猪肺炎支原体Mhp366-N蛋白多克隆抗体的制备及应用

【背景】猪肺炎支原体是猪的一种重要的病原。该菌的研究工具较少,特别是缺少开展其致病机制研究需要的抗体。【目的】制备猪肺炎支原体Mhp366-N蛋白抗体并确定其应用范围和使用时的最佳稀释倍数。【方法】Escherichia coli BL21(DE3)-pET28a(+)-mhp366-N重组菌诱导表达Mhp366-N蛋白并纯化。纯化的蛋白免疫小鼠制备多克隆抗体。用免疫印迹和免疫荧光方法检测猪肺炎支原体AH株感染3D4/21细胞后的Mhp366蛋白,确定2种方法中Mhp366-N多克隆抗体的最佳稀释倍数;之后检测临床采集的猪肺泡巨噬细胞中的猪肺炎支原体;最后以免疫组化试验检测猪肺炎支原体感染的肺细胞。【结果】纯化的Mhp366-N蛋白纯度超过85%,免疫小鼠制备的抗血清效价在1:128 000-1:512 000之间。在免疫印迹试验中Mhp366-N多克隆抗体的最佳工作浓度为1:100 000稀释,免疫荧光试验中Mhp366-N多克隆抗体的工作浓度范围在1:1 000-1:10 000 000,其可用于临床采集的猪肺泡巨噬细胞和细胞系中猪肺炎支原体的检测。免疫组化试验结果显示猪肺炎支原体能够进入猪肺泡巨噬细胞、Ⅰ型和Ⅱ型肺泡上皮细胞。【结论】制备的Mhp366-N多克隆抗体为猪肺炎支原体致病机制研究提供了良好的研究工具。     

防风种子发芽特性及促进发芽的试验研究

防风种子发芽率和发芽势均较低,其主要原因包括个体之间的差异及物种特性。种子发芽率较低首先表现在个体发芽率上的差异,试验所用15株防风发芽率从28.0%到92.0%,从整体 上表现发芽率较低。防风本身发芽率低,由于物种因素,种子刚刚采收后的休眠,个体之间解除休眠时间和进入衰老的时间不一致,致使所有的种子不能全部在同一时间进入发芽高峰,同时,防风发芽的启动日有所差异,15株防风启动日和发芽持续时间相差均为两周以上。采用5~50 mg·kg^-1 GA、1~10 mg·kg^-1 6-BA、1%KNO₃和3%H₂O₂可提高休眠防风种子的发芽率,GA、6-BA可除防风种子的休眠,而1% KNO₃和3% H₂O₂对解除休眠的种子无明显影响。采用多种微量元素,即10~100 mg·kg^-1 Mn²⁺、10 mg·kg^-1 Cu²⁺和1 mg·kg^-1 Mo对种子进行处理可显著提高防风种子的发芽率,提示在植株绿果期喷施该类元素也可提高防风种子的发芽率。采用GA、NAA处理幼果,也可以提高种子的发芽率。     

登革2型病毒乳鼠神经毒力相关位点的研究

pDVWS501为含有登革2型病毒全长cDNA的质粒, 可利用感染性转录体技术恢复为有活力的病毒MON501. 将MON501注射乳鼠脑内可引发脑炎症状, 其E蛋白的62, 203位分别为Glu, Asn. 采用OL-PCR方法把pDVWS501 E62位氨基酸突变为Lys, 得到质粒TB62; E203位氨基酸突变为Asp, 得到质粒TB203. 将pDVWS501, TB62和TB203酶切后体外转录得到全长登革2型病毒转录体, 应用电穿孔技术转染BHK-21细胞, 7天后收毒. RT-PCR证实有登革2型病毒存在, 接种C6/36细胞, 3~5 d可使其产生典型病变. 测定突变区域的序列, 结果表明得到了恢复病毒MON501和E62, E203位点突变的重组病毒HFT62, HFT203. 3株病毒均在其基因组5′端加“G”, 3′端则与登革2型病毒野生株相同. 分别将3株病毒稀释至10⁵~ 10² TCID₅₀, 经脑内途径注射1日龄乳鼠, 发现与MON501相比, HFT62, HFT203在同一稀释度发病乳鼠的个数减少, 发病时间延长且差异显著, 表明E62, E203可能是登革2型病毒乳鼠神经毒力相关位点.     

利用正交设计优化小苍兰ISSR-PCR反应体系

通过L₁₆(4⁵)正交试验,研究了镁离子浓度、dNTP浓度、模板DNA浓度、Taq DNA聚合酶浓度、引物浓度这5个因素在4个水平上对ISSR-PCR的影响,建立了适合于小苍兰ISSR-PCR的反应体系。优化体系为：25 μL PCR反应体系中含有1×Taq酶缓冲液(10 mmol·L^-1 KCl,8 mmol·L^-1(NH₄)₂SO₄,10 mmol·L^-1 Tris·HCl,pH 9.0,0.05% NP-40),2 mmol·L^-1 MgCl₂,0.06 U·μL^-1 Taq酶,0.4 μmol·L^-1引物,4.0 ng·μL^-1模板DNA,dATP、dCTP、dGTP、dTTP各0.6 mmol·L^-1。利用温度梯度PCR,确定了最适宜的退火温度为51.5℃。该优化体系的建立为下一步对小苍兰进行ISSR分子标记奠定了基础。     

广州市红树林和滩涂湿地生态系统与大气二氧化碳交换

在生物量调查和土壤温室气体排放量测定基础上,对广州市红树林和滩涂湿地生态系统与大气CO₂交换进行研究,分析湿地植被净生产力吸收CO₂的能力和不同积水状态下(常年积水、间歇积水、无积水)湿地碳汇功能.结果表明:红树林湿地植被净生产力吸收CO₂ 33.74 t·hm^-2·a^-1,土壤排放CO₂(包括CH₄折算成CO₂的温室效应量)12.26 t·hm^-2·a^-1,湿地每年净吸收大气CO₂ 21.48 t·hm^-2,说明红树林湿地是一个强的碳汇;滩涂湿地植被净生产力吸收CO₂ 8.54 t·hm^-2·a^-1,土壤排放CO₂ 5.88 t·hm^-2·a^-1,排放CH₄ 0.19 t·hm^-2·a^-1,若按碳素折算,湿地每年吸收大气中碳素2.33 t·hm^-2,土壤排放碳素1.74 t·hm^-2包括（CH₄中的碳),系统净固定碳0.59 t·hm^-2,说明滩涂湿地是一个弱的碳汇,若将CH₄的温室效应折算成CO₂量,则土壤排放CO₂ 9.78 t·hm^-2·a^-1,排放比吸收多1.24 t·hm^-2·a^-1,对大气温室效应而言,滩涂湿地是一个弱碳源;常年积水下排放的温室气体主要是CH₄,无积水下排放的温室气体主要是CO₂;常年积水湿地碳汇功能最大,无积水湿地碳汇功能最小.     

淡水驯化对桐花树光合生理特性的影响

以实验地全光照条件下淡水和人工海水培养种植的桐花树（Aegiceras corniculatum）幼苗为材料,采用Li 6400光合测定仪对不同月份桐花树幼苗的光合生理生态特性日动态进行测定,研究了桐花树的光合生理生态特性。结果表明：在7、10月份桐花树的净光合速率日变化呈双峰型,均出现“光合午休”现象。在7月份人工海水组和淡水组的最大净光合速率（P_max）分别为9.97和11.95 μmol·m^-2·s^-1;而10月份的P_max分别为12.2和12.9 μmol·m^-2·s^-1。而且淡水驯化下,桐花树的净光合速率较人工海水组高。由光响应曲线可知,桐花树人工海水组的最大净光合速率（P_max）、光饱和点（LSP）、光补偿点（LCP）和表观量子效率（AQY）分别为7 μmol·m^-2·s^-1,1 477 μmol·m^-2·s^-1,30 μmol·m^-2·s^-1,0.031 3;而淡水组为8.69 μmol·m^-2·s^-1,980 μmol·m^-2·s^-1,40 μmol·m^-2·s^-1,0.011。在所测的生理生态因子中,光合有效辐射和气孔导度是影响桐花树光合作用最为强烈的因子,与桐花树的净光合速率和蒸腾速率均有极显著的相关关系（p<0.01）。试验说明淡水驯化的桐花树对光强的利用范围变窄,但有较高的净光合速率,表明桐花树对淡水环境具有较强的适应性。     

元素硫和双氰胺对蔬菜地土壤硝态氮淋失的影响

采用温室盆栽淋洗试验,以NH₄HCO₃为氮肥源,研究了元素硫(S₀)和双氰胺（DCD）对种葱和不种作物土壤NO₃^--N淋失量和NO₃^--N、NH₄⁺-N浓度的影响.结果表明,在12周试验期间,与对照相比,S₀+DCD和S₀处理NO₃^--N淋失量分别低83%～86%和83%;NH₄⁺-N淋失量分别高16.8～21.0 mg·盆^-1和20.4～25.0 mg·盆^-1;而同期无机氮（NO₃^--N、NH₄⁺-N）淋失量则低60%.试验结束后,,S₀+DCD和S₀处理土壤无机氮含量分别比对照高79.9%～85.4%和74.9%～82.6%,以NH₄⁺-N为主.S₀+DCD处理无机氮淋失量比S₀和DCD处理分别低4.6%～14.4%和15.4%～30.1%;试验结束后土壤无机氮分别高6.1%和16.8～36.0%.在Na₂S₂O₃+DCD、Na₂S₂O₃和DCD处理中也发现类似结果.可见S₀施入土壤具有与DCD同样的氨稳定和硝化抑制作用.S₀与DCD配合施用可使DCD的硝化抑制性增强,其作用机理是S₀氧化中间体S₂O₃^2-、S₄O₆^2-,具有抑制硝化和DCD降解作用,延缓DCD硝化抑制效果.S0与DCD配合施用可用于延缓太湖流域蔬菜地土壤NH₄⁺-N向NO₃^--N转化,减少氮向水体迁移风险.     

氯化钠胁迫对不同品种南瓜幼苗阳离子含量的影响

选择19个不同类型南瓜品种,研究了300 mmol·L^-1 NaCl胁迫条件下,幼苗地上部和根系Na⁺、K⁺、Ca²⁺含量、Na⁺/K⁺、Na⁺/Ca²⁺、钠-钾和钠-钙运输选择性系数(S_Na⁺,K⁺和S_Na⁺,Ca²⁺值)的变化.结果表明：NaCl胁迫处理8 d后,不同品种南瓜幼苗Na⁺含量均明显增加,而K⁺含量下降,离子平衡被打破.青栗（Q₁）南瓜幼苗根系Na⁺含量、地上部Na⁺/K⁺、Na⁺/Ca²⁺、S_Na⁺,K⁺和S_Na⁺,Ca²⁺值均明显高于黑蜜南瓜（H₂）和黑籽南瓜（H₃）.不同品种南瓜幼苗体内Na⁺含量、地上部Na⁺/K⁺和Na⁺/Ca²⁺、S_Na⁺,K⁺和S_Na⁺,Ca²⁺值变化趋势与NaCI胁迫下不同品种南瓜幼苗盐害指数的结果基本一致,进一步验证了Q₁耐盐性强与NaCl胁迫下地上部Na⁺/K⁺、Na⁺/Ca²⁺、S_Na⁺,K⁺和S_Na⁺,Ca²⁺值较低以及K⁺、Ca²⁺含量较高有关;而H₂和H₃对盐敏感与NaCl胁迫下地上部Na⁺/K⁺、Na⁺/Ca²⁺、S_Na⁺,K⁺和S_Na⁺,Ca²⁺值较高,以及K⁺、Ca²⁺含量较低有关.     

多重抗性突变株L-氨酸发酵

以北京棒状杆菌(Corynebaetarium pakinan.ra)产L-缬氨酸突变株334(Ile-,α-AB^R)为亲株,经MNNG一次诱变,获得多重抗性突变株VT-405 (Ile-,α-B^R,2-TA^R NL^R NV^R)及其单菌落分离株125,产酸达28mg/ml。VT-405再经紫外线和LiCI复合处理,获A_r菌株。A_r保留其亲株的遗传特性,未再增加新的标记,产酸为30mg/ml1。通过2.6L自控罐发酵试验,证明L-缬氨酸发酵属发酵动力学II型。采用适宜的供氧和pH控制,进行补料分批发酵,平均产酸率高达36.78g/L。     

孝顺竹愈伤组织增殖培养基优化研究

为筛选适宜孝顺竹愈伤组织继代增殖培养基,控制褐变发生,提高再生体系效率,对培养基组成如5种基本培养基、6种有机添加物、7种糖类和5种大量元素等因子进行试验分析。结果表明：培养20 d,基本培养基以MS效果较好,愈伤组织增殖2.8倍,白至淡黄色,致密;有机添加物以1.0 g·L^-1脯氨酸效果较显著,愈伤组织增殖3.64倍,淡黄色,致密均一;碳源以30 g·L^-1麦芽糖效果较好,愈伤组织增殖2.96倍,白至淡黄色,致密。5种大量元素中NH₄NO₃对孝顺竹愈伤组织增殖的影响达到显著水平,以825 mg·L^-1为较佳浓度,培养29 d愈伤组织增殖可达5倍以上,部分出现根分化;适宜孝顺竹愈伤组织培养的大量元素组合为：KNO₃ 475 mg·L^-1+NH₄NO₃ 825 mg·L^-1+MgSO₄·7H₂O 185 mg·L^-1+KH₂PO₄ 340 mg·L^-1+CaCl₂·7H₂O 440 mg·L^-1。     

Mouse monoclonal antibodies to swine blood group antigens

Eight mouse hybridomas with haemagglutination capacity to swine blood group antigens were obtained, three of them producing antibodies capable of being used as blood group reagents. Two detected the Ba factor and another the Fa factor. The others gave non-specific and weak reactions or cross-reaction with antigens present in more than one system. We conclude that mouse monoclonal antibodies are also suitable for use in swine as a complement of polyclonal reagents.     

Characterization of swine short interspersed repetitive sequences

Swine genomic DNA segments containing repetitive sequences were isolated from a porcine genomic library using genomic DNA as a probe. Three fragments containing the repetitive sequences from two of the primary phage clones were subcloned for sequence analysis, which revealed six new PRE-1 repetitive families other than those reported earlier by Singer et al. (Nucleic Acids Research 15, 2780, 1987). The frequency of the repetitive sequences in the swine genome was estimated at 2 x 10(6) per diploid genome. Sequence analysis revealed similarities between these repetitive sequences and that of arginine-tRNA gene.     

The swine histo compatibility system SLA: serological studies in the common Swiss and Danish swine breeds

Alloantisera were produced in common Swiss and Danish swine breeds by immunization with leucocytes or skin-grafts. Out of the 126 antisera, 66 were chosen for further study based on titrations employing lymphocytes from unrelated pigs typed with French SLA antisera (Vaiman, Chardon & Renard, 1979). The 66 selected antisera and the French reagents defining 26 SLA specificities were used to type lymphocytes from 595 unrelated pigs of the common Swiss and Danish breeds. The reaction-patterns of the French, Swiss and Danish antisera were adequately correlated for the French SLA specificities Nos FJ 2, 3, 6, 7, 8, 9, 11, 14, 19, 20 and 24 and for the haplotypes FJ 15.1.18 and FJ 5.4. In addition, a cluster of correlated Danish and Swiss antisera characterized a new specificity, provisionally designated CPH 31. This specificity was frequent in the Danish Landrace pigs.
Using the reagents identified in this report, the segregation of SLA markers was studied. Back-cross families demonstrated segregation of 15 distinct SLA haplotypes of which 14 are common in French Landrace or Large White. Differences were found in haplotype frequencies in both the Swiss and the Danish Landrace and Large White breeds.     

猪支原体肺炎防治研究进展

本文列举了目前临床上用于防治猪支原体肺炎的药物与疫苗的名称和用法用量,并分析了其作用机制及使用效果。抗生素只能抑制支原体生长,无法完全清除,只能解决暂时性问题,且一旦停止用药容易复发,并容易使支原体产生耐药性。国外使用的主要是灭活疫苗,但它无法激发猪体全身免疫系统,需配合优良佐剂使用。国内研制的弱毒疫苗效果很好,但免疫接种方法难度大,不易推广。本文同时综述了目前实验室关于猪支原体肺炎基因工程疫苗的研究进展,并对该病进一步的综合防控措施提出了建议。     

African swine fever virus interaction with microtubules

The role of microtubules in intracellular transport of African swine fever virus (ASFV) and virus-induced inclusions was studied by immunofluorescence using anti-ASFV and anti-tubulin antibodies, by electron microscopy of infected Vero cells and by in vitro binding of virions to purified microtubules. MTC, a reversible colchicine analogue, was used to depolymerize microtubules. In cells treated with MTC multiple large inclusions containing ASFV antigens and particles were observed in the cytoplasm. Removal of the drug lead to migration and fusion of the inclusions at a perinuclear location. To study the effect of microtubule repolymerization on virus particle distribution, the particles were counted in thin sections of MTC treated cells and at different times after removal of the drug. In cells treated with MTC 6.8% and 3.6% of the virus particles were found respectively in the cytoplasm and at the cell membrane while 38% of the particles were located around the virosome. With reversal of the drug effect the number of virus particles around the virosomes progressively decreased to 10% at 2 h while the number of particles in the cytoplasm and at the cell membrane increased. At 2 h after removal of the drug 33.5% of the particles were found budding from the cell membrane. Virus particles were found closely associated with microtubules in cytoskeletons obtained by Triton X-100 extraction of taxol treated cells. The association of virus particles with microtubules was also observed in vitro using purified microtubules and virus particles. The results show that microtubules are involved in the transport of African swine fever virus particles from the assembly site to the cell surface and in the movement and fusion of the virus inclusions.     

A swine SINE (PRE-1 sequence) distribution in swine-related animal species and its phylogenetic analysis in swine genome

The distribution of PRE-1 sequence (a swine SINE) among the animal species related to Sus scrofa, i.e. Phacochoerus aethiopicus and Tayassu tajacu, was examined by dot-blot analysis using PRE-1 sequences as a probe. This revealed that Phacochoerus aethiopicus and Tayassu tajacu contained PRE-1 sequences, amounts of which in their genomes are almost the same as that in the swine genome, indicating that these species separated after PRE-1 sequences proliferated to diversify in the genome. In order to estimate the time when the PRE-1 started to diversify in the swine genome, PRE-1 sequences were extracted from GenBank DNA database by homology analysis using the PRE-1 consensus sequence as a probe. The 22 PRE-1 sequences obtained were aligned and their phylogenetic relation was calculated by the neighbour-joining method. The result of the calculation combined with the mutation rate of the pseudogenes (r = 4.6 times 10^-9 indicated that the PRE-1 sequence diversified at least 43.2 million years ago. Taken together, the period of time since the separation of the three species, Sus scrofa, Phacochoerus aethiopicus and Tayassu tajacu, is currently estimated to be less than 43.2 million years.     

非洲猪瘟防控及疫苗研发：挑战与对策

非洲猪瘟是由非洲猪瘟病毒引起的一种接触传染性、广泛出血性猪烈性传染病,最急性和急性感染死亡率高达100%。自2018年8月我国发生首起非洲猪瘟疫情后,3个多月内,已有18个省份累计暴发69起,给我国养猪业造成了沉重打击。从目前非洲猪瘟全球流行态势及世界各国防控经验来看,我国非洲猪瘟防控和根除面临的形势不容乐观,亟需安全有效的疫苗用于该病的防控。文中结合当前非洲猪瘟病原学最新研究成果,系统总结了非洲猪瘟防控策略、疫苗研究进展及其面临的挑战,重点分析了疫苗研发历程、存在的问题、未来发展方向以及商业化应用所面临的关键科学问题,以期为我国非洲猪瘟防控及病原和疫苗研究提供借鉴。     

非洲猪瘟在俄罗斯的流行与研究现状

非洲猪瘟(African swine fever,ASF)由非洲猪瘟病毒(ASFV)引起,是家猪和野猪的一种高度接触性、致死性传染病,可表现为最急性、急性、亚急性和慢性四种形式。猪感染后以发热、高病毒血症和出血性病变为特征。有的毒株可引起高发病率和高死亡率。自2007年ASF传入格鲁吉亚以来,该病在高加索地区(包括俄罗斯)逐步蔓延,造成多地大量家猪和野猪病死,经济损失惨重。2017年3月,ASF突然在远东地区伊尔库茨克州出现,疫点距中国北方最大陆路口岸满洲里仅约1 000 km,使得传入中国的风险空前提高。为此,本文对该病10年间在俄罗斯的流行状况和研究情况进行总结,以期为我国对该病的防控提供参考。     

非洲猪瘟病毒的生物学特性与疫苗研制的难点

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)引起的一种猪烈性传染病,是全球养猪业的"头号杀手",强毒株引发的超急性和急性感染死率高达100%。2018年8月ASF首次传入我国,截止2019年6月6日,已有32个省份累计暴发137起疫情,给我国社会、经济构成巨大威胁。ASF疫苗的研制始于20世纪60年代,但均以失败而告终,其主要原因是对ASFV生物学特性缺乏深入的研究。有效控制当前ASF疫情扩散、研制安全有效的疫苗将是我国面临的巨大挑战。本文对ASFV形态与基本结构、传播途径、致病机制、基因组及编码蛋白、入侵机制、免疫逃逸等生物学特性进行了概述,并分析了当前疫苗研制面临的难点,以期为我国有效控制ASF疫情及病原研究提供参考。     

Genome‐wide association with delayed puberty in swine

An improvement in the proportion of gilts entering the herd that farrow a litter would increase overall herd performance and profitability. A significant proportion (10–30%) of gilts that enter the herd never farrow a litter; reproductive reasons account for approximately a third of gilt removals, with anestrous and failure to conceive the most common reasons for culling. Tools to select gilts for reproductive longevity through genomics or alternative phenotypes would be of great benefit to the producer. Ninety‐one gilts that failed to display behavioral estrus by 240 days (cases) and 127 pubertal littermates (controls) were genotyped with the Illumina Porcine SNP60 Beadchip. One hundred and seventy‐four SNPs with the most significant associations were genotyped in an additional 86 cases and 103 controls. Twelve of these associations were significant in the final analysis. The most significant (P < 1.5 × 10^?14) region associated with failure to attain puberty was on chromosome 4 surrounding the NHLH2 gene. Delayed pubertal development and age at first estrus have been associated with NHLH2 in mice. Because attainment of puberty is a complex trait, identifying genes that affect pubertal age would greatly contribute to our knowledge of reproductive development as well as overall fertility.