Development and Application of a Novel Neutralization Test for Echovirus 25

目的:建立一种新型的快速、高通量的埃可病毒25型(ECHO25)中和抗体检测方法,并初步评价其在ECHO25中和抗体筛选和血清流行病学调查中的应用价值。方法:应用免疫荧光方法筛选ECHO25高亲和性抗体并将其作为检测单抗,结合酶联免疫斑点检测技术(ELISPOT)建立ECHO25中和抗体检测方法;使用不同效价的血清评价该方法的准确性;采用所建立的中和方法对ECHO25单克隆抗体、临床血清样品进行检测。结果:建立了快速检测ECHO25中和抗体的Nt-ELISPOT方法,以ECHO25单克隆抗体5B9作为检测抗体;相比经典的中和实验方法 Nt-CPE,该方法可显著缩短检测时间(从5~7 d缩短至1 d以内),检测结果具有较好的一致性;采用所建立的Nt-ELISPOT方法首次筛选获得3株对ECHO25具有较好中和能力的单克隆抗体;临床血清样品检测结果显示厦门地区可能存在ECHO25的流行。结论:该方法可以应用于中和抗体筛选和血清学的临床辅助诊断,为ECHO25的防治研究提供支持。Objective: To establish a rapid and high-throughput neutralization test for echovirus 25(ECHO25),and evaluate its application in neutralizing antibody screening and seroepidemiological surveys. Methods: Immuno-fluorescence assay was applied to screen a high affinity antibody, which was used as the detection antibody forECHO25, and a rapid neutralization test was established based on enzyme- linked immunospot assay(Nt-ELISPOT). The accuracy of this method was evaluated by detecting serum samples with different titer. Monoclonalantibodies against ECHO25 and clinical serum samples were detected via the established neutralization test. Results: A rapid method to detect neutralizing antibody against ECHO25 was established and an anti-ECHO25 anti-body, 5B9, was used as the detection antibody. The detection period could be shortened significantly comparedwith the classical neutralization test(Nt- CPE)(from five to seven days to less than one day), and the Nt-ELISPOT had good consistency with the Nt- CPE. Meanwhile, three neutralizing antibodies for ECHO25 werescreened firstly by this method. The detection results of clinical serum samples showed that infection of ECHO25 might be popular in Xiamen. Conclusion: This method can be used in neutralizing antibody screening and seroepi-demiological surveys, and it may provide support for the control of ECHO25.国家自然科学基金(81371817,81401669

Preparation and Application of Soluble Human Squamous Cell Carcinoma Antigen Expressed by Escherichia coli

旨在建立基于大肠杆菌表达系统的高效可溶性表达人鳞状上皮细胞癌抗原(SCCAg)方法,获得具有较好活性的重组SCCAg抗原并应用于建立抗原检测方法; 。基于pGEX-6P-l载体和大肠杆菌E. coli; ER2566菌株开展重组SCCAg抗原可溶性表达纯化方法研究,评价纯化抗原活性,筛选特异性单克隆抗体,初步建立并评价SCCAg抗原检测方法。结果; 显示,pGEX-6P-l载体和E coli; ER2566菌株可用于建立较高效的可溶性表达和纯化SCCAg抗原的方法,获得了具有较高纯度和活性的重组SCCAg抗原,筛选获得特异性单克隆抗体并; 初步建立了 SCCAg管式化学发光检测方法。建立了有效的基于大肠杆菌表达系统的可溶性表达和纯化SCCAg的方法。The aims of this study are to establish a method for efficient soluble; expression of human squamous cell carcinoma antigen(SCCAg ) based on; Escherichia coli expression system and obtain the recombinant SCCAg; antigen in fine activity, then apply it in the detection method; establishment of antigen. The study on the method of soluble expression; and purification of recombinant SCCAg antigen was conducted based on; pGEX-6P-l vector and E. coli ER2566 strain. The activity of the purified; antigen was evaluated by Abbott Kit and the specific monoclonal antibody; was screened by indirect ELISA. It was proved that PGEX-6P-1 vector and; E. coli strain ER2566 could be used to establish efficient soluble; expression and purification method for recombinant SCCAg antigen.; Moreover, the recombinant SCCAg antigen was proved to be in high purity; and activity. Thus,the SCCAg detection method of chemical luminous tube; was established with the specific monoclonal antibodies. In conclusion,; an effective method for the expression and purification of SCCAg, which; is based on the E. coli expression system, is established.国家高技术研究发展计划(863计划

Effects of Salt and Drought on Winter Wheat in Seedling Stage Under Different Nitrogen Rates

为探究不同浓度盐胁迫和水分胁迫及两者互作对小麦幼苗生理特性的影响,于2017年3月至5月布置盆栽试验,分别设置两个NaCl胁迫(S1,NaCl 1.9g/kg;S2,NaCl 2.9g/kg)和两个水分处理水平(W1,78%田间持水量;W2,47%田间持水量),测定了冬小麦幼苗地上部和地下部干物质量、全氮、叶绿素和可溶性糖含量。结果表明:①在本试验盐胁迫范围内,单一盐胁迫下盐分含量的上升会显著抑制小麦的生长,冬小麦各部分干重、全氮、叶绿素含量明显下降,渗透物质可溶性糖含量会上升;②低盐干旱胁迫互作改善冬小麦幼苗生长状况,叶绿素含量、各部分干物质累积、氮积累量以及可溶性糖含量最大,呈现出对盐旱复合胁迫的适应性;③高盐干旱胁迫互作会加剧对小麦幼苗的生长限制。因此,低盐胁迫下对冬小麦进行适度的干旱刺激可以促进小麦幼苗适应复合胁迫,有利于小麦幼苗生长

Development of a Neutralization Test for Enterovirus D68 Using the Enzyme-linked Immunospot Assay

针对肠道病毒68型(EV-D68)建立一种新型快速、高效的中和试验方法,并初步评价其在EV-D68流行病学研究中的应用价值。以灭活EV-D68病; 毒免疫Balb/c小鼠,利用杂交瘤技术制备其单克隆抗体;筛选反应性好、特异性强的抗EV-D68单克隆抗体作为检测抗体,基于酶联免疫斑点检测技术(; ELISPOT)建立EV-D68高效中和试验方法;进一步比较该方法与传统中和试验方法Nt-CPE的一致性,并使用该方法对临床血清样本进行检测。共; 获得10株分泌抗EV-D68单克隆抗体的杂交瘤细胞株,选取一株性能最优的15C5进行生产纯化,鉴定其为IgG3亚型;以15C5为检测抗体,建立了; 快速检测EV-D68中和抗体的Nt-ELISPOT方法;与Nt-CPE方法比较,Nt-ELIS-POT方法将检测时间由5~7d缩短至ld以内,并; 且两种方法检测结果一致性良好;应用所建立的Nt-ELISPOT方法对厦门地区146份人群血清样本进行检测,显示血清样本EV-D68中和抗体阳性率; 为98. 6%; (144/146),提示厦门地区可能存在过EV-D68的流行。本研究基于ELISPOT建立的新型EV-D68中和试验方法快速可靠,适合通量检测,; 可以应用于EV-D68中和抗体水平的血清学调查,为EV-D68感染的防控工作提供帮助。We wished to establish a novel, rapid and efficient neutralization test; for enterovirus D68 (EV-D68) and evaluate its application in; seroepidemiological studies. Balb/c mice were immunized with inactivated; EV-D68. Monoclonal antibodies against EV-D68 were prepared by the; hybridoma method. Anti-EV-D68 monoclonal antibodies with high affinity; and specificity were selected as detection antibodies for the; establishment of a rapid neutralization test for EV-D68 using the; enzyme-linked immunospot (ELISPOT) assay. Also, the consistency between; the established method and conventional one, neutralization test based; on the inhibition of cytopathic effects (Nt-CPE),was investigated.; Finally, the established method was evaluated with clinical serum; samples. Ten hybridoma cell lines secreting monoclonal antibodies; against EV-D68 were obtained, and the best of them, 15C5, was selected; for the production and characterization of monoclonal antibodies.; Results showed that 15C5 was an immunoglobulin(Ig) G3 subclass. A rapid; method for the detection of neutralizing antibodies against EV-D68,; Nt-ELISPOT, was established using 15C5 as the detection antibody.; Nt-ELISPOT could shorten the detection time substantially from 5~7 days; in conventional Nt-CPE to <1 day, and there was good consistency between; the two methods. The established Nt-ELISPOT was applied to 146 clinical; serum samples from Xiamen, China, and the positive detection rate was; 98. 6% (144/146),suggesting that EV-D68 infection might be prevalent in; Xiamen. The established novel neutralization test for EV-D68 based on; ELISPOT was rapid and reliable. This method could be used to detect; EV-D68 neutralizing antibodies in seroepidemiological studies, thereby; providing support for the prevention and control of EV-D68 infection.国家自然科学基金; 国家自然科学基金; 福建省自然科学基