新疆猪粪便戊型肝炎病毒RNA的检测及序列分析

从戊型肝炎病毒(Hepatitis Evirus,HEV)IgG检测阳性的新疆某猪场采集70份猪粪便,利用逆转录套式聚合酶链方法(RT-nPCR),检测HEV RNA,其中13份为阳性,阳性率18．57％。将PCR扩增产物克隆到pMD18-T载体上,构建成重组质粒并测序,结果表明,13株猪源HEV分离株在HEV ORF2 348bp核苷酸序列的同源性为97．1％～100％,为同一基因型;与HEVⅠ、Ⅱ、Ⅲ、Ⅳ的同源性分别为74．1％-77．6％,71．6％-74．1％,73.3％～78．2％和82．8％-91.4％,与ⅣA亚型的同源性同源性最高达89．4％-91．4％。以该核苷酸片段绘制的基因进化树显示13株猪源HEV与HEV Ⅳ T1株在同一分支上,属基因Ⅳ型;与国内其他猪源HEV分离株该片段核苷酸序列的同源性为82．6％-91．3％,提示中国猪源HEV的基因型比较一致,同属HEV Ⅳ型。     

新疆猪粪便戊型肝炎病毒RNA的检测及序列分析

从戊型肝炎病毒(Hepatitis E virus,HEV)IgG检测阳性的新疆某猪场采集70份猪粪便,利用逆转录套式聚合酶链方法(RT-nPCR),检测HEV RNA,其中13份为阳性,阳性率18.57%.将PCR扩增产物克隆到pMD18-T载体上,构建成重组质粒并测序,结果表明,13株猪源HEV分离株在HEV ORF2 348bp核苷酸序列的同源性为97.1%～100%,为同一基因型;与HEV Ⅰ、Ⅱ、Ⅲ、Ⅳ的同源性分别为74.1%～77.6%,71.6%～74.1%,73.3%～78.2%和82.8%～91.4%,与ⅣA亚型的同源性同源性最高达89.4%～91.4%.以该核苷酸片段绘制的基因进化树显示13株猪源HEV与HEVⅣT1株在同一分支上,属基因Ⅳ型;与国内其他猪源HEV分离株该片段核苷酸序列的同源性为82.6%～91.3%,提示中国猪源HEV的基因型比较一致,同属HEVⅣ型.     

Ⅲ型中国株HCVE2/NS1基因与已知分离株的同源性比较

利用逆转录套式ＰＣＲ扩增Ⅲ型中国株ＨＣＶＥ２／ＮＳ１基因片段,将其克隆到ｐｃＤＮＡ３载体上．采用双脱氧链终止法测定插入片段的核苷酸序列．并与已知分离株的相应区域进行同源性比较．首次克隆出Ⅲ型中国株ＨＣＶＥ２／ＮＳ１基因（ＨＣ－Ｗ１４）,其核苷酸序列与Ⅲ型日本株ＨＣＶ（ＨＣ－Ｊ６）该区域同源性为８８．３７％,其推定的氨基酸同源性为８９．２９％．而与已知的非Ⅲ型株ＨＣＶ该区域相比,核苷酸及氨基酸的同源性均相对较低．Ⅲ型中国株ＨＣＶ与Ⅱ型中国株ＨＣＶ在Ｅ２／ＮＳ１区域有较大的变异,揭示研制我国的ＨＣＶ疫苗应该考虑这种基因型之间的变异性．     

猪圆环病毒2型分离毒株全基因组的克隆及序列分析

根据GenBank中猪圆环病毒2型（PCV－2）基因序列,设计两对特异性引物,用PCR方法从接种疑似PMWS仔猪病料的细胞中分段扩增2个分离毒株全基因组。将扩增片段克隆入pGEM-T easy载体,筛选获得含有相应片段的阳性重组质粒。对质粒中的插入片段进行测序拼接,获得2株均为1768bp的全基因组序列。应用DNA star序列分析软件,对所测PCV－2序列与GenBank中的国内外PCV毒株进行同源性比较,并绘制系统发生树,结果发现：所测两毒株之间的核苷酸序列同源性极高,可达99．8％,亲缘关系密切;与PCV－2参考毒株的同源性介于95．3％－99．7％之间,其中与美国的一株PCV－2同源性最高,分别为99．5％和99．7％,亲缘关系很近;与国内两分离株同源性分别为96．4％和98．8％－98．9％;与PCV－1参考毒株同源性仅为77．1％－77．5％。     

弓形虫上海株的棒状体蛋白ROP2的克隆表达及纯化

根据已发表基因序列(GenBank登录号为Z36906)设计引物,以弓形虫(Toxoplasma gondii)上海本地株的基因组DNA为模板,扩增编码ROP2(rhpotry protein2)蛋白的基因片段,定向克隆至表达质粒pET32a(+),重组质粒经限制性酶切鉴定后测序,结果表明插入片段长度为1044bp,与GenBank上登录的序列相比,同源性为96%-100%,其中与弓形虫RH株的rop2基因同源性为100%。重组原核表达质粒pET32a-rop2转化至大肠杆菌BL21(DE3),经诱导可表达分子量约60.9kD的融合蛋白,能被感染弓形虫RH株的绵羊阳性血清识别。     

抗—HCV阴性献血员中丙型肝炎病毒RNA检测及序列分析

对95份抗—HCVIgG阴性献血员采用逆转录聚合酶链反应法（PCR）检测丙型肝炎病毒RNA,结果8次中有6次其检测出17份阳性标本（17／95,17．9％）,复查抗—HCVIgG仍为阴性。对其中8份阳性产物中高变区1的序列分析结果表明均为不同株HCV序列．排除了PCR污染的可能性。对其中2份阳性产物测定了全序列并与HCV各基因型代表株的相应序列比较,与HCVⅡ型相应序列的核苷酸同源性为77％～79％。而与HCVⅠ、Ⅲ、Ⅳ相应序列间的同源性为62％～69％,表明为HCVⅡ型序列。结果提示献血员抗—HCVIgG筛选不能完全排除HCV感染者,漏检不是由于HCV基因序列变异．而是检测方法本身缺陷所致。     

汉坦病毒的基因分型及其序列分析

为了探讨从核苷酸水平耐汉坦病毒进行分型,设计两对型特异性引物,采用反转录和聚合酶链式反应（ＲＴ－ＰＣＲ）,对亚太地区１８株汉坦病毒进行了扩增鉴定,并对其中７株汉坦病毒的ＰＣＲ产物进行了测序分析。ＰＣＲ的分型结果表明,Ⅰ型引物只能扩增血清Ⅰ型病毒的ｃＤＮＡ;Ⅱ型引物也只能扩增血清Ⅱ型病毒,其间无交叉反应。采用巢式ＰＣＲ和限制性内切酶验证了ＰＣＲ产物的特异性。序列分析结果表明,Ｒ３６Ｍ片段Ｇ１区的核苷酸序列与血清Ⅰ型病毒代表株７６－１１８的同源性为７８．４％,而与血清Ⅱ型病毒Ｒ２２的同源性为６８．１％;Ｒ３６与汉坦病毒序列同源性的成对比较结果也表明,Ｒ３６与血清Ⅰ型病毒的同源性均高于血清Ⅱ型病毒;Ｌｅａｋｅｙ虽然能被Ⅱ型引物扩增,但其序列与血清Ⅱ型病毒Ｒ２２的同源性仅为４４．９％,故不属于血清Ⅱ型病毒。上述研究结果表明,反转录聚合酶链反应能对多数汉坦病毒准确分型,但最终结果尚有赖于序列分析。     

禽Ⅰ型副粘病毒f基因克隆及序列分析

用RT—PCR一步法对云南省不同禽类(鸡、鸽子)3株禽Ⅰ型副粘病毒F基因进行扩增和克隆,并对其f基因片段核苷酸序列进行分析,结果表明,云南省禽Ⅰ型副粘病毒各毒株同源性为88．1％--94．9％,与疫苗株LaSota和强毒株F48E9的同源性为85．6％。所分离两株新城疫病毒在F蛋白裂解位点区(112—117aa)的氨基酸序列与强毒株在这一区域的序列完全相同,表明为强毒株。鸽Ⅰ型副粘病毒F蛋白裂解位点区的氨基酸序列与PPMVZQ98—1株在这一区域的序列完全相同,揭示为中强毒株。以1662bp核苷酸绘制系统发育树,表明云南地方新城疫病毒届于基因Ⅶ型,鸽Ⅰ型副粘病毒届于基因Ⅵ型。     

成熟志贺毒素全长与截短A亚单位在E.coli中的表达及纯化

应用PCR技术从Ⅰ型痢疾志贺菌(Shigella dysenteriaetype I)中,扩增出约895 bp的成熟ShT-A基因片段,克隆至pGEM-T载体中,经蓝白斑筛选、PCR和双酶切鉴定正确后,命名为pGEM-TA2。测序结果表明,ShT-A与GenBank中ShT-A序列完全一致。用BamHⅠ和KpnⅠ双酶切克隆质粒,得到为895 bp成熟ShT-A与pQE30原核重组表达载体连接,构建原核重组表达质粒。经IPTG诱导,SDS-PAGE电泳观察,没有目的蛋白表达。DNAsis软件分析ShT-A基因序列,发现513 bp处有HindⅢ位点,故以ShT-A上游引物的BamHⅠ和HindⅢ从pGEM-TA2切出一个513 bp的截短片段,重新插入pQE30表达载体,得到pQE30-A513重组表达质粒,转化E.coliM15,经IPTG诱导,SDS-PAGE电泳观察,在20 ku处出现1条特异性的表达蛋白带,与截短ShT-A513分子量相符,表达量约占菌体总蛋白的37.4%,表达形式为包涵体。为抗体的制备提供了必要的物质基础。     

表达狂犬病病毒糖蛋白的重组犬2型腺病毒的构建

本试验以犬2型腺病毒全基因组重组质粒pPolyⅡCAV 2及其E3 区重组质粒pVAX E3 为基础,通过DraⅢ和SspⅠ双酶切,缺失第25097bp 26141bp共1044bp的E3区片段,按与编码链相同转录方向插入由CMV启动子、狂犬病病毒SRV9 株糖蛋白基因、SV40 polyA基因构成的总长2424bp的表达盒,获得重组基因组质粒pPolyⅡCAV 2 CGS(34.7kb)。以AscⅠ和ClaⅠ双酶切,游离重组基因组(32.7kb),在脂质体LipofectamineTM 2000 介导下,转染MDCK细胞系,获得了E3 缺失区携带狂犬病病毒糖蛋白表达盒的重组犬2 型腺病毒CAV 2 CGS。Western印迹试验表明,CAV 2 CGS表达了狂犬病病毒糖蛋白。初步接种试验显示,重组病毒可以诱导犬产生狂犬病病毒特异性抗体。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.