口蹄疫病毒结构蛋白VP1上B细胞表位的筛选鉴定

以口蹄疫病毒株AF72 RNA为模板,反转录并扩增结构蛋白VP1基因,PCR纯化产物与pGEM-T easy载体连接并转化JM109菌株,对经凝胶电泳、PCR和EcoRⅠ酶切法鉴定为阳性的重组质粒进行测序,通过序列比对获得AF72 VP1的核苷酸序列和推导氨基酸序列,综合分析结构蛋白VP1的亲水性、可塑性、抗原指数以及表面可能性等参数,预测其潜在B细胞抗原表位并人工合成表位肽段,利用间接ELISA对潜在表位肽段进行筛选鉴定,结果显示,表位VP1a和VP1d为病毒株AF72结构蛋白VP1的优势B细胞表位,该结果为进一步的FMDV多表位疫苗研究提供有价值的参考依据.     

毛细管电泳-电化学方法测定干红枣Vc·VP·VE的含量

[目的]建立采用毛细管电泳-电化学法分析干红枣中维生素含量的测定方法。[方法]采用自组装的毛细管电泳电化学检测系统,对干红枣中Vc、VP、VE的含量进行分离检测,对测定时不同的工作电位、分离高压、pH值和有机溶剂进行最佳选择。[结果]在选用pH值为8．0的硼砂-硼酸缓冲液,CTAB为0．5mmol／L、分离高压为20kV、检测电位为0．8V的测定条件下,Vc的线性范围为0．10～5．10mg／L,r=0．9991,检出限为1．37mg／L;VP的线性范围为1．30～17．70mg／L,r=0．9996,检出限为1．23mg／L;VE的线性范围为0．21—9．60mg／L,r=0．9990,检出限为0．27mg／L。测得的干红枣Vc、VP、VE的含量分别为424．64、412．16、312．39mg／100g,平均回收率分别为97．7％、98．1％和96．7％。[结论]毛细管电泳-电化学测定法快速、灵敏度高、选择性强,是适合干红枣维生素含量快速检测的方法。     

O型口蹄疫病毒VP1基因原核表达及蛋白纯化

以O型FMDV重组质粒pMD18T-O-VP1为模板,利用PCR技术扩增得到O型FMDV-VP1基因片段,将此基因片段与原核表达载体pET32a连接构建重组表达载体pEq32a-O-VP1,经PCR和测序鉴定后,用WIG诱导归1基因的表达,收集不同诱导时间的菌液,进行SDS-PAGE电泳,摸索掌握最佳诱导时间,切取最佳诱导时间电泳胶片做Westem-blotting,分析检验表达产物与其抗血清的反应性。结果显示,分子量约为45ku的蛋白条带反应显著,能被口蹄疫阳性血清识别,表明FMDV-VP1基因在大肠杆菌中得到高效表达,纯化复性的表达蛋白有望开发为诊断抗原和多肽疫苗。     

利用Asia1型口蹄疫病毒VP1蛋白的单克隆抗体建立单抗竞争ELISA方法

应用纯化的Asia1型口蹄疫病毒VP1蛋白作为抗原,采用过碘酸钠法标记纯化的单抗,建立了单抗竞争ELISA,检测10份阳性猪口蹄疫血清及200多份阴性血清,试验结果与样品的背景相一致。敏感性试验和重复试验结果表明,该方法具有稳定和敏感等特性,适合Asia1型猪口蹄疫病毒抗体的监测。     

猪细小病毒BQ-C毒株VP2基因的鉴定及表达

本研究从临床发病猪采集病料,以PK15传代细胞进行病毒分离,通过PCR、血液凝集试验(HA)和电镜鉴定为1株猪细小病毒(porcine parvovirus,PPV),命名为BQ-C。对分离的毒株进行测序,结果显示该毒株与GenBank登录的PPV BQ株同源性为100%。为获得该毒株结构蛋白VP2基因的表达产物,将VP2基因片段插入到原核表达载体pET-30a(+),得到表达重组质粒。经双酶切和测序鉴定,将重组质粒转化大肠杆菌BL21(DE3)中进行表达。SDS-PAGE结果表明,获得的重组蛋白分子质量约为71.5 ku,与预期大小相符。Western blotting结果显示获得的重组蛋白能与PPV阳性血清特异性结合,表明重组蛋白具有良好的反应原性。结果表明,本研究成功分离了1株PPV BQ-C株,且表达的VP2重组蛋白可用于PPV血清学诊断和疫苗的研发。     

4株鸡传染性法氏囊病病毒VP2基因序列分析

试验从发病鸡场分离获得4株鸡传染性法氏囊病病毒（infectious bursal disease virus,IBDV）,分别为河北株（HB）、湖北株（HUB）、山东株（SD）、山西株（SX）。通过特异性引物扩增VP2基因并进行测序,序列分析结果显示,4株病毒中,HUB、SD和SX的七肽区为S-W-S-A-S-G-S（aa 326S-332S）,其222（A）、256（I）、294（I）和299（S）位是传染性法氏囊病病毒超强毒株（vvIBDV）的4个特征性氨基酸,确定这3株病毒均为超强毒株;而HB株七肽区为S-W-S-A-R-G-S（aa 326S-332S）,其222（P）、256（V）、294（L）和299（N）位氨基酸显示该毒株属于弱毒株。进化树分析结果显示,HUB、SD和SX 3株病毒与2011年中国分离的大部分IBDV毒株亲缘关系较近,同源性在96%以上,同时与经典毒株Cro-Po/00、超强毒株GX和UK661亲缘关系很近;而HB株与减毒疫苗株D78株和B87株的亲缘关系近。本试验结果表明,IBDV目前在中国流行呈混发型,但以超强毒株为主。     

利用SUMO表达系统高效表达猪O型口蹄疫病毒VP0、VP1、VP3基因

根据GenBank中公布的猪O型口蹄疫病毒(foot and mouth disease virus,FMDV)全基因合成了FMDV结构蛋白前体蛋白P1基因,同时设计了扩增FMDV结构蛋白VP0、VP1和VP3基因的引物。以P1基因为模板,分别经PCR扩增获得FMDV VP0、VP1和VP3基因。扩增产物克隆于Blunt载体中,酶切后将目的片段连接到原核表达载体SUMO中,构建重组表达质粒SUMO-VP0、SUMO-VP1和SUMO-VP3,将重组质粒转化大肠杆菌BL21(DE3)plysS进行诱导表达。经SDS-PAGE电泳可见融合蛋白均获得高效表达,融合蛋白表观分子质量分别约为55、48和40 ku。在IPTG浓度为1.0 mmol/L,温度为37 ℃,诱导5 h时融合蛋白表达量最大。Western blotting结果表明,融合蛋白均可被FMDV阳性血清识别,反应原性良好。     

CARDIAC MAGNETIC RESONANCE IMAGING OF PATENT DUCTUS ARTERIOSUS IN THREE DOGS

Patent ductus arteriosus (PDA) is the most common congenital cardiovascular disorder in dogs and requires an accurate diagnosis for an appropriate treatment. Cardiac MRI (cMRI) has been reported as a method for characterization of canine thoracic vasculature. However, to the authors’ knowledge, no published studies describe evaluation of canine PDA through cMRI. Three dogs were selected for this exploratory study. Electrocardiogram gating and breath‐hold techniques were performed using a 3T MR scanner. Both black blood imaging and bright blood cine acquisitions were performed. Quantification of stroke volume (SV) and shunting volume were calculated using a stack of short‐axis cine images. Additional 4D (three‐spatial dimensions plus time)‐TRAK (time‐resolved MR angiography with keyhole) sequences were conducted in patient 2 to verify other vasculature abnormality. Black blood images clearly depicted the course of the ductus from the descending aorta to the pulmonary artery in all three dogs. Morphological evaluation of PDA classified patients 1 and 2 as Type 2a and patient 3 as Type 1. Patient 2 was confirmed to have a concurrent persistent left cranial vena cava. Left ventricular SV, right ventricular SV, and left‐to‐right SV ratio were 12.4 ml, 3.36 ml, and 3.704, respectively, in patient 1; 6.85 ml, 1.22 ml, and 5.60 in the patient 2; and 3.67 ml, 2.14 ml, and 1.702 in patient 3. Findings indicated that cMRI is a feasible method for characterizing the morphology of PDA and extracardiac vasculature anomalies in dogs.     

Epitope Mapping and Immuno-detection of VP7 Protein of Bluetongue Virus Serotype 1

In this study,in order to map the key amino epitope of VP7 protein of blue tongue virus serotype 1 (BTV1),we used monoclonal antibody against VP7 protein of BTV1 to screen 7 mer phage display random peptide library. The selected phages including VP7 protein consensus sequence were amplified and purified,immunoreactivity between epitope of selected phages and monoclonal antibody against VP7 of BTV1 was analyzed by ELISA and competitive ELISA (c-ELISA).The result showed that the epitope of phages including LNWPMVR sequence could specially combine monoclonal antibody against VP7 of BTV1,and the combination could be inhibited or blocked by BTV1,it demonstrated that 7 mer phage simulated the antigenic determinant of BTV protein which could combine monoclonal antibody against VP7 of BTV1.The results suggested that the amino acides 163 to 189(LNAGARGDVQQIFQGRNDPMMLYLVWR) were the specific epitope of VP7 protein of BTV.     

Prokaryotic Expression and Immunoreactivity Analysis of VP2 Protein of Bluetongue Virus Serotype 1

The study was aimed to test the immunoreactivity of the VP2 protein of bluetongue virus serotype 1 (BTV-1) in vitro. Based on the published BTV-1 L2 gene of Y863 strain, specific cloning PCR primers were designed and synthesized. The L2 gene was amplified through RT-PCR method and then was purified and cloned into the expressing vector pEASY-Blunt E1. The cloned recombinant plasmids were identified. The positive recombinant L2 plasmid was cloned into BL21(DE3) competent cells to express VP2 protein. The acquired purified recombinant BTV-1 VP2 protein was analyzed through the methods of Western blotting, ELISA and blocking ELISA. The results showed that:BTV-1 VP2 protein was expressed as the inclusion bodies in the pEASY-Blunt E1 vector; 160 and 200 mmol/L glyoxaline were the best condition to wash down the expressed protein. The molecular weight of this purified recombinant protein with N-terminal His-tag was about 105 ku. Through the results of Western blotting, ELISA and blocking ELISA, it had been proved that this recombinant protein could combine with BTV-1 specific antibody and this combination could be blocked by BTV-1 virus. The study showed that the recombinant BTV-1 VP2 protein, expressed through the prokaryotic expression vector pEASY-Blunt E1, possessed good immunoreactivity and this study had established foundation for locating the serotypic epitopes of the BTV-1 VP2 protein.