海南山蛭对土壤湿度和气温的适应

在室内以土壤湿度梯度作为海南山蛭Haemadipsa hainana的栖息试验表明,海南山蛭栖息于含水量的15.1％－16.77％水分的土壤表面;野外调查表明,海南岛橡胶林内海南山蛭栖息于16.54％－17.66％含水量的土壤表面;热带雨林者栖息于19.92%-25.73%含水量的土壤,室内实验表明,海南山蛭爬向30－35℃温度范围,占试验蛭数79.15％,在8－9℃不活动,10℃能山蛭活动,随温度升高活动蛭数增加,而15℃试验蛭答部活动,活动蛭数与温度关系为Y－18.6Z-172.3,R-0.976,P=0.01。在橡胶林内8－9℃山蛭不活动;10℃开始有山蛭活动,且随温度升高活动蛭数增加,当17℃时所有山蛭活动,活动蛭数与温度关系Y＝4.73X-3.97,R=0.93,P=0.1。首次使用土壤湿度梯度测定山蛭对土壤湿度的要求,这是一个新的有普遍意义的方法,并讨论了海南山蛭对土壤湿度气温的适应。     

不同农药对海南山蛭毒力测定及其防治研究

在室内,利用速灭杀丁,叶蝉散,杀虫双,乐果,三氯杀螨,草甘膦等6种农药对海南山蛭（Haemadipsa hainane)进行毒力测定,比较各种农药LK50表明,速灭杀丁＞叶蝉散＞三氯杀螨醇＞杀虫双＞乐果,除草剂草苷膦对海南山蛭完全无毒杀作用,草甘膦＋速灭杀丁对海南山蛭仍有很强的毒杀作用,海南山蛭对速灭杀丁的敏感性为,成体＞幼体＞亚成体,在田间小区试验表明,1．5％速灭杀丁防治效果为95．08％,大面积应用表明“草苷膦药液＋0．5％速灭杀丁”防治效果最好,达94．74％,“草甘膦药液＋1％叶蝉散乳油”防治效果次之,达90．5％,草甘膦＋灭蛭农药是防治山蛭好药剂,达到灭蛭除草的目的。     

河南省的山蛭及其吸血习性观察

河南省的山蛭及其吸血习性观察陈广文陈晓虹和振武许人和朱东明（河南师范大学生物系新乡４５３００２）关键词河南山蛭吸血习性１分类地位与地理分布山蛭俗称山蚂蟥,在动物分类上属环节动物门蛭纲（Ｈｉｒｕｄｉｎｅａ）、颚蛭目（Ｇｎａｔｈｏｂｄｅｌｉｄａ）、山蛭科...     

海南山蛭种群数量动态与气象因素关系研究

在海南岛橡胶林内,每月观测海南山蛭Ｈａｅｍａｄｉｐｓａ ｈａｉｎａｎａ种群数量Ｂａ,用逐步回归分析方法研究了１０个气象因子对海南山６种数量的影响,结果表明,１）每年海南山蛭种群数量不同,６ａ间影响海南山蛭种群数量的主要气候因素是Ｘ１（月雨量）、Ｘ３（月雨日）和Ｘ５（月有露日数）;２）海南岛５～１０月份为雨季,海南山蛭这种群数量明显增大,影响山蛭种群数量的主要气候因素是Ｘ５和Ｘ１６（月最大风速和）和     

中国动物生态地理类群特征及其防护医用研究进展

综述中国蛭类研究如下内容：（1）生态学。山蛭生态学,包括山蛭的生态分布,海南山蛭（Hhainana）的种群动态,对温度、土壤湿度和pH值的适应,以及对温、光、湿的综合反应和人类经济活动对其种群数量的影响;山蛭行为生态学,山蛭的运动包括慢缩短、快缩短、身体摆动和转动、洗刷运动、亲吻运动、觅食行为6程序,影响山蛭行为的一些因素以及对环境因素刺激的生态学意义;淡水水蜂生态学,包括浙江水田蛭类生活习性、广州水牛光润金线蛭种群数量动态与水体化学因子关系、广州水田吸血菲牛蛭生活水体化学环境;山蛭和水生吸血菲牛蛭的觅食、生长动态、生命周期和生殖生物学;（2）形态学、分类学和动物地理学。形态学包括山蛭机能组织学、山蛭器官系统解剖;分类学,中国蛭类动物有2亚纲（蛭蚓亚纲、真蛭亚纲）、3目（蛭蚓目、吻蛭目、无吻目）、9科、33属、111种,占世界蛭类物种数约1／6;动物地理学,包括世界山蛭科属动物地理,中国山蛭科动物地理、中国医蛭科动物地理。（3）蛭类的防治和驱避,淡水吸血蛭类防治所用农药种类,不同农药对海南山蛭的毒力（LD50、LD95）及使用,并比较了12种驱避剂对海南山蛭的驱避效果。（4）蛭类的医学利用,蛭素是蛭类唾液腺分泌的一种抗凝物质,蛭素有水蛭素（Hirudin）、山蛭素（Haemadin）和吻蛭素（Hementin）。记述了蛭素的分离、纯化和功能以及有关分子生物学内容。     

中国蛭类动物生态地理类群特征及其防护医用研究进展

综述中国蛭类研究如下内容:(1)生态学.山蛭生态学,包括山蛭的生态分布,海南山蛭(H. hainana)的种群动态,对温度、土壤湿度和pH值的适应,以及对温、光、湿的综合反应和人类经济活动对其种群数量的影响;山蛭行为生态学,山蛭的运动包括慢缩短、快缩短、身体摆动和转动、洗刷运动、亲吻运动、觅食行为6程序,影响山蛭行为的一些因素以及对环境因素刺激的生态学意义;淡水水蛭生态学,包括浙江水田蛭类生活习性、广州水生光润金线蛭种群数量动态与水体化学因子关系、广州水田吸血菲牛蛭生活水体化学环境;山蛭和水生吸血菲牛蛭的觅食、生长动态、生命周期和生殖生物学;(2)形态学、分类学和动物地理学.形态学包括山蛭机能组织学、山蛭器官系统解剖;分类学,中国蛭类动物有2亚纲(蛭蚓亚纲、真蛭亚纲)、3目(蛭蚓目、吻蛭目、无吻目)、9科、33属、111种,占世界蛭类物种数约1/6;动物地理学,包括世界山蛭科属动物地理,中国山蛭科动物地理、中国医蛭科动物地理.(3)蛭类的防治和驱避,淡水吸血蛭类防治所用农药种类,不同农药对海南山蛭的毒力( LD50、LD95)及使用,并比较了12种驱避剂对海南山蛭的驱避效果.(4)蛭类的医学利用,蛭素是蛭类唾液腺分泌的一种抗凝物质,蛭素有水蛭素(Hirudin)、山蛭素(Haemadin)和吻蛭素(Hementin).记述了蛭素的分离、纯化和功能以及有关分子生物学内容.     

薄层等电聚焦电泳技术应用于山蛭的分类研究

山蛭属于环节动物门蛭纲的一种嗜吸人畜血液的动物。广泛分布于东南亚的亚热带丛林及我国华东、华南、西南各省。在我国,迄今已发现15种山蛭。对于这些山蛭的形态、分类及生态等生物学方面的研究曾先后有过报道。其中仅盐源山蛭的生化成分被作过初步分析。至于采用较先进的电泳技术分析山蛭种间的蛋白质特征及差     

海南岛山蛭生态分布的调查研究

为了解决山蛭危害问题,我们对海南岛山蛭分类生态做了调查研究。 一、调查方法 有关山蛭密度的调查方法并无借鉴。考虑到山蛭是在地面杂草上活动,是不均匀的点状分布。水平分布的调查方法是,在山蛭最多的林段内采用慢步行走20分钟,同时用120cm长的小木棍边走边打动周围的杂草、诱山蛭活动,把所看到的山蛭分成体、亚成体、幼体计数记录。在水平分布的基础上,选择四大林区(黎母山、吊罗山、尖峰岭、坝王岭、顺序下同)进行山蛭垂直分布的调查研究。在四个调查点都选择山     

盐边县北部林区山蛭综合治理初步研究

四川省盐边县城之北部和西北部,林密草茂,草场宽阔,是该县的主要林牧区,也是山蛭的适生区。在海拔1450—3650米之间的七百多平方公里之不同部位分布着五种山蛭:盐源山蛭(Haemadipsa yanyuanensis)、广川山蛭(H.guangchuane)、盐边山蛭(H.yanbianensis)、森林山蛭(H.sylvestris)和百灵山蛭(H.bailingnensis)。山蛭的分布密度与其栖息的场所环境条件和人畜活动频度成正相关。山蛭集中分布在海拔1500—2800米之间,因为这一区间具备山蛭最活跃的环境条件,又是人畜活动最频繁的地带。山蛭在山凹地段多于山顶和山坡;阴山、半阴山多于向阳山;同一…     

荧光定量PCR检测重组新蛭素中毕赤酵母基因组DNA的残留量

目的:建立real-time PCR定量检测毕赤酵母基因组DNA残留量的方法,提高重组新蛭素的产品安全性。方法:选择拷贝数高且分布广泛的毕赤酵母5S rRNA基因为靶标基因设计扩增引物,提取酵母基因组DNA,稀释后作为扩增模板。以罗氏荧光定量PCR_LightCycler480平台为基础,建立基于SYBR GreenⅠ荧光染料的real-timePCR的检测方法,并考察用该方法检测重组新蛭素中毕赤酵母基因组残留量的灵敏度、精密度和回收率。结果:该法检测宿主DNA残留量灵敏度高,DNA浓度为0.1~1000 pg/μL范围内呈现良好的线性关系,其标准曲线的误差值小于0.2;用该法对5批注射用重组新蛭素(酵母)产品中宿主基因组DNA残留量进行了测定,结果分别为0.03、2.3、0.2、0.6、0.2 pg/mg。结论:该方法具有操作简便、灵敏度高等优点,可用于重组产品中酵母基因组残留DNA的定量测定。     

Re-usable DNA template for the polymerase chain reaction.

DNA covalently bound to an uncharged nylon membrane was used for consecutive amplifications of several different genes by PCR. Successful PCR amplifications were obtained for membrane-bound genomic and plasmid DNA. Membrane-bound genomic DNA templates were re-used at least 15 times for PCR with specific amplification of the desired gene each time. PCR amplifications of specific sequences of p53, p16, CYP1A1, CYP2D6, GSTM1 and GSTM3 were performed independently on the same strips of uncharged nylon membrane containing genomic DNA. PCR products were subjected to restriction fragment length polymorphism analysis, single-strand conformational polymorphism analysis and/or dideoxy sequencing to confirm PCR-amplified gene sequences. We found that PCR fragments obtained by amplification from bound genomic DNA as template were identical in sequence to those of PCR products obtained from free genomic DNA in solution. PCR was performed using as little as 5 ng genomic or 4 fg plasmid DNA bound to membrane. These results suggest that DNA covalently bound to membrane can be re-used for sample-specific PCR amplifications, providing a potentially unlimited source of DNA for PCR.     

Development of DNA diagnostic methods for the detection of new fish iridoviral diseases

A new disease of epidemic proportions caused by fish viruses within the Iridoviridae family inflicts serious damage on red sea breams (Pagrus major) and striped jack (Caranx delicatissimus) populations grown in aquacultures in Japan. A partial segment of the fish iridoviral DNA was directly amplified using the polymerase chain reaction (PCR) with synthetic primers designed from well conserved nucleotide sequences between the frog virus 3 (Ranavirus) and the silkworm iridescent virus type 6. The deduced amino acid sequence from the nucleotide sequence of the PCR fragment demonstrates a high correlation with a partial sequence from the frog virus 3. Using the PCR method with specific primers, we could detect three of four different known types of fish iridoviruses in diseased fishes. To construct more reliable detection methods specific for this viral family, DNA fragments which can specifically hybridize with all of the four known iridoviridae viral DNAs were screened from the genomic library of one iridoviridae strain. The hybridization assay, using a specific fragment which contains regions which are highly homologous with a characterized partial sequence from the frog virus 3, proved to be a reliable diagnostic tool for fish iridoviral diseases.     

Detection of small sequence differences using competitive PCR: molecular monitoring of genetically improved, mercury-reducing bacteria

A quantitative PCR approach is presented to detect small genomic sequence differences for molecular quantification of recombinant DNA. The only unique genetic feature of the mercury-reducing, genetically improved Pseudomonas putida KT2442::mer73 available to distinguish it from its native mercury-resistant relatives is the DNA sequence crossing the border of the insertion site of the introduced DNA fragment. The quantification assay is a combination of specific PCR amplification and temperature gradient gel electrophoresis (TGGE). Gene quantification is provided by a competitively co-amplified DNA standard constructed by point mutation PCR. After computing the denaturation behavior of the target DNA stretch, a single base difference was introduced to achieve maximum migration difference in TGGE between the original target DNA and the modified standard without altering the PCR amplification efficiency. This competitive PCR strategy is a highly specific and sensitive way to detect small sequence differences and to monitor recombinant DNA in effluxes of biotechnological plants.     

Preliminary development of a diagnostic test for Brucella using polymerase chain reaction

A highly sensitive and specific diagnostic test for Brucella based on polymerase chain reaction is under development in our laboratory. A commercially available PCR kit was used to create primers that allowed the amplification of a 635 bp fragment of a 43 kDa outer membrane protein gene from Brucella abortus strain 19. We successfully amplified the cloned gene present in the pMS64 plasmid and genomic Brucella S19 DNA. The amplified DNA was easily detected by agarose gel electrophoresis. Using both the pMS64 plasmid and Br. abortus S19 purified DNA as template each component of the PCR reaction was adjusted for the optimum amplification of the DNA sequence. Optimum specific amplification resulted when the primer annealing temperature was 60 degrees C. The gene fragment was amplifiable in 25 different Brucella species and strains. To test the specificity of the reaction, DNA extracted from 17 micro-organisms possibly associated with cattle were tested. No amplification was observed. The sensitivity of the reaction was determined with different concentrations of genomic Brucella strain 19 DNA. As little as 0.1 pg DNA (less than 100 brucella cells) could be detected. The specificity and sensitivity of PCR combined with its simplicity and speed suggests the potential of this technique for routine diagnosis of brucellosis.     

Preliminary development of a diagnostic test for Brucella using polymerase chain reaction

A highly sensitive and specific diagnostic test for Brucella based on polymerase chain reaction is under development in our laboratory. A commercially available PCR kit was used to create primers that allowed the amplification of a 635 bp fragment of a 43 kDa outer membrane protein gene from Brucella abortus strain 19. We successfully amplified the cloned gene present in the pMS64 plasmid and genomic Brucella S19 DNA. The amplified DNA was easily detected by agarose gel electrophoresis. Using both the pMS64 plasmid and Br. abortus S19 purified DNA as template each component of the PCR reaction was adjusted for the optimum amplification of the DNA sequence. Optimum specific amplification resulted when the primer annealing temperature was 60C. The gene fragment was amplifiable in 25 different Brucella species and strains. To test the specificity of the reaction, DNA extracted from 17 micro-organisms possibly associated with cattle were tested. No amplification was observed. The sensitivity of the reaction was determined with different concentrations of genomic Brucella strain 19 DNA. As little as 0.1 pg DNA (less than 100 brucella cells) could be detected. The specificity and sensitivity of PCR combined with its simplicity and speed suggests the potential of this technique for routine diagnosis of brucellosis.     

毛囊蠕形螨与皮脂蠕形螨基因组DNA的RAPD分析和序列比对

【目的】分析毛囊蠕形螨Demodex folliculorum（D.f.）和皮脂蠕形螨D. brevis（D.b.）基因组DNA的多态性, 对相关条带进行测序分析。【方法】采用改良小昆虫DNA提取法提取两种人体蠕形螨基因组DNA, 选择RAPD技术对其进行多态性分析, 将相关条带分别与pMD18-T载体连接, 克隆、测序后进行酶切鉴定和分析。【结果】毛囊蠕形螨共扩增15条带, 皮脂蠕形螨共扩增12条带;两种蠕形螨既有共有条带, 又有特异性条带;根据条带差异计算得到两种间的遗传距离为0.5556. 毛囊蠕形螨约800 bp处特异性条带测序结果显示, 序列片段长度为855 bp（GenBank登录号为FI277970）;特异性引物扩增和酶切鉴定均为毛囊蠕形螨所特有. 序列比对显示与阿糖胞苷DNA区域结合蛋白有46%的序列相似度。两种人体蠕形螨约300 bp处共有条带序列分析显示, 碱基序列均为341 bp（GenBank登录号分别为D.f. FI520176;D.b. FI520175）, 在第84和第165位点有2个碱基不同, 分别是A/G和C/T互换, 同源性高达99.4%. 但未发现有开放阅读框和相似度高的序列。 【结论】序列片段为855 bp的特异性条带为毛囊蠕形螨所特有;341 bp碱基序列为毛囊蠕形螨和皮脂蠕形螨所共有, 同源性高达99.4%. RAPD技术可用于两种人体蠕形螨基因组DNA的多态性分析和物种鉴定。     

重组人脑源性神经营养因子在大肠杆菌中的可溶性表达及纯化

按照人脑源性神经营养因子(hBDNF)基因成熟肽编码序列设计合成引物,从人基因组DNA中扩增出360bp的片段,插入到改构载体pTIG-trx上,获得了pTIG-trx—BDNF原核表达重组质粒,限制性酶切分析和DNA序列测定均证实该克隆插入片段为hBDNF基因成熟肽编码序列。将该重组质粒转化大肠杆菌BL21(DE3),经IPTG诱导表达,在大肠杆菌表达系统中获得了高效可溶表达,并对表达产物进行了分离纯化,得到纯度大于83％的样品,Western杂交证实该蛋白具有hBDNF抗原活性。     

A型肉毒神经毒素基因的PCR检测

目的:建立快速筛查A型肉毒毒素的PCR方法。方法:根据GenBank中报道的肉毒毒素基因序列,综合应用多种生物软件分析设计特异的检测引物,从提取的基因组DNA、热裂解产物和菌液等不同形式的模板中扩增大小为457bp的A型肉毒毒素特异基因片段,以肉毒梭菌其他血清型及破伤风梭菌为对照。结果:检测方法无交叉反应,灵敏度可达10pgDNA,3×103个菌。结论:建立的检测方法特异性强、灵敏度高,可以用于A型肉毒毒素基因的快速筛查。     

新疆绵羊种布鲁氏菌HtrA基因的克隆与序列分析

根据已发表的牛流产型布鲁氏菌(B.abrotus)HtrA(High temperature requirment A)基因的核酸序列设计引物,从新疆绵羊种布鲁氏菌(B.ovis)基因组中扩增到了大约1600bp的片段。将该片段纯化后克隆到PBS-T载体上,对所得到的重组质粒进行PCR鉴定、酶切分析后,对克隆的片段进行测序表明,新疆绵羊种布鲁氏菌HtrA基因与发表的牛流产型布鲁氏菌、马耳他布鲁氏菌(B.melitensis)、猪种布鲁氏菌(B.suis)的HtrA基因核苷酸序列的同源性分别为99.68%、99.81%、99.55%,推导的氨基酸序列也存在很高的同源性。     

Polymerase chain reaction amplification and sequence analysis of human mutant adenine phosphoribosyltransferase genes: the nature and frequency of errors caused by Taq DNA polymerase

Using the polymerase chain reaction (PCR) with Taq DNA polymerase, we have amplified a 2.4-kb fragment of genomic DNA containing the adenine phosphoribosyltransferase (APRT) gene from patients with APRT deficiency. Several clones from each patient were sequenced after subcloning the PCR product into M13mp18. Selected regions of the amplified fragment were also sequenced directly. This enabled us to distinguish PCR-induced errors from endogenous mutations and polymorphisms in each clone. 44 PCR errors were found in a total of 57,94 kb of DNA sequenced from 25 clones from 7 patients. All the errors were due to the PCR process and not to subcloning, as shown by sequence analysis of 5 APRT-positive clones isolated from a phage genomic library.