副猪嗜血杆菌Apd-ELISA抗体检测试剂盒的制备和初步应用

旨在对副猪嗜血杆菌（Haemophilus parasuis,HPS）的黏附素蛋白（autotransporter passenger domain,Apd）ELISA抗体检测方法进行参数优化、临界值确定和应用评价,获得性能稳定的副猪嗜血杆菌病抗体检测试剂盒。首先通过猪的免疫攻毒试验获得HPS阴、阳性对照血清,优化Apd-ELISA的反应参数;然后用背景确认的阴、阳性血清确定其临界值,并对封闭液及酶标板的包装方式进行选择;最后用Apd-ELISA试剂盒对临床样品进行检测并与荷兰Biocheck的OppA-ELISA试剂盒进行比较。结果表明,抗原包被质量浓度为0.5 μg·mL^-1、被检血清以1：200倍稀释后反应45 min以及二抗稀释度为1：15 000时,Apd-ELISA的反应背景下降,区分度提高。根据背景确认的27份阳性和40份阴性血清的OD_{630 nm}值以及临界值计算公式（S-N）/（P-N）,得到Apd-ELISA的阴阳性临界值是0.33。证实用封闭液K封闭和真空包装的酶标板可以在50℃保存4 d（相当于在4℃保存22个月）。用Apd-ELISA试剂盒检测1 179份临床血清,表明母猪、后备和育肥猪以及仔猪的HPS血清阳性率分别为83.76%、61.09%和27.48%。与荷兰的Biocheck试剂盒进行比较,发现Apd-ELISA与Biocheck试剂盒（OppA-ELISA）对HPS临床阴性血清和疫苗免疫血清检测的符合率为100%,对HPS临床阳性血清的符合率仅为4.76%（6/126）。然而,Apd-ELISA与OppA Western blot的阳性符合率可达到73.02%（92/126）。本研究获得了能有效区分HPS阴、阳性血清和稳定保存的Apd-ELISA抗体检测试剂盒,可用于HPS灭活疫苗免疫后的抗体检测和副猪嗜血杆菌病的血清抗体检测。     

伪狂犬病病毒gG基因缺失通用转移载体的构建

对克隆有伪狂犬病病毒Ea株基因组DNA SphI 15kb片段的质粒pBSA用SphI和KpnI消化，将含gG全基因及其上游PK基因，下游gD基因部分编码区的约2．6kb的片段亚克隆到消除了EcoRI位点的载体pUC19中，获得重组质粒pUSK(E)。以pEGFP-N1为模板，通过PCR扩增约0．3kb的SV40Poly(A)片段，并将其克隆到杆状病毒转座载体pFastBac1的NotI和PstI位点，利用pFastBac1的BamHI和PstI将含有9个酶切位点以及SV40Poly(A)的片段亚克隆到pUSK(E)的相应位点，在插入的同时导致gG基因5‘端编码区约9个酶切位点以及SV40Poly(A)的片段亚克隆到pUSK(E)的相应位点，在插入的同时导致gG基因5‘端编码区的400bp的缺失，构建了由gG启动子驱动gG部分编码区缺失的通用转移载体pgG-Uni。序列分析进一步证实：有7个单一酶切位点可供外源基因直接插入。上述结果为构建以伪狂犬病病毒作载体的多价基因工程疫苗以及利用标记蛋白探讨伪狂犬病病毒在体内的增殖与分布奠定了基础。     

The two-component regulatory system CiaRH contributes to the virulence of Streptococcus suis 2

Two-component regulatory systems (TCSs) are widely distributed among bacteria and enable the organisms to make coordinated changes in gene expression in response to a variety of environmental stimuli. In this work, we constructed a mutant strain of the TCS CiaRH and measured its virulence in vitro and in vivo. Compared with the wild type strain, the mutant strain exhibited a significant decrease in adherence to epithelial cells Hep-2 and PIEC. Furthermore, the deletion of CiaRH not only enhanced the bactericidal activity of RAW264.7 macrophage against Streptococcus suis 2, but also increased blood clearance of S. suis 2 in vivo. More importantly, the mutant was attenuated in vivo in CD1 mice and pigs, with reduced mortality, morbidity and impaired bacterial growth observed in specific organs. These results suggest that the CiaRH is required for S. suis 2 virulence.     

乳胶凝集试验检测鸡新城疫病毒血清抗体的研究

用硫酸铵沉淀、透析浓缩的鸡新城疫病毒（ＮＤＶ）抗原致敏乳胶,然后所鸡新城疫阳性血清进行方阵滴定,选择出最佳致敏条件并制成新城疫病毒乳胶抗原,由此建立超乳胶凝集试验（ＬＡＴ）,用来检测新城疫血清抗体;用所建立的乳胶凝集试验（ＬＡＴ）和血凝抑制试验（ＨＩ）同步检测６９份鸡血清,结果乳胶凝集试验阳性６２份,阴性７份;血凝抑制试验阳性６６份,阴性３份,两者的总符合率为９４．２％（６５／６９）,且两者的检出率基本一致。结果表明,乳胶凝集试验具有操作简便、快速、敏感性高、特异性强且可用于现场检测等优点,是一种适合基层单位用来检测鸡新城疫病毒血清抗体的新方法。     

含伪狂犬病毒完整UL49基因及部分UL48基因片段的克隆与序列分析

以地高辛标记的伪狂犬病毒（PRV）Ea株UL49.5基因片段为探针，同SalI、KpnI、BamHI消化的PRV Ea株基因组DNA进行Southern杂交，确定SalI2.8kb、KpnI17.0kb、BamHI25.0kb片段中含UL49基因。将SalI2.8kb片段克隆到pUC119中，利用其中的BamHI进行亚克隆，获得重组质粒pUC2.0。测定约2.0kb片段的全序列并进行序列分析，发现该片段包含UL50基因部分编码区，UL49.5基因和UL49基因的完整编码区，并且在UL49基因下游，还存在一段约369个碱基的序列，经与人单纯疱疹病毒I型（HSV－1）、牛单纯疱疹病毒I型（BHV－1）的UL48基因进行比对，具有较高的同源性，推测为PRV UL48基因的部分编码区。     

阴山北麓旱作区主要作物热能值及结构调整研究

阴山北麓农牧交错地带,气候干旱冷凉,种植结构单一,生产能力低而不稳。农业生产不仅要考虑作物综合生产能力,同时要考虑其水分消耗数量。作物的热能产值反映了其综合生产能力。文中采用能值研究方法,通过田间试验、实验室分析测试对该地区三年主要作物的热能产值、耗水量及其生物产量和经济效益进行了系统研究,结果表明,当地主要作物的热能值大小依次为:向日葵>玉米>马铃薯>草玉米>草谷子>草莜麦>油菜>莜麦>小麦>胡麻>箭筈碗豆>山黧豆。考虑到热能值、生物产量、经济效益和水分生产能力,该地区种植结构调整首先应考虑马铃薯的种植,其次是向日葵,然后是饲草和杂粮。优化作物布局,可以获得作物综合生产能力和环境资源保护的双赢,能够促进资源合理利用与农牧业的可持续发展。     

基于产量及环境友好的玉米氮肥投入阈值确定

为了寻求河套灌区玉米高产与环境友好双赢的氮肥投入阈值,该文采用田间试验和室内分析化验相结合的方法,在内蒙古五原县连续三年定位研究了不同施氮水平对河套灌区玉米产量、土壤N素残留量及氮平衡的影响。结果表明:随着施氮量的增加,籽实产量呈先增加后下降的趋势,2 m土壤矿质氮质量分数呈指数增加趋势。随着施氮量及施氮年限的增加,土壤剖面N素含量呈增加趋势。随着土壤深度的增加,在0~80 cm土层间土壤N素含量呈下降趋势。盈余率为0时,施氮量为237 kg/hm2,籽实产量为13.7 t/hm2,2 m土壤矿质氮为478 kg/hm2,土壤氮素回收率为24%,植株氮素回收率为41%,土壤-玉米系统总回收率为65%;95%最高产量到最高产量为13.2~13.9 t/hm2,对应施氮量为193~291 kg/hm2,2 m土壤矿质氮为419~563 kg/hm2,氮素盈余率为-19%~23%,土壤氮素回收率为21%~26%,植株氮素回收率为41%,土壤-玉米系统总回收率为62%~67%。施氮量193~291 kg/hm2是既保证玉米产量又满足土壤氮素盈余较少、土壤-玉米系统氮素回收较高的合理施氮阈值。该研究为河套灌区玉米合理施用氮肥提供了科学依据。     

Development of a specific latex agglutination test based on a recombinant hemagglutinin protein to detect antibodies to H5 avian influenza viruses

A latex agglutination test (LAT) was developed for rapid detection of antibodies to H5 avian influenza viruses (AIVs). The hemagglutinin protein of H5 AIV was covalently linked to carboxylated latex by ethyl-dimethyl-amino-propyl carbodiimide to prepare the sensitized latex beads. The LAT was evaluated with the hemagglutination inhibition (HI) assay as the reference test. The H5-LAT showed a sensitivity of 87.0% and specificity of 88.9% in detecting 126 serum samples from experimentally infected chickens and a sensitivity of 82.5% and specificity of 86% in detecting 587 field chicken serum samples from mostly vaccinated chickens. The agreement ratio between H5-LAT and HI was found to be 87.3% and 83.1% for the two groups of samples, respectively. Difficulty with background agglutination in stored chicken sera was overcome by serum pretreatment with either dried chicken liver powder or dilution buffer containing detergent Tween-20. The H5-LAT has advantages over a previously reported whole-virus LAT in terms of biosafety in preparation, chemical stability, and higher specificity. It is a rapid and simple test suitable for field monitoring of antibodies to H5-type AIV.     

Evaluation of recombinant proteins of Haemophilus parasuis strain SH0165 as vaccine candidates in a mouse model

The recently completed genome sequence of Haemophilus parasuis strain SH0165 allowed us to screen putative OMPs for the development of recombinant vaccines. The objective of this study was to evaluate the immunogenicity and protective efficacy of three OMPs of H. parasuis. Three putative OMPs (SmpA, YgiW and FOG) were cloned, expressed and purified by Ni affinity chromatography using nitriloacetic acid resin. Mice were immunized either individually (individual protein, IP) or synergistically (synergistic protein, SP) with the recombinant proteins. A significant increase in IgG titer was detected in all protein-immunized mice. Isotyping studies revealed that the antibodies produced were predominantly IgG2a-type, indicating a predominant Th1 response. A significant increase was observed in IL-2, IL-4 and IFN-γ levels in the culture supernatants of splenocytes isolated from immunized mice. Furthermore, mice were challenged intraperitoneally with 6×10(9)CFU (5×LD(50)) of highly virulent homologous serovar 5 strain (SH0165) or 7.0×10(9) CFU (5×LD(50)) of the heterologous serovar 4 strain (MD0322) at fourteen days after the last immunization. All of the recombinant proteins enhanced survival and reduced histopathological lesions. Our results indicated that the three OMPs showed protection both individually and synergistically against infection with the highly virulent H. parasuis in mice.     

Cloning, expression and characterization of a cell wall surface protein, 6-phosphogluconate dehydrogenase, of Haemophilus parasuis

Haemophilus parasuis (H. parasuis) is a swine pathogen responsible for the Glässer’s disease. In order to understand the pathogenesis of the H. parasuis infection, the gnd gene encoding a cell surface protein, 6-phosphogluconate-dehydrogenase (6PGD) of H. parasuis was inducibly expressed in Escherichia coli BL21 with a hexahistidyl N-terminus to permit its purification. Western blotting using the r6PGD-specific antiserum showed that the 6PGD protein is on the cell surface of H. parasuis. The characterization of 6PGD in H. parasuis pathogenesis involved as an adhesion and its immunogenicity in mice was further investigated. The adherence assay with H. parasuis and swine alveolar epithelial cells (SJPLC) pre-incubated with (His)₆6PGD and non-incubated SJPLC showed a noticeable reduction in the adhesion of H. parasuis in the (His)₆6PGD pre-incubated SJPLC compared to the non-incubated SJPLC. Further, the r6PGD protein induces the production of IL-8 and IL-6 by SJPLC. Furthermore, immunization with the r6PGD protein can provide the protective efficacy by 75% following intraperitoneal administration of a 5 × LD₅₀ dose of H. parasuis SH0165, and elicited a good protective immune response, which demonstrated the importance of 6PGD to bacterial pathogenesis. Identification and characterization of the role of H. parasuis 6PGD in adhesion and immunogenicity will allow us to use this protein to develop new antimicrobial therapies and/or vaccines.