating from seven provinces were submitted to this Institute for rabies diagnosis between August 1, 1963 and December 31, 1967. Brain tissue was examined by the fluorescent antibody technique and the mouse infectivity test was carried out with brain, salivary gland, interscapular adipose tissue and kidney samples. Rabies virus was detected in 44 bats, 29 of which were from Ontario, 12 from British Columbia and three from Manitoba. Most of the positive cases were diagnosed in summer months. Seven species were represented among the specimens found to be rabid; there were 32 big brown bats, three hoary bats, three silver-haired bats, two little brown bats, one eastern pipistrelle, one Keen myotis and one red bat. Another bat which was not identified also proved to be infected with rabies.     

不同疫区家犬携带狂犬病毒的比较研究

目的比较河南和陕西两省外观健康的家犬狂犬病毒携带率,为加强当前犬只管理、控制我国狂犬病不断上升的疫情提供参考依据。方法调查河南和陕西两省近年来人间狂犬病疫情;分别采集河南和陕西两省外观健康的家犬脑组织样品121份和645份,以免疫荧光实验（IFA）、小鼠颅内接种试验（MIT）以及逆转录聚合酶链式反应（RT—PCR）检测样品带毒情况;以MEGA及DNAStar等生物信息学软件对病毒核蛋白基因进行分析。结果从河南省121份犬脑样品中栓出阳性结果9份,陕西省645份样品无阳性;9株狂犬病毒与我国人用精制狂犬病疫苗CTN株系统发育关系较近。结论外观健康的家犬能够携带狂犬病毒;犬养殖量大、免疫率低以及狂犬病毒携带率高仍是当前我国狂犬病流行的主要原因。     

HPV16 E5蛋白原核表达、鉴定及真核表达稳定株的筛选

目的研究HPV16 E5蛋白的生物学特性及其细胞转化机制,对HPV16 E5蛋白原核和真核表达质粒进行构建、表达和鉴定。方法以临床确诊的HPVl6感染患者子宫颈细胞DNA为模板,采用PCR方法扩增HPV16 E5基因,经BamHⅠ和HindIⅡ双酶切后插入相同酶切的pET32a（＋）载体,转染JMl09感受态细胞,并进行阳性克隆筛选。经IPTG对重组质粒进行蛋白诱导表达,SDS-PAGE和Wlescem blotting检测目标蛋白表达情况。将BamHⅠ和XhoⅠ双酶切pET32（＋）,E5质粒后取得的E5全基因片段转入pcDNA3．1（＋）空质粒,构建pcDNA3．1（＋）m5真核表达质粒并对NIH3T3细胞进行转染,最后用G418进行稳定表达株的筛选,并采用RT．PCR鉴定细胞内HPV16 E5基因表达情况。结果成功构建了原核表达质粒pET32/E5,在1mmol／LPTG、28℃诱导条件下BL21（DE3）菌体中HPVl6E5．TRX融合蛋白占菌体总蛋白的10％左右。真核表达质粒pcDNA3．1（＋）／E5在成功转染NIH3T3细胞后于250μg/mlG418浓度时筛选21d得到了E5基因稳定表达株,RT-PCR产物测序得到了HPV16 E5基因全序列。结论成功构建了pET32／E5原核和pcDNA3．1（＋）／E5真核表达质粒,HPV16 E5蛋白在大肠杆菌和NIH3T3细胞中的稳定表达．这些结果为进一步深入研究HPV16 E5蛋白的生物学特性及其致细胞转化的作用奠定了坚实基础。     

安徽省阜阳市狂犬病街毒株G基因序列分析

目的了解安徽省阜阳市流行的狂犬病毒株与人用、兽用狂犬病疫苗株在G基因核苷酸和氨基酸水平的差异,为有效控制我国狂犬病疫情提供初步科学依据。方法在安徽省阜阳市收集犬脑组织162份,用酶联免疫吸附试验（ELISA）和小鼠颅内接种试验（MIT）检测样品带毒情况,对阳性样品用RT—PCR扩增G基因并测序,以TOPALi和DNAStar软件对G基因序列进行分析。结果从安徽省阜阳市162份犬脑样品中检出阳性样品15份;这15株病毒与我国现在使用的各种疫苗株在G基因的核苷酸和氨基酸水平上均存在不同程度的变异,与我国人用疫苗株CTN同源性较高。结论15株狂犬病毒为基因Ⅰ型狂犬病毒,在核苷酸或氨基酸水平上,与疫苗株CTN之闻的同源性要高于与其它疫苗株之间的同源性。