猪繁殖与呼吸综合征病毒GZ-KY1株的分离及ORF5基因的克隆分析

本试验首次采用PK-15细胞,通过同步接种的方法,成功分离到了1株猪繁殖与呼吸综合征病毒(PRRSV,GZ-KY1株),并扩增克隆了该毒株的ORF5基因。试验结果表明,该毒株ORF5基因全长603 bp,系美洲型,与国内强毒株的氨基酸一致性高于弱毒株,与经典美洲型毒株及国内弱毒株亲缘关系较远。     

H9N2亚型禽流感病毒TJ1株的分离与鉴定

从发病蛋鸡群分离到H9N2亚型禽流感病毒TJ1株,对其进行了系统的生物学特性鉴定.结果表明,分离毒TJ1株具有血凝性,且凝集不被新城疫和产蛋下降综合征抗血清抑制,而被H9亚型禽流感病毒标准阳性血清抑制,禽流感琼扩试验结果为阳性,在电镜下呈典型的流感病毒粒子形态,证明该毒株为A型流感病毒;通过致病力试验,分离毒TJ1株对6周龄SPF鸡无致病性,为低致病性毒株;用其制备成油乳剂灭活疫苗免疫雏鸡,能很快产生对H9特异的血凝抑制抗体,于免疫后3周攻毒,可以保护雏鸡抵御同病毒攻击的感染,表明分离毒TJ1株具有良好的免疫原性.     

PRV LA株gD基因的序列测定及其重复高变区的发现

对猪伪狂犬病病毒鲁A株(PRVLA株)gD基因进行了克隆和序列测定，结果表明：在测序的l453bp的DNA序列中包括着1个l203bp的ORF(即gD基因)，它编码400个氨基酸组成的多肽；在整个gD基因的ORF内PRVLA株与PRV Ea株、Hulbei株、Rice株、NIA-3株、Kaplan株的gD基因比较，核苷酸的同源性分别为98．3％、98．3％、98．0％、98．1％、98．6％，氨基酸的同源性分别为97．8％、97．8％、97．5％、98．1％、98．6％；发现PRVLA株gD基因与Ea株、Hubei株、Rice株、NIA-3株、Kaplan株的gD基因均在802～837nt处有1个C(A)GGCCC的重复高变区，其对应的是gD267～279位氨基酸残基Arg—Pro的重复高变区。正是该重复高变区的碱基缺失或插入使得PRVgD的ORF在l194～l215nt间变化，gD前体的氨基酸残基为398～404个。     

猪圆环病毒2b亚型的分离鉴定及基因组序列分析

为分离鉴定流行于重庆某猪场的猪圆环病毒2型,本研究从断奶仔猪多系统衰竭综合征猪的淋巴结病料中进行病毒学检测,病毒利用PK-15细胞增殖,其基因组序列经克隆、测序、拼接获得,并完成对全序列的生物信息学分析鉴定。结果获得了1株猪圆环病毒2型(命名PCV-2CQ1),该毒株的全序列大小为1 767bp,同源性分析发现该病毒与国内公布的PCV-2毒株同源性超过了90%,尤其与广西分离株同源性达100%;系统进化分析本分离病毒与浙江株(EU257511)、黑龙江株(HM038032)聚类到一起,形成一个进化分支,同属于PCV-2b型;对编码的衣壳蛋白分析发现,PCV-2CQ1Cap氨基酸发生了较大变异,与强毒株同源性较高,考虑到本病毒源自PMWS病猪,推测该毒株属于强毒株。     

Studies on the interactions of two different serotypes of Actinobacillus pleuropneumoniae

In vitro and in vivo interactions of various field strains of Actinobacillus pleuropneumoniae of serotypes 1, 2, 5 and 7 were studied. There was no influence of one serotype over the other when strains belonging to two serotypes were cultivated together in vitro. In vivo interactions showed predominance of serotype 1 over other serotypes when a strain of serotype 1 was inoculated together with a strain of serotype 2 or 5 in mice. Serotype 1 strain remained predominant irrespective of whether it was inoculated before or after the inoculation of serotype 2 strain. The mortality caused by the inoculation of two strains was higher than the mortality caused by a single strain. Early mortality was observed on inoculation of strains of serotype 2, 5 or 7 along with a strain of serotype 1. Both serotypes could be detected in the blood on cultural examination of mice infected with mixed serotypes.     

一株具有产酸能力的芽孢杆菌的筛选及性能检测

 为了分离具有产酸能力的芽孢杆菌,使用菌种分离培养基从牛粪中分离得到一株具有产酸能力的芽孢菌,命名为GF1。根据该菌株的生理生化特性鉴定和16S rDNA序列测定分析, 初步鉴定该菌为蜡样芽孢杆菌。对GF1发酵液中的代谢产物进行液相色谱分析,发现GF1可代谢产生乳酸、乙酸等有机酸。对GF1进行液体发酵,通过在沸水中加热10 min灭活菌体,检测GF1最终芽孢率可达到90%以上,对GF1进行耐热、耐酸和耐胆盐处理,发现GF1具有良好的耐热性能和较强的耐酸耐胆盐特性,以小鼠做安全性试验,结果成活率为100 %,表明GF1安全无毒。GF1不仅能够产酸,还可以产生芽孢,可以突破乳酸菌在颗粒料中添加的瓶颈。     

羊种布鲁氏菌WadC基因缺失株的构建与鉴定

试验旨在分析糖基转移酶编码基因WadC影响布鲁氏菌胞内存活的作用。以羊种布鲁氏菌Rev.1基因组为模板,通过同源重组方法获得WadC基因上、下游同源臂融合片段,并与载体pUC19-SacB连接,构建pUC19-SacB-ΔwadC重组载体,电转至羊种布鲁氏菌Rev.1,构建ΔwadC缺失株（Rev.1ΔwadC）,检测菌株Rev.1ΔwadC的遗传稳定性,比较分析亲本株Rev.1和缺失株Rev.1ΔwadC的生长特性及其在BMDC和RAW264.7细胞中的生存能力。结果显示,试验成功构建基因缺失株,连续传代30次未发现基因回复突变;在体外相同培养条件下,缺失株Rev.1ΔwadC与亲本株Rev.1生长趋势相似,均在20 h到达对数生长期,44 h进入平台期;侵染BMDC细胞48和72 h时,其胞内存活率显著低于亲本株（P<0.05）;而侵染小鼠RAW264.7巨噬细胞试验显示,亲本菌株和基因缺失株无显著性差异（P>0.05）。综上所述,本试验成功构建并获得了具有良好遗传稳定性的布鲁氏菌WadC基因缺失株,该缺失株在体外培养条件下与亲本株生长趋势相似;但该缺失株在BMDC细胞内的存活能力显著变弱,为深入研究布鲁氏菌WadC基因功能奠定基础。     

Evidence for the porcine origin of equine rotavirus strain H-1

Equine group A rotavirus (RVA) strain H-1 (RVA/Horse-tc/GBR/H-1/1975/G5P9[7]) was found to have VP4, VP6-7, NSP1 and NSP4 genes of porcine origin. In order to obtain conclusive information on the exact origin and evolution of this unusual equine strain, the remaining six genes (VP1-3, NSP2-3 and NSP5 genes) of strain H-1 were analyzed in the present study. By whole genomic analysis, strain H-1 exhibited a porcine RVA-like genotype constellation (G5-P[7]-I5-R1-C1-M1-A8-N1-T1-E1-H1), different from those of typical equine RVA strains. The VP2-3 and NSP2-3 genes of strain H-1 were found to originate from porcine RVAs. On the other hand, it was difficult to pinpoint the exact origin of the VP1 and NSP5 genes of strain H-1, though phylogenetically, these genes appeared to be possibly derived from porcine or Wa-like human strains. Taken together, at least nine (VP2-4, VP6-7 and NSP1-4 genes) of the 11 gene segments of strain H-1 were found to be of porcine origin, revealing a porcine RVA-like genetic backbone. Therefore, strain H-1 is likely a porcine RVA strain that was transmitted to horses.     

Construction and Identification of Brucella WadC Gene Deletion Strain

The purpose of the test was to analyze the role of the glycosyltransferase-encoding gene WadC in affecting the intracellular survival of Brucella.Using the Brucella sheep Rev.1 genome as template,the fusion fragments of the homologous arms of the upper and lower arms of WadC gene were obtained by homologous recombination,and ligated to the vector pUC19-SacB to construct the pUC19-SacB-ΔwadC recombinant vector,which was transferred to sheep species Brucella Rev.1,constructing a ΔwadC deletion strain (Rev.1ΔwadC),testing the genetic stability of the strain Rev.1ΔwadC,comparing and analyzing the growth characteristics of the parental strain Rev.1 and the deletion strain Rev.1ΔwadC and the BMDC and RAW264.7 viability of cells.The results showed that the gene-deficient strain was successfully constructed in the experiment,and no genetic back mutation was found in 30 consecutive passages.Under the same culture conditions in vitro,the growth trend of the deleted strain Rev.1ΔwadC was similar to that of the parental strain Rev.1,and both reached logarithmic growth period at 20 h and reached plateau period at 44 h.When the BMDC cells were infected at 48 and 72 h,the intracellular survival rate was significantly lower than that of the parent strain (P<0.05).The RAW264.7 macrophage test of infected mice showed that the parent strain had no significant difference with the gene deletion strain (P>0.05).To sum up,this experiment successfully constructed and obtained a strain of Brucella WadC gene with good genetic stability.The deletion strain had similar growth trend with the parent strain under in vitro culture conditions;However,the survival ability of the deletion strain in BMDC cells was significantly weakened.This study laid a foundation for further study on the function of WadC gene of Brucella.     

一起鸭甲肝病毒1型和3型混合感染的研究报道

自山东省某疑似鸭肝炎发病鸭肝脏中分离到两株病毒,命名为TA1和TA2,分离毒分别回归3日龄雏鸭,可复制出鸭肝炎的典型症状和病理变化。利用鸭甲肝病毒1型（DHAV-1）和3型（DHAV-3）特异性引物进行RT-PCR 扩增,结果为阳性,经测序证实为DHAV-1和DHAV-3。分别扩增分离毒的VP1基因,经序列测定及遗传进化关系分析发现,分离毒TA1和DHAV-3毒株之间有较高的核苷酸序列相似性,与DHAV-3遗传距离最近,属于基因C型;分离毒TA2和DHAV-1毒株之间有较高的核苷酸序列相似性,与DHAV-1的遗传距离最近,属基因A型。     

An Argentine equine herpesvirus strain with special restriction patterns protect mice challenged with a pathogenic strain

Equine herpesvirus 1 (EHV-1) was first isolated in Argentina in 1979. This strain SPv has special restriction patterns, but a previous study demonstrated that SPv did not modify its growth in cell culture. In addition, it showed low virulence in the mouse respiratory model consistently with results found in female BALB/C at different state of gestation. This study evaluates in a mouse respiratory model, if primary infection with SPv strain protects animals from subsequent challenge with a pathogenic strain. Body weight loss was not observed in mice intranasally inoculated with SPv strain and challenged with HH1 Japanese strain. The SPv primary infection does not completely prevent clinical presentation by HH1 infection but the SPv inoculated animals recovered more quickly, with less intense and less persistent histological lesions. The challenge infection caused a rapid and prolonged increase in anti-EHV-1 antibodies in the mice previously infected with SPv, along with a more rapid reduction of viral titres in lungs. In this work it was demonstrated that this EHV-1 strain constitute a good immunogen. These results show that this SPv strain could be considered to produce an EHV-1 vaccine.     

产几丁质酶菌株的筛选、鉴定及其对玉米秸秆中优势霉菌的抑制作用

本试验旨在研究利用产几丁质酶菌株抑制玉米秸秆中优势霉菌的生长,为提高秸秆利用率提供理论依据。利用胶体几丁质培养基从玉米秸秆样品中筛选出一株产几丁质酶菌株BS-1,同时将优势霉菌进行分离、纯化。通过对BS-1菌株和优势霉菌形态学观察以及16 S r DNA或18 S r DNA序列测定进行菌种鉴定。利用3,5-二硝基水杨酸比色法测定BS-1菌株发酵24、48、72、96、120、144、168 h的几丁质酶活性,以及牛津杯法检测BS-1菌株发酵液对玉米秸秆中优势霉菌的抑制作用。结果显示,玉米秸秆样品中分离出的BS-1菌株经鉴定为枯草芽孢杆菌(Bacillus subtilis),4株优势霉菌分别为卷枝毛霉(Mucor circinelloides)、尖孢镰刀菌(Fusarium oxysporum)、米曲霉(Aspergillus oryzae)和黑曲霉(Aspergillus niger)。BS-1菌株在37℃下培养120 h产几丁质酶活性达到最高值3.23 U/m L。BS-1菌株发酵液对玉米秸秆中4株优势霉菌均有明显抑制作用,抑菌圈直径分别为18.13、18.48、17.55、15.68 mm。由此可见,筛选到的产几丁质酶枯草芽孢杆菌BS-1能够有效抑制玉米秸秆中的卷枝毛霉、尖孢镰刀菌、米曲霉、黑曲霉4株优势霉菌生长。     

鸽源Ⅰ型禽副黏病毒HN基因序列测定和分析

以分离自云南省的一株鸽源禽I型副黏病毒YN-P1株的基因组RNA为模板,通过RT-PCR的方法扩增出HN基因片段,然后将其克隆至T载体中。经PCR鉴定后,对阳性克隆进行核苷酸序列测定。测序后拼接得出HN基因的全序列。测序结果显示,YNP1毒株HN基因为1 716 nt。通过与参考毒株比较HN基因序列,发现所研究毒株YN-P1与TW95核苷酸序列同源性最高为88.3％;与ZJ1／Go氨基酸序列同源性最高为91.6％。     

Characterization of a lentogenic Newcastle disease virus isolated from broiler chickens in Japan

Newcastle disease virus (NDV), named MET95, was isolated from a non-vaccinated broiler flock in Japan in 1995. The MET95 strain was determined to be a lentogenic NDV. The strain has the properties of eluting rapidly at 4 C and has low thermostability in hemagglutinating activity with chicken erythrocytes. In these studies, no difference could be found between the MET95 strain and the Hitcher B1 vaccine strain. However, the chickens inoculated with the MET95 strain, as well as chickens that they were in contact with, had a much higher hemagglutination-inhibition antibody response than those inoculated with the B1 strain. Accordingly, the MET95 strain is thought to be a promising candidate as a live ND vaccine strain. In Japan, this is the first report on the isolation of lentogenic NDV from chickens since the paper on the Ishii strain isolated in 1966.     

First genetic characterization of equine adenovirus type 1 (EAdV-1) in Turkey

Equine adenovirus type 1 (EAdV-1) is a cause of repiratory tract infection in equids. In present study for the first time in Turkey, the prevalence of EAdV-1 in nasal swab samples obtained from horses showing respiratory symptoms was investigated by polymerase chain reaction (PCR), and molecular characterization of the hexon gene detected in the Turkish (TR) strain was performed. Overall, the prevalence of EAdV-1 was found low (1.4%) as indicated by a positive PCR reaction from the nasal swab extracts tested. Phylogenetic analysis based on the partial sequences of the hexon gene of a TR-EAdV-1 strain with those of previously isolated AdVs from different mammals and an EAdV-1 M1 strain showed that the EAdV-1 strains were placed into a unique cluster. Although the TR-EAdV-1 strain was closely related to CAV-1, CAV-2 and bat adenovirus reference strains, larger-scale studies are necessary to better understand the molecular epidemiology and population structure of EAdV-1 in Turkey.     

一株类NADC30猪繁殖与呼吸综合征病毒的分离及遗传变异分析

为了解广西地区的猪繁殖与呼吸综合征病毒(PRRSV)的遗传变异及流行情况,本实验室从广西某发病猪场采集到的猪肺脏组织中检测到1株PRRSV,命名为GXNN1839,并对该毒株进行病毒的分离鉴定、GP5和Nsp2的测序分析.结果 显示:该毒株可在PAM细胞上分离增殖,有明显的细胞病变,通过IFA试验可以检测到细胞内PRR...     

表达无毒性大肠杆菌ST1-LTB融合蛋白基因工程菌株的构建

利用基因突变技术，将形成ST1分子内二硫键的半胱氨酸碱基进行突变，使ST1失去本身毒性，进而将其与含有LTB基因的pET-28b( )连接，转化至受体菌BL21(DE3)，重组菌株BL21(DE3)(pXST3LTB)经IPTG诱导后，其表达产物免疫的小鼠能够抵抗大肠杆菌强毒菌的攻击并且消除了ST1的毒性，表明构建的工程菌株BL21(DE3)(pXST3LTB)可作为预防幼畜大肠杆菌性腹泻基因工程菌苗的候选菌株。     

鸿雁源鸭甲肝炎病毒Ⅰ型的分离与鉴定

为了对一起死亡率高达91．3％、急性死亡的鸿雁病例进行病原学分析,通过细菌分离排除法和病毒分离方法获得致鸭胚死亡病毒（暂命名为FJ-017株）。该病毒无血凝活性,用鹅细小病毒、番鸭细小病毒、鸭呼肠孤病毒、鸭甲肝炎病毒1型、鸭瘟病毒、鸭坦布苏病毒特异引物分别进行扩增,电泳结果显示,仅鸭甲肝炎病毒1型引物可扩增出条带。将扩增产物回收后克隆,序列分析表明FJ-017株与22株DHAV-1参考株的同源率为93．5％-99％,而与DHAV-2和DHAV-3参考株的同源性均为79．9％。遗传进化分析表明FJ-017株与DHAV-1关系密切,在进化树中共处一分支,表明FJ-017株为鸭甲肝炎病毒1型,此为国内外首次报道。     

1株Ⅶ型新城疫病毒融合蛋白基因的克隆与生物信息学分析

为了研究Ⅶ型新城疫病毒（Newcastle disease virus,NDV）CY1株的进化情况,分析其遗传变异和基因功能,试验应用RT-PCR扩增NDV辽宁分离株CY1株的融合蛋白(fusion,F)基因,进行测序分析,构建系统进化树,并用生物信息软件对蛋白结构功能进行分析。结果显示,NDV F基因全长1662 bp,编码553个氨基酸。生物信息学分析结果表明,F蛋白的分子质量为58991.0 u,理论等电点为8.48,半衰期为30 h（哺乳动物类网状细胞）,不稳定系数31.62,脂肪指数为108.12,总平均疏水性0.174,最大疏水性为3.078,最小疏水性为-2.578,信号肽序列为第1-31位氨基酸残基,跨膜区结构分析结果表明该蛋白为跨膜蛋白,预测可能含有6个N-糖基化作用位点、9个酪蛋白激酶Ⅱ磷酸化位点、7个蛋白激酶C磷酸化位点、14个N-豆蔻酰化位点、1个酰胺化位点、1个亮氨酸拉链、1个微体C端靶信号位点、1个双向核定位信号。系统进化分析结果表明,NDV CY1株与zj1株进化距离最近。     

自然发酵泡菜汁中植物乳杆菌的分离鉴定与体外益生特性研究

利用MRS固体培养基从自然发酵的泡菜汁中分离到1株优势乳酸菌,将其命名为菌株R1,经16S rRNA基因序列分析,并结合生理生化特性和糖发酵试验将其鉴定为植物乳杆菌(Lactobacillus plantarum)。菌株R1具有很强的产酸能力,24 h内可使MRS液体培养基p H从6.14降为3.59。菌株R1的发酵上清液对痢疾志贺氏菌、金黄色葡萄球菌、铜绿假单胞菌、鸡肠炎沙门氏菌、大肠埃希氏菌均有很好的抑制作用。体外益生试验表明,菌株R1能耐受0.3%的胆盐、p H 3.0的酸度以及60℃、30 min的热处理,对人工胃液和人工肠液也有很好的耐受性。菌株R1对头孢类和青霉素类抗生素较敏感,对诺氟沙星、卡那霉素、链霉素等抗生素不敏感。