目的 对一株分离自广西兴安的猪源A(2009/H1N1)流感病毒进行遗传演化分析及致病性研究,为流感疫情防控提供科学依据.方法 通过SPF鸡胚进行流感病毒分离,对8个基因进行RT-PCR扩增、测序及进化分析;以6周龄雌性BALB/c小鼠为感染模型进行病毒滴定,分析临床数据评估病毒致病性.结果 分离的甲型H1N1病毒株A/swine/Guangxi/18/2013(H1N1) HA、NS和NP基因属于古典型H1N1,NA和M基因源于类禽型H1N1,PA和PB2基因归属禽源,PB1基因来自人源H3N2.HA裂解位点序列为PSIQSR↓ GLF,具有低致病性特征.以50μl 106 TCID50感染小鼠后其体重发生明显变化,最高体重平均变化率为86.98％,死亡率为12.5％;感染第3d测定肺、鼻甲病毒滴度(Log10 TCID50/ml)分别为5.25±0.28和3.89±0.47.结论 A/swine/Guangxi/18/2013(H1N1)为2009/H1N1流感病毒,能感染小鼠而造成体重下降并产生明显的临床症状.该病毒能在小鼠肺与鼻甲中进行复制,具有低致病性特征.

In order to prepare the magnetic chitosan microspheres with uniform particle size, SPG membrane emulsification and in-situ method were effectively combined. The membrane emulsification pressure, the content of the emulsifier, the disperse phase ratio, the stirring rate, the content of the crosslinking agent, the pH condition of the solution, and the mass ratio of chitosan to FeCl2 ·4H2 O were investigated. The optimal conditions were as follows: the acetic acid of 3%, emulsifier Span-80 content of 4 mL, emulsification pressure of 200 kPa, speed of 400 r/min, glutaraldehyde of 2 mL, solution pH of 7. 5, mass ratio of chitosan to FeCl2 ·4H2 O of 1:1. Under these conditions, the magnetic chitosan microspheres with average particle diameter of 6. 78 μm and saturation magnetization of 25. 69 emu/g were obtained. The uniform magnetic microspheres could be produced by the membrane emulsification combining in-situ method. Due to the easy operations and the mild conditions, this method displays great potential in the preparation of magnetic micro-spheres.

探讨广西地区近期流行猪伪狂犬病病毒(PRV) gE基因的变异规律,以及与国内外毒株的基因差异性,为制定有效的猪伪狂犬病防控措施提供科学依据.对广西不同地区流行的PRV gE基因进行克隆和序列测定,并与国内外PRV毒株的gE基因进行同源性比对分析,构建遗传进化树.广西地区流行的PRV与广东Fa株、湖北HD株的gE基因核苷酸的同源性与编码氨基酸序列同源性最高,分别为99.8％和99.7％;广西地区流行毒株间gE基因同源性达99.5％～99.8％,推导氨基酸序列同源性为98.8％～99.7％,广西地区近期流行PRV毒株的变异不明显.4株PRV毒株与近年国内分离的毒株ZK、NY、QBA、HNXX、MZ1处于同一分支上,遗传关系较近;而与国内经典株Ea、GDSH遗传关系稍远,与国外分离株Yangsan、NiA3、Becker、Rice处于不同的遗传分支,保持较远的亲缘关系.4株PRV gE基因的48位点氨基酸和其中的GXNN1、GXBH1的496位点氨基酸增加1个天冬氨酸的插入,具有变异株的特征.广西地区流行的PRV毒株属于目前我国主要流行的变异株,其氨基酸的插入将对变异株的分子流行病学调查提供重要的参考.

[目的]明确2型猪链球菌的枯草杆菌素样丝氨酸蛋白酶(SspA)基因序列特征及其原核表达重组蛋白的免疫原性,为2型猪链球菌病的诊断及疫苗研究奠定基础.[方法]针对SspA基因上、下游序列分别设计1对引物,从112株2型猪链球菌临床分离株中扩增其相应片段,并进行测序分析;同时以SspA基因的重组质粒pET28a-SspN、pET28a-SspM和pET28a-SspC分别转化感受态大肠杆菌BL21(DE3)后经IPTG诱导表达,再以SDS-PAGE检测融合蛋白表达情况,Western blotting鉴定其免疫原性.[结果]SspA基因上游片段较保守,而下游片段变异区间较大.在IPTG诱导下,含有重组质粒pET28a-SspN、pET28a-SspM和pET28a-SspC的菌株表达出3段融合蛋白,其中,SspN和SspC重组蛋白存在于破碎菌体的上清液中,而SspM重组蛋白以包涵体形式存在于沉淀中,其大小为:SspN重组蛋白66 kD,SspM重组蛋白65 kD,SspC重组蛋白32 kD.Western blotting鉴定结果显示,3段重组蛋白均能与2型猪链球菌感染血清发生反应,且以SspN重组蛋白反应最强烈,而与以全菌灭活苗制备的猪血清未发生特异性反应.[结论]2型猪链球菌SspA基因上游片段较保守,经大肠杆菌重组表达的SspN蛋白免疫原性较强,且具备鉴别2型猪链球菌感染与免疫的潜力,可作为研究猪链球菌病血清学诊断的候选抗原片段.

[目的]了解广西地区流行猪瘟病毒(CSFV)E2基因的变异规律与趋势,及其与疫苗毒株基因的差异性,为制定猪瘟防控措施提供科学依据.[方法]对广西地区流行CSFV毒株的E2基因进行克隆及序列测定,并与兔化弱毒(HCLV)、石门(Shimen)毒株的E2基因进行同源性比对分析,绘制遗传进化树.[结果]广西地区流行CSFV毒株与 HCLV株、Shimen株的核苷酸序列同源性为81.9％～83.3％和81.1％～82.4％,推导氨基酸序列同源性为89.3％～90.6％和87.9％～89.5％;不同广西地区流行毒株间的核苷酸序列及其推导氨基酸序列同源性分别为90.8％～99.5％和94.1％～99.5％,且均属于S2基因群的S2.1亚群,其中广西玉林株GXYL1、GXYL2、GXYL3、GXYL4、GXYL5均属于S2.1b子群,而广西贺州株GXHZ1、GXHZ2与广西北海株GXBH1、GXBH2株属于S2.1c子群.与HCLV株、Shimen株的E2基因编码氨基酸序列相比,9株广西地区流行CSFV毒株共有49处氨基酸发生变异,其中与抗原特性有关的变异有5处,分别为G713E、T724Y、E727H、I732S和S734R位点.[结论]广西地区流行CSFV毒株随时间推移的变异不明显,但其遗传变异总趋势朝着偏离疫苗株的方向发展.

本研究对从广西5种不同动物体内分离到的禽Ⅰ型副粘病毒毒株,进行了HN基因的扩增、克隆和序列分析.5个APMV-Ⅰ分离株HN基因的核苷酸序列同源性为94.7％～98.4％,推导氨基酸序列同源性为96.3％～99％,同源性较高；5个毒株的HN基因与国内广东、山东、福建等地的当前流行株同源性较高,但与经典毒株同源性较低；遗传分析表明HN基因相对保守,都源于同一个基因亚群.

为了解禽Ⅰ型副黏病毒的跨种传播以及对HN基因进行序列分析,运用RT-PCR方法,扩增马源禽Ⅰ型副黏病毒广西分离株M10株的HN基因,将其克隆至PMD 18-T载体中,进行序列测定,序列分析表明,M10株的HN基因片段长度约为2.0kb,ORF为1716个碱基,编码571个氨基酸;ORF区与参考毒株的核苷酸同源性为80.9％-99.2％,推导的氨基酸同源性为88.6％-98.6％.在同源性比较的基础上,进一步绘制禽Ⅰ型副粘病毒HN基因的系统进化树,分析表明,M10株与参考毒株YG03、NDV-03、FP1等亲缘关系较近.

【Objective】This study was aimed to understand the biological characteristics of horse origin avian paramyxovirus serotype I and provide references for preventing and controlling the paramyxovirus interspecies transmission.【Method】 In Baise of Guangxi,causative agents were isolated from suspicious samples of horse using chicken embryos inoculation and cultured.After serological experiment and pathogenicity identification,F gene of isolates was amplified using RT-PCR and sequenced.【Result】The isolated strain could agglutinate chicken erythrocytes and was inhibited by Newcastle disease virus antiserum.Its titer of HA and HI was 27and 210,respectively.The MDT and ICPI of chicken embryos was 72 h and 1.48,respectively.The isolated strains,which was moderate susceptible,belonged to genotype Ⅶ,sharing 95.9% and 95.1% nucleotide sequence homologies with NDV04-21 and TW-96p,respectively.According to the phylogenetic tree of F gene,the isolates,NDV04-21 and TW-96p belonged to the same branch,which were APMV-I genotype Ⅶ.Amino acid sequence of the isolate fusion(F) protein cleavage site 112-117 was 3'-R-R-Q-K-R-F-5',which was in accordance with standard virulent strain HER33 and F48E9.【Conclusion】The results indicated that APMV-I infected horses.It was reconfirmed that hosts of APMW-1 were developing in Guangxi area.