Random cDNA probes to infectious bursal disease virus

Viruses from three commercially available modified-live infectious bursal disease virus vaccines were propagated in tissue culture. Following this, a series of 32P-labeled probes was generated using the entire RNA genome as template for formation of randomly primed cDNAs. These probes were tested against dot blots of the three vaccine strains, as well as the USDA standard challenge strain and one field-origin strain. Dot blots were made of both crude tissue extract and LiCl-precipitated RNA genome. All three probes detected the standard challenge and field strains. Although differences in probe binding could be quantified among the strains, cross-hybridization indicated considerable homology within genomic regions preferentially transcribed under the experimental conditions.     

Baculovirus virions displaying infectious bursal disease virus VP2 protein protect chickens against infectious bursal disease virus infection

Infectious bursal disease (IBD) is an acute and contagious viral infection of young chickens caused by IBD virus (IBDV). The VP2 protein of IBDV is the only antigen for inducing neutralizing antibodies and protective immunity in the natural host. In the current study, we have succeeded in construction of one recombinant baculovirus BacSC-VP2 expressing His6-tagged VP2 with the baculovirus envelope protein gp64 transmembrane domain (TM) and cytoplasmic domain (CTD). The His6-tagged recombinant VP2 was expressed and anchored on the plasma membrane of Sf-9 cells, as examined by western blot and confocal microscopy. Immunogold electron microscopy demonstrated that the VP2 protein of IBDV was successfully displayed on the viral surface. Vaccination of chickens with the VP2-pseudotyped baculovirus vaccine (BacSC-VP2) elicited significantly higher levels of VP2-specific enzyme-linked immunosorbent assay antibodies and neutralizing antibodies than the control groups. IBDV-specific proliferation of lymphocytes was observed in chickens immunized with the recombinant BacSC-VP2. An in vivo challenge study of the recombinant baculovirus BacSC-VP2 showed effective protection against a very virulent (vv) IBDV infection in chickens. In addition, mortality and gross and histopathological findings in the bursa demonstrated the efficacy of the vaccine in reducing virulence of the disease. These results indicate that the recombinant baculovirus BacSC-VP2 can be a potential vaccine against IBDV infections.     

利用杆状病毒表达系统表达鸡传染性法氏囊病病毒VP2蛋白

从GenBank下载传染性法氏囊病病毒(IBDV)VP2基因序列,根据昆虫细胞偏爱密码子对IBDV VP2基因进行密码子优化,化学合成优化后的VP2基因序列,然后构建杆状病毒表达载体pTri Ex-4-VP2,构建成功的重组杆状病毒pTri Ex-4-VP2转染sf9细胞,制备病毒原种,采用SDS-PAGE和Western blot方法鉴定VP2的免疫原性。结果显示:细胞感染Bac-VP2后,其细胞裂解蛋白在58 ku附近有1个条带,且该条带与IBD阳性血清发生特异性反应;间接免疫荧光试验(IFA)结果显示VP2基因能在Sf9细胞中表达;动物攻毒保护试验中,2次免疫重组VP2蛋白后能诱导14日龄SPF鸡产生对IBDV强毒攻击,保护率为60%。本研究为进一步研究IBDV VP2基因工程疫苗提供了物质基础。     

The use of biotin-labeled cDNA probes for the detection of infectious bursal disease viruses

A cDNA library was prepared from the double-stranded RNA genome of the infectious bursal disease virus (IBDV) strain ST-C. The cDNA molecules were annealed into the plasmid pUC9 and used to transform Escherichia coli strain JM107. A cDNA clone that contained IBDV-specific nucleotide sequences was selected and designated STC-1. Radiolabeled probes were prepared from STC-1 and hybridized to genome segment A of ST-C in a northern blot hybridization assay. The STC-1 cDNA was 448 base pairs in length, and its nucleotide sequence indicated that it is located near the VP-2/VP-4 junction in IBDV genome segment A. Biotin-labeled probes were prepared from STC-1 and used in a dot-blot hybridization assay to detect IBDV. Under relatively low stringency conditions of hybridization, the biotinylated probes detected four subtypes of IBDV serotype 1 and a serotype 2 IBDV isolate.     

鸡传染性法氏囊病毒江苏地方株的分离及其VP2基因分析

用SPF鸡胚从江苏溧水某鸡场病死鸡的法氏囊组织中分离到1株法氏囊病毒(IBDV-LS株),该病毒不能凝集鸡的红细胞,琼扩试验证明与IBDV标准阳性血清发生反应,应用RT-PCR扩增IBDV VP2基因,测序后并与GenBank中的IBDV已知序列进行比较。结果表明,分离的IB-DV-LS与国内外超强毒的核苷酸同源性在94.6%以上,推导氨基酸同源性96.6%,并与超强毒株处于进化树同一分支;说明IBDV-LS属IBDV超强毒株,而亲水区内个别氨基酸的替换,提示该毒株已发生一定的变异。     

传染性法氏囊病安徽近期毒株VP2基因在昆虫细胞中的表达

为真核表达传染性法氏囊病病毒(IBDV)VP2基因,将2007年12月从安徽境内发生的传染性法氏囊病(IBD)免疫预防失败的病鸡法氏囊组织中克隆的IBDV VP2基因插入到pFastBac1供体质粒中,转化E.coilDH10Bac感受态细胞,经抗性筛选,获得重组杆状病毒表达质粒rBac-VP2,用脂质体法转染SD细胞.对rBac-VP2感染的Sf9细胞,用间接免疫荧光试验(IFA)检测,具有特异性荧光;用western blot分析,在50 ku处出现1条特异蛋白条带;电镜观察重组VP2蛋白能够自组装成病毒样颗粒.本实验为研制针对近期流行IBDV的新型病毒样颗粒疫苗和新型检测试剂奠定了基础.     

鸡传染性法氏囊病病毒的快速检测

随着 IBDV变异株的出现 ,发病鸡常不产生法氏囊的肉眼病变 ,由于传染性和非传染性因素亦可引起淋巴细胞减少和法氏囊坏死 ,所以 ,采取组织病理学方法诊断 IBDV感染并不完全可靠。由于主动性抗体应答需要一定的时间及大多数雏鸡都带有母源抗体 ,因此血清学早期诊断也不完全可靠。该试验采用本所研究的 IBD快速试纸与经典的琼扩试验 ,在对 IBDV的检测效果上进行了详细的比较 ,介绍如下。1　材料与方法1 .1　 IBD快速检测试纸　由河南省农业科学院生物技术研究所制备提供。1 .2　试验鸡　 1日龄试验鸡 30 0只由河南农业大学试验鸡场提…     

传染性腔上囊病病毒VP3蛋白模拟表位的筛选

为了研究传染性腔上囊病病毒（IBDV）的致病机理及研制有效的IBDV疫苗，利用噬菌体展示技术对IBDV VP3抗原表位进行了筛选。以4株IBDV VP3单克隆抗体HRB-3F、HRB-7B、HRB-7C和HRB-10E作为筛选分子，对噬菌体随机15肽库进行3轮吸附-洗脱-扩增淘洗，从每株单克隆抗体筛选到的噬菌斑中随机挑取20个单克隆噬菌斑，通过间接ELISA和竞争抑制ELISA检测，共选出13个单克隆噬菌斑，经噬菌体gⅢ部分基因的核苷酸序列测定，确定了8个15肽为IBDV抗原表位。这8个15肽在一级结构上没有3个以上连续氨基酸与IBDV GX（Gen—Bank登录号：AY444873）VP3的氨基酸序列相同，但二级结构上均以β折叠为主，并且与单抗的结合可被VP3蛋白有效地抑制。证实，筛选的是IBDV VP3的模拟表位。     

Serological survey of infectious bursal disease virus serotypes 1 and 2 in California turkeys

The prevalence of two infectious bursal disease (IBD) viruses--serotype 1, of chicken origin, and serotype 2, of turkey origin--was studied in California turkeys. Serum samples were collected from 15 turkey flocks representing nine counties. The virus-neutralization test was used. Overall, only 15% of 342 samples were positive for serotype 1, whereas 89% were positive for serotype 2. However, 12 out of 15 flocks had a least one sample positive for serotype 1, and 14 out of 15 flocks had at least one sample positive for serotype 2. Flocks with antibodies to serotype 1 had low geometric mean titers (GMTs) (1.5 to 8.8) and low-to-medium prevalence rates (5 to 53.3%). Flocks with antibodies to serotype 2 had high GMTs (36.8 to 2048) and high prevalence rates (60 to 100%). Results of this study lead us to question the efficacy of IBD vaccination in turkeys.     

传染性法氏囊病超强病毒株VP2基因原核表达及其免疫原性检测

为检测传染性法氏囊病超强毒株(vvIBDV)重组VP2蛋白的免疫原性,本研究利用RT-PCR方法扩增vvIBDV的结构蛋白VP2基因,并将其克隆到表达载体pGEX-4T-3中,构建重组表达质粒pGEX-VP2。将其转化受体菌E.coli BL21(DE3)plysS,经IPTG诱导后,SDS-PAGE电泳和western blot分析表明,表达的重组蛋白约69 ku,并以包涵体形式存在。表达的重组蛋白经纯化后,免疫6周龄BALB/c小鼠制备免疫血清,ELISA分析表明制备的血清效价在1∶5 120以上,表明vvIBDV VP2具有良好的免疫原性,为建立vvIBDV的ELISA检测方法提供了试验依据。     

鸡传染性腔上囊病病毒VP2基因在昆虫细胞中的表达

为了深入研究鸡传染性腔上囊病病毒（IBDV）VP2基因的结构和功能,利用Bac-to-Bac系统研制出含有VP2基因的重组杆状病毒rBac-VP2,将rBac-VP2感染Sf9细胞,并用抗IBDV VP2特异性单克隆抗体经间接免疫荧光试验检测,证实感染重组病毒Sf9细胞能高效表达IBDV VP2基因产物;Western-blotting分析结果表明,VP2基因表达产物的分子质量约为40 ku.     

鸡传染性法氏囊病病毒VP2蛋白可溶性原核表达及免疫原性分析

为在大肠杆菌中表达出高可溶性的VP2-LS3蛋白笼纳米颗粒并检测其免疫原性,将扩增的7种融合标签(GST、NusA、MBP、PpiB、γ-crystallin、ArsC和Grifin)片段连在VP2-LS3基因的N端,构建7个带有不同标签的重组原核表达载体。再将其分别转化至大肠杆菌BL21(DE3),用IPTG诱导表达,表达产物经SDS-PAGE电泳分析,筛选出显著促进VP2-LS3蛋白可溶性表达的融合标签,并进行大量诱导表达、纯化。纯化得到的VP2-LS3重组蛋白分别进行TEV酶酶切、电镜观察分析及免疫家兔,并对获得的兔抗VP2血清进行Western blot和间接ELISA分析。结果显示,与其他6个标签相比,MBP标签促进VP2-LS3蛋白的可溶性表达具有显著性,可溶性表达水平达到69.5%;TEV酶可以把His6-MBP标签蛋白与VP2-LS3蛋白分开,获得高纯度的VP2-LS3蛋白;镜检显示形成了LS3蛋白笼纳米颗粒;Western blot结果表明,表达的VP2-LS3重组蛋白可与兔抗血清发生特异性反应,而不与阴性对照血清发生反应;间接ELISA检测制备的兔抗VP2血清效价达到1∶12 800。本研究获得了高可溶性的VP2-LS3蛋白笼纳米颗粒,为传染性法氏囊病病毒(IBDV)基因工程疫苗的研发奠定基础。     

Failure of maternally derived infectious bursal disease antibodies to serotypes 1 and 2 to protect against heterologous virus

Chicks with maternally derived infectious bursal disease antibodies to either serotype 1 or 2 were challenged at intervals between day-old and 7 weeks of age using the homologous and the heterologous viruses. With both serotypes good protection was demonstrated against the homologous challenge, but not against the heterologous virus.     

Double-antibody sandwich ELISA for detection of infectious bursal disease virus.

A double-antibody sandwich ELISA was employed for detection of IBD virus in bursal suspension. Fifty IBD-free white leghorn chickens aged 5 weeks were experimentally infected with IBD virus. Bursae were collected 4, 8, 12, 24, 36 and 48 hours and 3, 4 and 5 days post-infection. An equal number of chickens acted as appropriate controls. The colour difference between a positive and a negative reaction was clearly distinguished with the naked eye. The cut-off level between ELISA negative and ELISA positive absorbance values was estimated at mean absorbance of negative controls plus three times the standard deviation.     

传染性法氏囊病病毒JS株VP2基因真核表达载体的构建及其应用

应用RT-PCR方法从鸡传染性法氏囊病病毒(IBDV)JS株中扩增出VP2基因,并克隆入T-easy载体。序列测定分析结果表明IBDVJS株的VP2基因与国际标准强毒株的核苷酸序列同源性达98%,氨基酸序列同源性达99%以上。随后将VP2基因克隆入真核表达载体pcDNA3.1/zeo( ),构建成功真核表达质粒pcD-VP2,pcD-VP2体外转染COS-1细胞,能在COS-1细胞中表达。利用pcD-VP2质粒进行动物实验,结果表明雏鸡免疫14d后在体内可检测到特异性抗体,pcD-VP2的真核表达质粒二次免疫诱导鸡产生对IBDV强毒攻击的保护率为67%,这一结果提示VP2基因具有重要开发应用价值。     

鸡GM-CSF基因对传染性法氏囊病病毒VP2 DNA疫苗的免疫调节作用

为了探讨鸡粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因对鸡传染性法氏囊病病毒(IBDV)VP2 DNA疫苗诱导的免疫反应的影响,将pTriEx-4-VP2-GM-CSF质粒免疫SPF鸡,加强免疫2周后,进行IBDV强毒攻击,检测机体免疫相关指标。结果表明:鸡GM-CSF基因能够增强VP2 DNA疫苗诱导的特异性免疫反应,影响细胞因子TNF-α、IFN-γ、IL-2、IL-4、IL-5、IL-6、IL-10的表达水平,对外周血淋巴细胞的增殖没有显著影响。这一结果提示鸡GM-CSF基因对IBDV VP2 DNA疫苗具有一定的免疫调节作用。     

鸡传染性法氏囊病病毒vp2基因在杆状病毒表达系统中的表达及免疫原性研究

将鸡传染性法氏囊病病毒超强毒Gx株vp2基因克隆到载体pFastBac HTA中,构建重组转座载体pFVP2,然后将其转化DH10Bac感受态大肠杆菌,将vp2基因整合到Bacmid穿梭载体中,获得重组穿梭载体BacmidVP2;通过脂质体转染将其转染Sf9昆虫细胞,获得重组杆状病毒rBacVP2。用Western blot和间接免疫荧光试验分析表明IBDV VP2蛋白在Sf9昆虫细胞获得正确表达,所表达的重组VP2蛋白分子量约50 Ku。以rBacVP2感染Sf9细胞裂解物免疫3周龄SPF鸡,在免疫后7 d可检测到ELISA抗体;免疫后14 d可检测到琼脂免疫扩散抗体。攻毒试验表明,初次免疫后14 d对IBDV超强毒株的攻击保护率为75%,2次免疫后14 d对其的攻击保护率为100%。