牛冠状病毒重组N蛋白间接ELISA检测方法的建立

为建立牛冠状病毒(BCV)检测方法,利用构建的pET30a-N重组质粒高效表达了BCV重组N蛋白.western blot检测该重组蛋白具有很好的免疫活性.以纯化的蛋白作为包被抗原,通过方阵试验确定了抗原的最适包被浓度为1.75 μg/mL,血清最佳稀释倍数为1:200,酶标二抗最佳稀释倍数为1:8 000,建立了检测BCV抗体的间接ELISA方法.用该法对黑龙江一些地区采集到的256份牛血清样品进行检测,结果阳性率为65.23%,与病毒中和试验方法的检测结果符合率达95.31%.本研究建立的间接ELISA方法为BCV的检测和区域流行病学调查提供了一种快速简便的血清学诊断方法.     

载副猪嗜血杆菌PLGA Omp16蛋白微球的制备及其免疫效果的初步研究

为研究载副猪嗜血杆菌(H. parasuis)外膜蛋白Omp16 PLGA微球的生物学特性,本研究以H. parasuis外膜蛋白Omp16为芯材,聚乳酸-羟基乙酸共聚物(PLGA)为壁材,通过复乳溶剂挥发法制备PLGA微球疫苗。通过扫描电镜测定微球的形态、激光粒径仪测定微球的粒径分布。结果显示,PLGA微球呈球性,表面圆整,粒径分布较窄,平均粒径为5.2μm;采用BCA法测定微球的包封率和载药率,结果显示,微球的包封率为70.5%,载药率为5.78%;以透析释药法测定微球的体外释放特性,结果显示,微球体外释放42 d累计释放抗原95.3%。用该微球0.2 mg/次,免疫小鼠二次(间隔14 d)后,采用ELISA法测定小鼠抗H. parasuis IgG水平,结果显示,微球佐剂组小鼠H. parasuis IgG抗体水平显著高于灭活疫苗组(p0.01);腹腔注射H. parasuis LY02株(血清5型)进行攻毒保护试验,结果显示,微球佐剂组小鼠攻毒保护率(81.8%)显著高于灭活疫苗组(63.6%)(p0.05)。以上试验结果表明,制备的PLGA微球具有良好的缓释性能,能增强小鼠体液免疫应答,提高H. parasuis攻毒保护率。本研究为H. parasuis亚单位微球疫苗的制备奠定基础。     

盐酸多西环素壳聚糖微球的制备与评价

制备盐酸多西环素(doxycycline hydrochloride,DH)缓释微球,并对其进行评价。通过Design-expert软件进行试验设计,以载药量和包封率为考察标准进行优化,采用乳化交联的方法制备壳聚糖包载DH缓释微球,运用紫外-可见分光光度计(UV-VIS Spectrophotometer)、扫描电子显微镜(SEM)、傅里叶变换红外光谱(FT-IR)、热重分析(TGA)、X射线衍射(XRD)及拉曼光谱(Raman spectra)对微球的结构、性能和形态进行评价。结果显示,在最佳制备条件下,即壳聚糖为20 g/L,体系内DH质量为0.3 g,转速为910 r/min,液体石蜡12 mL时,所制备载药微球的载药量为56.49%,包封率为61.41%。FT-IR表明壳聚糖包载DH主要以物理作用为主;热失重表明微球物理包合后热稳定性较差;XRD结果表明DH被包载后晶体结构未发生变化。结果表明,本试验成功制备表面光滑、粒径整齐、载药量和包封率较高的DH壳聚糖微球。     

甲壳素对猪传染性胃肠炎免疫调节作用的研究

研究了甲壳素对猪传染性胃肠炎疫苗的免疫调节作用；用浓碱法处理甲壳素可获得吸附能力强的佐剂，但物理性状不适合用于疫苗佐剂：壳聚糖可用作制备具有良好水溶性的疫苗佐剂，经与白油和蜂胶等佐剂比较，表明该佐剂对猪传染性胃肠炎具有一定的免疫调节作用。即4种佐剂的疫苗在免疫后14d，壳聚糖疫苗组抗体效价显著高于其它组；免疫后30～120d油乳剂疫苗组抗体效价显著高于其它组。     

副猪嗜血杆菌微球疫苗制备及免疫效果研究

副猪嗜血杆菌感染是猪场主要细菌性疾病之一,而疫苗免疫为控制副猪嗜血杆菌感染的重要手段。本研究以壳聚糖和海藻酸钠为微球壁材,以副猪嗜血杆菌LY02株(血清5型)裂解蛋白为抗原,通过乳化交联法制备了副猪嗜血杆菌微球疫苗。微球平均粒径为2.58μM,微球的平均包封率为68.3%,平均载药率为3.56%。体外释放试验结果显示,微球具有良好的缓释性能,抗原释放时间超过30 d。小鼠免疫试验显示该微球疫苗对血清5型副猪嗜血杆菌的攻毒保护率达90%。该微球疫苗的研究为副猪嗜血杆菌病疫苗开发提供了一个新的思路。     

REV Gp90亚单位疫苗联合核酸缓释佐剂对雏鸡的免疫保护作用

主要探讨禽网状内皮组织增生症病毒(REV)亚单位疫苗联合CpG-ODN/Poly(I:C)缓释佐剂对雏鸡的免疫保护作用。根据已发表的REV囊膜蛋白Gp90的基因序列,设计引物,从感染REV的细胞中扩增出目的基因,构建原核重组表达载体,在Rosseta(E.coli)菌中诱导表达,对表达蛋白进行纯化和Western blot鉴定;制备的重组蛋白与CpG-ODN/Poly(I:C)缓释佐剂联合接种7日龄雏鸡,2次加强免疫后第2周进行REV攻毒,接种后每周检测血清抗体和攻毒后检测REV病毒血症,评价免疫保护效果。结果显示,成功构建重组原核表达质粒,并获得相对分子质量为51 000的重组表达产物,该产物能够与REV单克隆抗体特异性结合;与CpG-ODN/Poly(I:C)缓释佐剂联合接种后能够诱导雏鸡产生较单一免疫Gp90蛋白抗体效价显著增加的血清抗体,经二次加强免疫全部鸡只产生抗体;攻毒后接种雏鸡病毒血症为0%(0/13),而对照组和单一免疫Gp90蛋白组分别为69%(9/13)和15%(2/13)。结果表明,本研究制备的REV Gp90重组蛋白联合CpG-ODN/Poly(I:C)缓释佐剂对雏鸡具有完全的保护力,这为开展REV疫苗防控提供科学数据。     

壳聚糖-海藻酸钠载药微球制备工艺研究

为研究制备壳聚糖-海藻酸钠载药微球的新方法,研究采用D相乳化法,结合微乳及胶体制备技术,以硫酸小檗碱为模型药物,以药物包封率为评价指标,应用单因素试验考察了氯化钙溶液浓度、海藻酸钠溶液浓度、壳聚糖溶液浓度,以及初次凝胶时间对微球性能的影响。应用响应面法筛选优化了载药微球的制备工艺。结果表明,在微球制备及药物包封率的影响因素中,海藻酸钠溶液浓度、氯化钙溶液浓度对评价指标的影响最大,其次是壳聚糖溶液浓度和初次凝胶时间,筛选优化的载药微球生产工艺条件为海藻酸钠溶液浓度1.57%,氯化钙溶液浓度2.13%,壳聚糖溶液浓度0.86%,初次凝胶时间30.71min,该条件下制备微球的平均粒径为329nm,药物包封率94.09%。验证试验证实,本工艺可制备优良的硫酸小檗碱壳聚糖-海藻酸钠微球,且制备工艺简便,生产效率高,本技术为微球制剂的产业化生产奠定了一定基础。     

纳米氢氧化铝吸附禽流感病毒灭活疫苗的研制及其对肉鸡抗体效价的影响

自制纳米氢氧化铝佐剂,通过吸附H5N1灭活抗原,制备了纳米铝佐剂疫苗,按照中华人民共和国农业行业高致病性禽流感免疫技术规范标准,免疫肉鸡。经检测发现纳米铝佐剂疫苗组肉鸡产生有效保护抗体时间较油佐剂疫苗和常规铝佐剂疫苗提前3d和7d,抗体峰值达到时间较油佐剂疫苗和常规铝佐剂疫苗提前14d。结果表明,纳米氢氧化铝作为高致病性禽流感H5N1的免疫佐剂,可以更早的刺激动物产生有效保护抗体,纳米铝佐剂禽流感疫苗具有广泛的应用价值。     

ALV-Jgp85重组蛋白与免疫佐剂联合接种雏鸡诱导免疫保护的研究

为研究原核表达J亚群禽白血病病毒(ALV-J)gp85囊膜蛋白诱导雏鸡产生保护性抗体及抗ALV-J感染的作用,本研究采用ALV-J gp85重组蛋白与不同免疫佐剂接种雏鸡,检测其免疫后血清抗体变化,并在二免后第35 d进行攻毒,检测鸡体内病毒血症。结果显示单一gp85重组蛋白只能诱导少部分雏鸡产生抗体,抗体效价较低、维持时间短;而弗氏佐剂(F)和CpG(C)佐剂联合重组蛋白能够使所有的免疫鸡产生抗体,效价较高,维持时间约56 d;二次免疫可以增强免疫效果,高效价抗体维持时间进一步延长。重组蛋白免疫组减少病毒血症的免疫保护率为33.3%,而F佐剂组及F和C佐剂与重组蛋白联合免疫组免疫保护率分别为53.8%和66.7%;另外,F和C佐剂与重组蛋白联合免疫明显增强机体对病毒的抵抗力。以上结果表明使用F和C佐剂可以增强gp85重组蛋白的免疫原性,产生较好免疫保护作用,为开发有效的ALV-J亚单位疫苗提供实验依据。     

牛病毒性腹泻/黏膜病(1型+2型)、牛传染性鼻气管炎、牛副流感(3型)三联灭活疫苗(E2蛋白+C1株+HB01株)的研制与免疫效果评价

采用CHO细胞表达牛病毒性腹泻/黏膜病病毒1型（BVDV1）E2蛋白,采用杆状病毒重组表达牛病毒性腹泻/黏膜病病毒2型（BVDV2）E2蛋白,采用MDBK细胞微载体悬浮培养技术培养牛传染性鼻气管炎病毒（IBRV）和牛副流感病毒3型（BPIV3）,收获蛋白表达产物和细胞培养物,经纯化、灭活后与605佐剂混合,制备牛病毒性腹泻/黏膜病(1型+2型)、牛传染性鼻气管炎、牛副流感(3型)三联灭活疫苗(E2蛋白+C1株+HB01株)。将疫苗免疫健康易感牛进行免疫效果评价,结果表明该产品免疫效果良好,免疫牛IBRV和BPIV3中和抗体效价均可达到1∶77以上;BVDV1和BVDV2 E2蛋白琼扩抗体效价均可达到1∶32以上;免疫牛攻毒保护率均可达到4/5以上。     

Mucosal and systemic isotype-specific antibody responses to bovine coronavirus structural proteins in naturally infected dairy calves.

Blood, feces, nasal secretions, and tears were collected weekly from 5 randomly selected 1- to 8-week-old calves in a large commercial dairy herd. Clinical signs and bovine coronavirus (BCV) shedding from the respiratory and enteric tracts of calves were monitored through the 8-week period by direct immunofluorescence of nasal epithelial cells, protein A-gold immunoelectron microscopy on feces, and ELISA on nasal secretions and feces. All samples were analyzed for antibody isotypes to BCV structural proteins by immunoblotting. All calves had BCV respiratory tract infections and 4 of 5 calves shed virus in feces. Several calves had multiple or prolonged periods of BCV respiratory tract or enteric tract shedding or both. All calves (except 1) had passive IgG1 antibodies to some BCV proteins (mainly the E2 and E3 proteins) in their serum when they were 1 week old. The presence of these passive serum antibodies (mainly to the E2 and E3 BCV proteins) was associated with decreased or delayed systemic and mucosal antibody responses in calves, in particular IgA responses in nasal secretions and tears to the E2 and E3 BCV proteins, but not to the N protein. Moderate amounts of maternal BCV E2- and E3-specific antibodies in serum did not prevent BCV enteric tract or respiratory tract infections in calves, but may have delayed the development of active antibody responses to these BCV proteins. However, calves with BCV respiratory tract or enteric tract infections had no detectable passive antibodies to any BCV proteins in nasal secretions or feces.     

Isotype-specific antibody responses to bovine coronavirus structural proteins in serum, feces, and mucosal secretions from experimentally challenge-exposed colostrum-deprived calves.

Five newborn isolation-reared colostrum-deprived calves were inoculated orally and intranasally when they were 20 to 30 hours old and challenge exposed when they were 21 days old with a suspension of virulent bovine coronavirus (BCV). Blood, feces, nasal swabs, tears, saliva, and bronchoalveolar lavage (BAL) fluids were collected from each calf prior to inoculation and then weekly for 5 post-inoculation weeks. An ELISA was used to quantitate the immunoglobulin isotype titers of BCV antibodies in all samples. An immunoblot assay was used to determine the antibody isotype responses to BCV structural proteins in all the samples, except saliva. At postinoculation days 2 to 3, all calves had severe watery diarrhea, shed BCV in their feces, and had evidence of BCV replication in their upper respiratory tract. After challenge exposure, no calves became ill and no evidence of BCV replication in the respiratory or intestinal tracts was detected. At postinoculation week 1, IgM responses to the N protein were seen in mucosal secretions (except nasal fluid) and feces. At postinoculation weeks 2 and 3, IgA was predominant in mucosal secretions and feces directed toward all the BCV proteins (except the E2 protein in BAL fluid). After challenge exposure, an increase (or failure to decrease) in most IgA and some IgG1 titers to BCV proteins was seen. The increases in IgA titers were to all viral proteins in all mucosal secretions and feces, except to the N and E1 viral proteins in feces. The IgG1 titer increases were to the E2 proteins in tears and BAL fluid and to the E3 protein in BAL fluid.(ABSTRACT TRUNCATED AT 250 WORDS)     

A longitudinal study of bovine coronavirus enteric and respiratory infections in dairy calves in two herds in Ohio

This prospective longitudinal study examined the epidemiology and disease syndrome associated with bovine coronavirus (BCV) infections in a cohort of 8 conventional calves from 0 to 120 days of age, in two dairy herds in Ohio. The periods of respiratory shedding of BCV were determined by direct immunofluorescent (DIF) staining of nasal epithelial cells and ELISA of nasal swab supernatant fluids. The periods of fecal shedding of BCV were determined by ELISA and immunoelectron microscopy (IEM). The isotype-specific antibody titers to BCV in serum (at selected intervals between 0 and 120 days of age) and the post-suckling (24 to 48 h after birth) total immunoglobulin levels were examined by ELISA and zinc sulfate turbidity tests, respectively. Of the 8 calves studied, 4 had evidence of BCV respiratory (by DIF or ELISA) or enteric infections (by IEM or ELISA) in association with diarrhea or rhinitis, even though 7 of 8 calves showed increases in one or more serum antibody isotypes to BCV and 6 of 8 calves showed BCV respiratory or enteric antigen shedding by ELISA. Serological antibody titer increases occurred in 3 calves before 30 days of age and in 4 calves after 30 days of age; two of the latter calves had a second rise in serum antibody titers to BCV after the initial rise. A serological antibody titer increase was not observed in one calf. This suggests that BCV infections may be very common in a closed herd and may occur in older calves, although many may be subclinical and some may be recurrent. There were no statistically significant correlations between total serum immunoglobulin levels or BCV antibody isotype titers in serum (24-48 h after birth) and clinical disease or infection by BCV; however, calves with low levels of IgA BCV antibodies in serum (24-48 h after birth) had a significantly greater average number of days with diarrhea than those calves having high levels of IgA BCV-specific antibodies in serum.     

Fatal winter dysentery with severe anemia in an adult cow

An adult dairy cow fatally affected with winter dysentery was investigated pathologically and virologically. The cow had severe anemia and diarrhea with massive blood. Pathologically, the loss of surface epithelial cells and necrosis of crypt epithelial cells in the large intestine were observed. Bovine coronavirus (BCV) antigen was observed in necrotic crypt epithelial cells of the large intestine. Virus particles were found in the necrotic epithelial cells of the large intestine. Virologically, BCV was isolated from the feces of the dead cow. The dead cow had no serum antibody against BCV although the co-habitants did. These suggest that severe infection of BCV in the cow without the BCV antibody accompanied by severe hemorrhagic anemia resulted in the cow's death.     

猫传染性腹膜炎病毒N蛋白在昆虫细胞-杆状病毒系统中的表达与鉴定

本研究旨在利用昆虫细胞-杆状病毒表达系统表达猫传染性腹膜炎病毒(Feline infectious peritonitis virus,FIPV)N蛋白,并制备抗该蛋白的多克隆抗体,用于FIPV临床抗原/抗体诊断及N蛋白功能研究。参考GenBank中FIPV的N基因序列,选择FIPV毒株N基因(登录号:KC461235.1),并对该N基因进行密码子优化、基因合成,酶切后将N基因连接至pFastBac1载体,转化大肠杆菌DH5α感受态细胞,经氨苄西林抗性筛选阳性克隆,提取质粒经酶切及测序鉴定正确后,转化大肠杆菌DH10Bac感受态细胞,经蓝白斑筛选及PCR鉴定后,最终成功构建重组杆状病毒质粒Bacmid-N,转染Sf9细胞包装杆状病毒,于28℃温箱培养4 d后收集感染Sf9细胞上清,并通过镍离子亲和层析纯化获得重组N蛋白。经SDS-PAGE及Western blotting鉴定结果表明,本研究成功利用昆虫细胞-杆状病毒表达系统表达出大小约为51 ku的FIPV重组N蛋白。将该蛋白与弗氏佐剂按一定比例混合后,免疫6周龄BALB/c小鼠。用间接ELISA方法检测小鼠血清抗体效价可达1∶102400;利用Western blotting和间接免疫荧光试验对N蛋白多克隆抗体进行检测分析,结果表明,真核表达的重组蛋白免疫原性良好,多克隆抗体具有良好的反应原性,可特异识别FIPV感染细胞。本研究为FIPV抗原/抗体诊断试剂盒的研发奠定了基础。     

鸡传染性支气管炎病毒特异性单克隆抗体的制备与鉴定

为获得鸡传染性支气管炎病毒(Avian infectious bronchitis virus, IBV)N蛋白的单克隆抗体,通过原核表达IBV N蛋白,纯化后作为免疫原免疫BALB/c小鼠,并按常规方法制备杂交瘤细胞。经ELISA方法筛选阳性杂交瘤细胞,经过3次亚克隆获得3株杂交瘤细胞株,命名为1#、18#、19#,并进行了抗体亚类的鉴定、Western-blot和IFA检测。结果显示：制备的3株单克隆抗体亚型均为IgG1,Western-blot和IFA试验结果表明单克隆抗体均能与IBV发生特异性反应而与其他禽病常见病毒均无交叉反应。本研究成功制备了IBV单克隆抗体,为进一步建立IBV检测方法和深入研究IBV的生物学特性奠定了基础。     

Reduced likelihood of bovine coronavirus and bovine respiratory syncytial virus infection on organic compared to conventional dairy farms

The prevalence of antibodies to bovine coronavirus (BCV) and bovine respiratory syncytial virus (BRSV) infections was studied in 20 conventional and 20 organic dairy herds. The organic farms had produced 'certified' milk for at least 2 years. On two occasions, with a 1-year interval, 699 serum samples from 624 peri-parturient cows were tested by ELISA for antibodies to BCV and BRSV. Accompanying data relating to the sampled animals were collected in order to identify potential factors associated with increased antibody prevalence. The antibody prevalence was high at both sampling times with approximately 85% and 80% of animals positive for antibodies to BCV and to BRSV, respectively. Conventional herds had a significantly higher mean antibody prevalence to BCV and BRSV than the organically managed herds (P<0.01). Animal age was significantly associated with increased antibody prevalence (P<0.001). The findings of this study suggest that organic farm management may be effective in reducing the seroprevalence of these viruses relative to conventional farming methods.     

猪繁殖与呼吸综合征病毒核衣壳蛋白的原核表达及其单克隆抗体的制备

试验通过RT-PCR法扩增出猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)核衣壳蛋白(N蛋白)基因并克隆到原核表达载体pet-30a中,转入到BL21感受态细胞,经IPTG诱导表达,使用SDS-PAGE和Western blotting分析表达产物的特性。利用Ni柱纯化该重组蛋白,采用NC膜皮下包埋法免疫Balb/C小鼠,取免疫后的脾细胞与SP2/0细胞进行融合后筛选杂交瘤细胞株,检测杂交瘤细胞株的特性并制备单克隆抗体。SDS-PAGE和Western blotting结果表明,该重组蛋白表达正确,约为20 ku,能被抗PRRSV的阳性血清特异性识别。超声后用Ni柱纯化,经SDS-PAGE分析可得单一的目的条带。NC膜皮下包埋法免疫效果良好,通过细胞融合、ELISA筛选获得3株能稳定分泌抗PRRSV N蛋白抗体的杂交瘤细胞株(H7、F7、C8),制备出高特异性的针对N蛋白的H7单抗。IFA与Western blotting结果显示制备的单抗可与病毒N蛋白产生特异性反应。亚型鉴定为IgG2b型,染色体分析证实杂交瘤细胞染色体数目正确。结果成功实现了N蛋白的原核表达,并获得高特异性的单克隆抗体,为PRRSV N蛋白抗原的检测奠定基础。     

猫传染性腹膜炎病毒N蛋白在昆虫细胞-杆状病毒系统中的表达与鉴定

本研究旨在利用昆虫细胞-杆状病毒表达系统表达猫传染性腹膜炎病毒（Feline infectious peritonitis virus,FIPV）N蛋白,并制备抗该蛋白的多克隆抗体,用于FIPV临床抗原/抗体诊断及N蛋白功能研究。参考GenBank中FIPV的N基因序列,选择FIPV毒株N基因（登录号：KC461235.1）,并对该N基因进行密码子优化、基因合成,酶切后将N基因连接至pFastBac1载体,转化大肠杆菌DH5α感受态细胞,经氨苄西林抗性筛选阳性克隆,提取质粒经酶切及测序鉴定正确后,转化大肠杆菌DH10Bac感受态细胞,经蓝白斑筛选及PCR鉴定后,最终成功构建重组杆状病毒质粒Bacmid-N,转染Sf9细胞包装杆状病毒,于28 ℃温箱培养4 d后收集感染Sf9细胞上清,并通过镍离子亲和层析纯化获得重组N蛋白。经SDS-PAGE及Western blotting鉴定结果表明,本研究成功利用昆虫细胞-杆状病毒表达系统表达出大小约为51 ku的FIPV重组N蛋白。将该蛋白与弗氏佐剂按一定比例混合后,免疫6周龄BALB/c小鼠。用间接ELISA方法检测小鼠血清抗体效价可达1：102 400;利用Western blotting和间接免疫荧光试验对N蛋白多克隆抗体进行检测分析,结果表明,真核表达的重组蛋白免疫原性良好,多克隆抗体具有良好的反应原性,可特异识别FIPV感染细胞。本研究为FIPV抗原/抗体诊断试剂盒的研发奠定了基础。