口蹄疫病毒非结构蛋白3AB双抗体夹心ELISA方法的建立

抗原纯净度是口蹄疫 (Foot-and-mouth disease,FMD) 灭活疫苗质量检验的一项重要内容,一般采用疫苗2–3次免疫动物后,检测非结构蛋白 (Non-structural protein,NSP) 抗体是否阳转,判断疫苗抗原的纯净度。文中旨在建立定量检测FMD灭活疫苗抗原中NSP 3AB含量的ELISA方法,为疫苗质量控制提供参考方法。利用口蹄疫病毒 (Foot-and-mouth disease virus,FMDV) NSP 3A单克隆抗体和辣根过氧化物酶 (Horseradish peroxidase,HRP) 标记的3B单克隆抗体,建立定量检测NSP 3AB含量的双抗体夹心ELISA检测方法。采用原核表达并纯化的3AB蛋白作为标准品,标准品系列稀释,绘制标准曲线,以标准品与未加抗原的阴性对照吸光值 (OD) 的比值大于2.0的标准品最低浓度为最低检测限。标准品浓度介于4.7–600.0 ng/mL之间时,测得的OD值与浓度呈线性相关,回归曲线呈直线,相关系数R2=0.99,确定最低检测限为4.7 ng/mL。检测12份未纯化灭活抗原中3AB蛋白含量介于9.3–200.0 ng/mL之间;而纯化后的病毒抗原中3AB蛋白残留量低于最低检测限;33份来自不同厂家的成品疫苗抗原中9份疫苗抗原3AB蛋白含量在9.0–74.0 ng/mL之间,其余24份疫苗抗原中3AB蛋白残留量低于最低检测限。检测3AB蛋白含量的双抗体夹心ELISA方法能够特异、敏感地检测疫苗抗原中的3AB蛋白含量,为疫苗质量控制与纯净度检验提供了一种可供选择的检测方法。     

口蹄疫病毒非结构蛋白3AB 的原核表达及应用

旨在建立一种检测口蹄疫病毒非结构蛋白抗体的敏感、特异的ELISA方法。克隆、表达了口蹄疫病毒非结构蛋白3AB基因,原核表达的重组蛋白经亲和层析法纯化及Western blotting鉴定后作为包被抗原,建立检测口蹄疫病毒非结构蛋白抗体的3AB间接ELISA方法,通过与商品化试剂盒3ABC-ELISA的比对试验对其进行评价。结果显示,重组蛋白3AB以包涵体形式表达;能与口蹄疫病毒感染血清发生特异性反应,而不能与疫苗免疫动物血清发生反应;在检测田间样品时,与3ABC-ELISA具有同样的特异性和敏感性 (P＞     

TSP1对高糖诱导的肾小管上皮细胞损伤的影响研究

目的探讨血小板反应蛋白1(TSP1)对高糖诱导的肾小管上皮细胞损伤及炎症因子分泌的影响。方法以肾小管上皮细胞为研究对象,通过转染TSP-1 shRNA(shTSP-1),沉默TSP-1基因,探讨下调TSP1对高糖条件下肾小管上皮细胞损伤的影响。肾小管上皮细胞依次分为:对照组(以5.6 mmol/L葡萄糖培养)、高糖组(以30 mmol/L葡萄糖培养)、NC+高糖组(对照慢病毒转染,以30 mmol/L葡萄糖培养)、干扰+高糖组(TSP1 shRNA慢病毒转染以30mmol/L葡萄糖培养)。Realtime PCR和Western Blot检测高糖对细胞中TSP1表达影响,同时检测TSP1 shRNA干扰效果。DCFH-DA法检测细胞中活性氧(ROS)水平,硫代巴比妥酸法检测培养液中丙二醛(MDA)含量,ELISA法检测培养液上清中肿瘤坏死因子-α(TNF-α)、白细胞介素-8(IL-8)含量,Annexin V-FITC/PI双染法检测细胞凋亡,Western Blot法检测细胞中活化的Caspase-3(c-caspase-3)蛋白水平。两组均数差异比较采用独立样本t检验,多组均数间差异比较采用单因素方差分析,组间两两比较采用SNK-q检验。结果高糖组细胞中TSP1mRNA和蛋白水平高于对照组(P 0.05)。干扰+高糖组细胞中TSP1 mRNA和蛋白水平低于高糖组(P 0.05)。与对照组比较,高糖组细胞中ROS水平升高,培养液中MDA、TNF-α、IL-8含量升高[(2.36±0.21)nmol/ml、(45.91±2.87)ng/ml、(25.42±3.26) ng/ml:(1.05±0.13)nmol/ml、(20.14±1.36)ng/ml、(12.98±1.63)ng/ml],差异有统计学意义(F=18.595,F=43.825,F=21.155,P 0.05);细胞凋亡率升高(P 0.05),细胞中c-caspase-3蛋白水平升高(P 0.05)。与高糖组和NC+高糖组比较,干扰+高糖组细胞中ROS水平降低,培养液中MDA、TNF-α、IL-8含量降低[(1.63±0.10)nmol/ml、(34.20±2.06)ng/ml、(18.75±1.62)ng/ml:(2.36±0.21) nmol/ml、(45.91±2.87)ng/ml、(25.42±3.26)ng/ml和(2.30±0.42)nmol/ml、(46.32±5.24) ng/ml、(26.91±2.74)ng/ml],差异具有统计学意义(F=18.595,F=43.825,F=21.155,P 0.05);细胞凋亡率降低,细胞中c-caspase-3蛋白水平降低(P 0.05)。结论高糖诱导肾小管上皮细胞中TSP1表达,下调其表达可以减少细胞分泌炎症因子,抑制高糖诱导的肾小管上皮细胞损伤。     

霍乱疫苗中残余霍乱毒素检测方法的建立及验证

采用间接酶联免疫法,即用神经节苷脂包被,加入待检样品,再加入兔抗霍乱毒素B亚单位抗体,用标准样品的吸光值(A值)对标准样品的浓度绘制4-参数拟合曲线,根据标准曲线计算出待测样品中的CT浓度。结果显示,在浓度范围(0.6～16)ng/ml之间,CT标准浓度和检测浓度成线性关系,r2=0.9986。精确度在浓度范围(0.6～16)ng/ml,CT的平均回收率在96.24%～114.44%之间。精密度:批内变异CV%≤12.98%,批间变异CV%≤18.48%。特异性CT浓度在10ng/ml时,平均回收率为102.6%;CT浓度在5ng/ml时,平均回收率为111.17%;CT浓度在2.5ng/ml时,平均回收率为123.83%。实验表明该方法可检测霍乱疫苗原液中CT的含量。     

猪O型口蹄疫病毒细菌样颗粒疫苗的制备与免疫原性鉴定

验证基于革兰氏阳性增强基质(Gram-positive enhancer matrix,GEM)展示口蹄疫病毒细菌样颗粒(Bacteria-like particles,BLP)疫苗的可行性。按照大肠杆菌偏好性密码子优化合成基于猪口蹄疫病毒Mya98株序列的3种抗原基因设计,并将其插入到含有锚钩蛋白基因的原核表达载体p QZ-PA,鉴定阳性后转入Escherichia coli BL21,进行诱导表达。利用SDS-PAGE与Western blotting对目的基因表达及产物的可溶性进行分析。利用GEM颗粒纯化目的蛋白,制备细菌样颗粒疫苗抗原;利用BCA试剂盒测定重组蛋白的浓度,将重组蛋白与白油佐剂乳化,制备疫苗,免疫5周龄小鼠,同时设商品化多肽苗对照与空白对照,免疫后不同时间采集试验小鼠血清,利用口蹄疫病毒多肽ELISA抗体检测试剂盒和O型口蹄疫抗体液相阻断酶联免疫(Enzyme-linked immunosorbent assay,ELISA)检测试剂盒检测免疫小鼠血清的抗体水平;利用噻唑蓝比色法(Methylthiazolyldiphenyl-tetrazolium bromide,MTT)测定淋巴细胞增殖情况;利用荧光定量PCR方法检测相关细胞因子表达,评价细胞免疫水平。SDS-PAGE结果表明,设计在大肠杆菌中的3种口蹄疫病毒抗原基因均以可溶形式获得高效表达;Western blotting结果显示,表达的重组蛋白能够与口蹄疫病毒阳性血清发生反应,利用GEM颗粒能够实现重组蛋白的一步离心纯化,制备BLP疫苗抗原;免疫试验结果表明,设计的重组抗原B(T1BT2)4B不但能够刺激免疫小鼠产生更高水平的多肽特异性ELISA抗体与口蹄疫特异性液相阻断抗体,而且产生了更高水平的脾淋巴细胞增殖及Th1型的细胞因子分泌。初步实验结果表明,本研究制备的BLP疫苗GEM-B(T1BT2)4B具有良好的免疫原性,为研究口蹄疫病毒基因工程亚单位疫苗开辟了一条新的思路。     

重组人睫状神经营养因子蛋白质定量检测方法的建立

采用ELISA双抗体夹心法,建立一种快速灵敏的定量检测rhCNTF成品蛋白含量的方法。结果显示,rhC-NTF抗原浓度在(0～25)ng范围内线性良好(r>0.99),灵敏度为0.3ng/ml,与其他重组细胞因子无交叉反应,样品的检测结果与理论含量相吻合,CV<15%,该方法检测速度快、重复性好、灵敏度高、特异性好。     

口蹄疫病毒结构蛋白P1-2A及蛋白酶3C基因的转基因番茄对豚鼠免疫反应的初步研究

构建了O型口蹄疫病毒China99株结构蛋白P1-2A、非结构蛋白3C以及部分2B基因(P1-2X3C)的植物双元表达载体pBin438/P1-2X3C,通过农杆菌介导法转化番茄子叶,经卡那霉素抗性筛选,获得40余株抗性植株,对得到的抗性植株进行分子生物学检测,65%的再生植株PCR检测阳性;RT-PCR结果证实P1-2X3C基因在转基因番茄中能够有效转录;ELISA和Western blot检测表明转基因植株中表达的目的蛋白具有免疫反应性。转基因番茄叶片蛋白粗提液经肌肉途径免疫豚鼠,于第3次免疫后28d用100ID50/0.2mL的同源强毒攻击,结果表明口蹄疫病毒P1-2X3C基因的转基因番茄表达产物具有良好的免疫原性,豚鼠3免后血清效价可达1:64~1:128,攻毒后两组免疫豚鼠保护率分别达3/5和5/5。     

不同浓度尼古丁对牙周膜成纤维细胞增殖及纤维结合蛋白合成的作用

目的:探讨不同浓度尼古丁对人牙周膜成纤维细胞(PDLFs)增殖及纤维结合蛋白(Fn)合成的作用。方法:不同浓度的尼古丁(50 ng/ml,250 ng/ml,500 ng/ml,1μg/ml,2μg/ml,3μg/ml)作用于PDLFs,MTT比色法检测细胞增殖活性,流式细胞仪检测细胞周期,酶联免疫吸附测定法(ELISA)检测Fn合成含量。结果:不同浓度尼古丁作用下:人PDLFs的增殖均被抑制,且呈浓度依赖性。浓度为250 ng/ml~3 ug/ml的尼古丁有明显抑制人PDLFs增殖的作用(P0.05),3 ug/ml尼古丁显示出最强的抑制增殖作用(P0.01);人PDLFs的G0-G1期、S期、G2-M期与对照组相比,G0-G1期细胞周期分布比例逐渐增高,S期和G2-M期逐渐降低,差异均有统计学意义,呈浓度依赖性;人PDLFs合成Fn逐渐减少,呈浓度依赖性。浓度为50 ng/ml~3μg/ml的尼古丁均有明显抑制人PDLFs合成Fn的作用(P0.05),其中3μg/ml抑制作用最强。结论:尼古丁抑制牙周膜细胞的增殖及Fn的合成,呈浓度依赖性,并影响其细胞周期的进程,进而影响牙周新附着的形成,加重牙周病的病情。     

一种实用的筛选病毒抗原表位方法的建立(英)

采用基因分段克隆、表达结合蛋白质印迹, 筛选到了口蹄疫病毒非结构蛋白3ABC上高结合力、保守的感染相关线性表位,分别位于3ABC蛋白上第106～155和156～190位氨基酸.这两个表位可与感染不同血清型口蹄疫病毒动物康复血清反应,但不与来自健康免疫动物和未接触病毒动物的血清发生反应.实验表明,用基因工程表达的多肽筛选抗原表位的方法是可行的.     

Vero细胞狂犬病疫苗在凝胶柱层析工艺中样品浓度对纯化效果的影响

为研究浓缩Vero细胞狂犬病疫苗蛋白浓度对纯化效果的影响。用超滤浓缩法对狂犬病疫苗进行不同倍数的超滤浓缩,将浓缩后不同倍数的样品分别用凝胶柱层析法进行纯化,结果样品浓度在40mg/ml以下时,狂犬病毒收集液中杂蛋白去除率可达99%以上,小牛血清含量在25-37.5ng/ml之间;浓缩超过40mg/ml时,并随着样品浓度的增加,狂犬病毒收集液中杂蛋白去除率逐渐下降,且有病毒丢失,因此采用凝胶柱层析法纯化狂犬病疫苗,蛋白浓度应低于40mg/ml。     

丙型肝炎病毒NS3蛋白的原核表达及多克隆抗体制备

提高对丙型肝炎患者实验室检测的灵敏度和特异性,对相关人群进行筛查和早期诊断,是控制丙型肝炎病毒(HCV)流行与传播的有效措施。为了建立更为可靠的HCV诊断方法,通过采用PCR方法从J6/JFH12a型病毒中克隆出HCV ns3基因片段,将其连接到p ET-28a载体上,重组载体p ET-28a-ns3转化大肠杆菌BL21(DE3)后诱导表达,以10%SDS-PAGE进行鉴定,获得表达的NS3重组蛋白分子量为72 k Da。将纯化的NS3蛋白免疫BALB/c小鼠,第4次免疫后采集血液并分离血清进行抗体活性鉴定,小鼠抗体效价为1∶256 000。进一步的Western blotting和间接免疫荧光结果显示,以重组NS3蛋白免疫小鼠制备的多克隆抗体可以很好地识别HCV感染Huh7.5.1细胞中的NS3蛋白,为下一步开展单克隆抗体制备和检测试剂盒研制工作奠定了基础。     

Development of surface plasmon resonance imaging biosensors for detection of ubiquitin carboxyl-terminal hydrolase L1

We have developed a new method for highly selective determination of the ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) concentration using a surface plasmon resonance imaging (SPRI) technique and two different biosensors. UCH-L1 was captured from a solution by immobilized specific rabbit monoclonal antibody or specific LDN-57444 inhibitor due to formation of receptor–UCH-L1 complex on the biosensor surface. The analytically useful dynamic response range of both biosensors is between 0.1 and 2.5 ng/ml. The detection limit is 0.06 ng/ml for the biosensor with antibody and 0.08 ng/ml for the biosensor with inhibitor. Biosensors based on both antibody and inhibitor were found to be suitable for quantitative determination of the UCH-L1 and exhibit good tolerance to the potential interferents. Both biosensors gave comparable results in the range of 0 to 0.20 ng/ml for plasma samples and 0.30 to 0.49 ng/ml for cerebrospinal fluid samples. To validate the new methods, comparative determination of UCH-L1 by the commercial enzyme-linked immunosorbent assay (ELISA) kit was performed. In general, in terms of UCH-L1 concentration, a good correlation between SPRI and ELISA was found. The developed biosensors can be used successfully for the determination of UCH-L1 in body fluids.     

Quantification of the secretory glycoproteins of the subcommissural organ by a sensitive sandwich ELISA with a polyclonal antibody and a set of monoclonal antibodies against the bovine Reissner’s fiber

The subcommissural organ (SCO) is an ependymal brain gland that releases glycoproteins into the ventricular cerebrospinal fluid where they condense to form the Reissner’s fiber (RF). We have developed a highly sensitive and specific two-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of the bovine SCO secretory material. The assay was based on the use of the IgG fraction of a polyclonal antiserum against the bovine RF as capture antibody and a pool of three peroxidase-labeled monoclonal antibodies that recognize non-overlapping epitopes of the RF glycoproteins as detection antibody. The detection limit was 1 ng/ml and the working range extended from 1 to 4000 ng/ml. The calibration curve, generated with RF glycoproteins, showed two linear segments: one of low sensitivity, ranging from 1 to 125 ng/ml, and the other of high sensitivity between 125 and 4000 ng/ml. This assay was highly reproducible (mean intra- and interassay coefficient of variation 2.2% and 5.3%, respectively) and its detectability and sensitivity were higher than those of ELISAs using exclusively either polyclonal or monoclonal antibodies against RF glycoproteins. The assay succeeded in detecting and measuring secretory material in crude extracts of bovine SCO, culture medium supernatant of SCO explants and incubation medium of bovine RF; however, soluble secretory material was not detected in bovine cerebrospinal fluid.     

Generation of monoclonal antibodies against non-structural protein 3AB of foot-and-mouth disease virus

To identify linear epitopes on the non-structural protein 3AB of foot-and-mouth disease virus (FMDV), BABL/c mice were immunized with the 3AB protein and splenocytes of BALB/c mice were fused with myeloma Sp2/0 cells. Two hybridoma monoclonal antibodies (mAbs) cell lines against the 3AB protein of foot-and-mouth disease virus (FMDV) were obtained, named C6 and E7 respectively. The microneutralization titer was 1:1024 for mAb C6, and 1:512 for E7. Both mAbs contain kappa light chains, and were of subclass IgG2b. In order to define the mAbs binding epitopes, the reactivity of these mAbs against FMDV were examined by indirect ELISA. The results showed that both mAbs can react with FMDV, but had no cross-reactivity with Swine Vesicular Disease (SVD) antigens. The titers in abdomen liquor were 1:5×10⁶ for C6 and 1:2×10⁶ for E7. In conclusion, the mAbs obtained from this study are specific for the detection of FMDV, can be used for etiological and immunological researches on FMDV, and have potential use in diagnosis and future vaccine designs.     

Sandwich capture ELISA by a murine monoclonal antibody against a genus-specific LPS epitope for the detection of different common serotypes of salmonellas.

A sandwich capture ELISA based on a murine monoclonal antibody against a genus-specific epitope in the outer core region of the Salmonella lipopolysaccharide is described for the detection of different common serotypes of salmonellas. Four h broth cultures of seven standard and 24 wild strains of salmonellas were all detected by the capture ELISA while overnight broth cultures of 21 non-salmonella standard strains were all negative. The capture ELISA detected 1 ng/ml of Ra lipopolysaccharide, 10(6)/ml of a smooth wild strain of Salm. typhimurium, and 1120 cells of Salm. heidelberg after enrichment culture for 4 h.     

Production and Characterization of Monoclonal Antibodies of Shrimp White Spot Syndrome Virus Envelope Protein VP28

BALB/c mice were immunized with purified White spot syndrome virus (WSSV). Six monoclonal antibody cell lines were selected by ELISA with VP28 protein expressed in E. coli. in vitro neutralization experiments showed that 4 of them could inhibit the virus infection in crayfish. Western-blot suggested that all these monoclonal antibodies were against the conformational structure of VP28. The monoclonal antibody 7B4 was labeled with colloidal gold particles and used to locate the VP28 on virus envelope by immunogold labeling. These monoclonal antibodies could be used to develop immun-ological diagnosis methods for WSSV infection.     

Production and characterization of monoclonal antibodies of shrimp White spot syndrome virus envelope protein VP28

BALB/c mice were immunized with purified White spot syndrome virus (WSSV). Six monoclonal antibody cell lines were selected by ELISA with VP28 protein expressed in E. coli. in vitro neutralization experiments showed that 4 of them could inhibit the virus infection in crayfish. Western-blot suggested that all these monoclonal antibodies were against the conformational structure of VP28. The monoclonal antibody 7B4 was labeled with colloidal gold particles and used to locate the VP28 on virus envelope by immunogold labeling. These monoclonal antibodies could be used to develop immunological diagnosis methods for WSSV infection.     

基于N蛋白单克隆抗体的小反刍兽疫病毒抗体检测胶体金试纸条的研制

本研究旨在建立一种简便、快捷、可直观检测小反刍兽疫病毒(peste des petits ruminants virus,PPRV)抗体的检测方法。将pET-32a-N重组质粒转化至大肠杆菌(Escherichia coli) Rosetta(DE3)感受态细胞中进行诱导表达,以纯化的PPRVN蛋白免疫8周龄BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,间接酶联免疫吸附试验(enzyme-linked immunosorbent assays, ELISA)筛选及亚克隆,获得了抗PPRV N蛋白的单克隆抗体。将PPRV N蛋白分别作为金标抗原及检测线(T线)包被抗原、单克隆抗体作为质控线(C线)包被抗体,组装成检测PPRVN蛋白抗体的胶体金免疫层析试纸条。结果显示：成功获得1株能稳定分泌抗N蛋白抗体的杂交瘤细胞株,命名为1F1;间接ELISA检测1F1腹水效价为1:128000;亚类鉴定结果为IgG1,轻链为kappa链。Westernblotting结果显示,1F1能与PPRV N蛋白特异性结合;间接免疫荧光(indirect immunofluorescent ass...