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.     

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%。     

IBDV H1株VP2基因的原核表达及其产物的复性

应用RT-PCR技术从传染性腔上囊病病鸡总RNA中克隆出1461bp的VP2基因，将其克隆于pET-28a载体，筛选并构建了pVP2表达载体。经IPTG诱导，在其宿主菌BL21（DE3）中成功表达了53．7ku的蛋白，SDS-PAGE和Western-blotting分析结果显示，VP2表达产物以包涵体形式存在，可与鸡IBDV抗血清及1株IBDV单抗发生特异性反应。将VP2表达产物进行纯化和复性，用复性前后的蛋白分别与特异性多抗进行Dot-ELISA检测，结果，复性后蛋白的反应活性比复性前增强了10倍；用复性后的蛋白与IBDV单抗进行Dot-ELISA，结果显示，VP2蛋白与单抗1H4、1E12、583、EA6、3c7反应强（＋＋＋）；与4E4、1H11反应中度（＋＋）；与4E5、3C4、1E11、3H9反应较弱；而与EC6、3D12、1A1无反应。     

Evaluation of modified vaccinia virus Ankara expressing VP2 protein of infectious bursal disease virus as an immunogen in chickens

A recombinant modified vaccinia Ankara (MVA) virus expressing mature viral protein 2 (VP2) of the infectious bursal disease virus (IBDV) was constructed to develop MVA-based vaccines for poultry. We demonstrated that this recombinant virus was able to induce a specific immune response by observing the production of anti-IBDV-seroneutralizing antibodies in specific pathogen-free chickens. Besides, as the epitopes of VP2 responsible to induce IBDV-neutralizing antibodies are discontinuous, our results suggest that VP2 protein expressed from MVA-VP2 maintained the correct conformational structure. To our knowledge, this is the first report on the usefulness of MVA-based vectors for developing recombinant vaccines for poultry.     

Protective immune responses of recombinant VP2 subunit antigen of infectious bursal disease virus in chickens

Infectious bursal disease virus (IBDV) is the causative agent of Gumboro disease and poses a huge threat to poultry industry. The risks associated with conventional attenuated viral vaccines make it indispensable to probe into the development of novel and rationally designed subunit vaccines which are safer as well as effective. VP2 is the major host-protective antigen found in IBDV capsid. It encompasses different independent epitopes responsible for the induction of neutralizing antibody. Here, we report the efficacy of the immunodominant fragment of VP2 which induces both humoral and cellular immunity against infectious bursal disease. A 366bp fragment (52-417bp) of the VP2 gene from an IBDV field isolate was amplified and expressed in Escherichia coli as a 21kDa recombinant protein. The efficacy of rVP2(52-417) antigen was compared with two commercial IBDV whole virus vaccine strains. The rVP2(52-417) induced significantly high antibody titres in chicken compared to commercial vaccines and the anti-rVP2(52-417) sera showed reactivity with viral antigens from both commercial strains (P<0.0001) and field isolates. Also, the chicken splenocytes from rVP2(52-417) immunized group showed a significantly high proliferation (P<0.01) compared to other groups, which implies that the rVP2(52-417) fragment contains immunogenic epitopes capable of eliciting both B and T cell responses. Further, rVP2(52-417) conferred 100% protection against vIBDV challenge in the immunized chickens which was significantly higher (P<0.001) compared to 55-60% protection by commercial vaccine strains. Hence, the study confirms the efficacy of the immunodominant VP2 fragment that could be used as a potent vaccine against IBDV infection in chicken.     

Adjuvant activity of chicken interleukin-12 co-administered with infectious bursal disease virus recombinant VP2 antigen in chickens

A recombinant fowlpox virus (rFPV/VP2) expressing infectious bursal diseases virus (IBDV) VP2 gene has been constructed. After purification and identification of rFPV/VP2, the adjuvant activity of the recombinant chicken IL-12 (rchIL-12), synthesized by our previous construct of rFPV/chIL-12, in rFPV/VP2-expressed rVP2 antigen was assessed in one-week-old specific-pathogen free chickens. The results indicated that rchIL-12 alone or rchIL-12 plus mineral oil (MO) co-administered with rVP2 antigen significantly enhanced the production of serum neutralization (SN) antibody against IBDV, compared to those with MO alone. The SN titers in groups receiving rVP2 antigen with MO alone were more inconsistent after vaccination. On the other hand, rchIL-12 significantly stimulated IFN-γ production in serum and in splenocyte cultured supernatant, suggesting that rchIL-12 alone or plus MO significantly induced a cell-mediated immune response. Finally, bursal lesion protection from very virulent IBDV (vvIBDV) challenge in chickens receiving rVP2 antigen with rchIL-12 alone or plus MO was much more effective than that with MO alone at two weeks after boosting. Taken together, rchIL-12 alone augmented in vivo the induction of a primary and also a secondary SN antibody production and a cell-mediated immunity against IBDV rVP2 antigen, which conferred the enhancement of bursal lesion protective efficacy from vvIBDV challenge. These data indicated that a potential for chIL-12 as immunoadjuvant for chicken vaccine development such as IBDV rVP2 antigen.     

Efficacy of VP2 protein expressed in E. coli for protection against highly virulent infectious bursal disease virus

The ability of a heat-inactivated whole virus from a highly virulent infectious bursal disease virus (hvIBDV) and VP2 protein from hvIBDV expressed in E. coli provided protection against a hvIBDV challenge in specific-pathogen-free (SPF) chickens. Six out of seven chickens that were injected three times with crude VP2 protein developed significant antibody titer against IBDV. However, only four out of the seven chickens survived the hvIBDV challenge. Despite showing low antibody titer profiles, all chickens immunized with the heat-inactivated whole virus also survived the challenged with hvIBDV. However, all of these chickens had bursal atrophy and mild to moderate depletion of lymphocytes. Thus, antibodies raised against IBDV VP2 protein expressed in E. coli and denatured IBDV proteins induced some degree of protection against mortality but not against bursal damage following challenge with hvIBDV.     

传染性法氏囊病病毒株VP2基因在重组腺病毒中的表达

用RT-PCR方法从传染性法氏囊病(IBD)免疫预防失败的病鸡法氏囊组织AH1与AH2中扩增传染性法氏囊病病毒(IBDV)VP2基因。序列分析结果显示,AH1与AH2病毒VP2基因长度均为1350nt,编码450aa,核苷酸和氨基酸序列的同源性分别为98.2%、99.3%,七肽基序均为SWSASGS,在222、253、256、279、284、294和299位上的氨基酸残基分别是A、Q、I、D、A、I和S,具有IBDV强毒的分子特征。进一步将VP2基因克隆入人5型腺病毒穿梭载体(pShuttle-CMV),与腺病毒骨架载体(pAdEasyTM)共转化大肠杆菌BJ5183进行同源重组并转染HEK-293A细胞,经多次亚克隆获得了重组腺病毒rAd-(IBDV)VP2。利用Western-blot、IFA等方法检测IBDVVP2蛋白的体外表达情况,结果证明VP2基因在腺病毒中获得了表达。     

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。     

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。     

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.     

Lysis of myelocytes in chickens infected with infectious bursal disease virus

In specific-pathogen-free chickens infected with the highly virulent HPS-2 strain or virulent reference GBF-1 strain of infectious bursal disease virus (IBDV), pathologic changes of the bone marrow were investigated. On histologic examination, bone marrow lesions were prominent in the HPS-2 group but only mild in the GBF-1 group. The bone marrow of the HPS-2 group showed severe lysis and depletion of heterophil myelocytes with pyknotic nuclear alteration 2-3 days after inoculation. On examination with an electron microscope, heterophil myelocytes were characterized by shrinkage of the cytoplasm and peripheral condensation of nuclear chromatin. IBDV particles were not detected in altered myelocytes. A terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling method demonstrated a positive reaction in only heterophil myelocytes. In contrast, nucleosomal DNA fragmentation in HPS-2-infected bone marrow cells was indiscernible by agarose gel electrophoresis. These findings indicate that lysis of bone marrow cells is selectively induced in heterophil myelocytes at an early stage after IBDV infection and independent of virus replication.     

鸡传染性法氏囊病的免疫接种对策

鸡传染性法氏囊(简称IBD)是一种高度接触性传染性且对生产危害极大的免疫抑制性疾病.3～6周龄的雏鸡最易感染,且发病迅速,病情较重.另外,由于法氏囊发炎,使其免疫功能受到破坏,因而雏鸡经接种MD、ND等疫苗后不能产生理想的免疫反应,甚至免疫失败.据统计,IBD发病率一般为80%～95%,高的可达100%,死亡率为20%～35%.有的高达50%,呈尖峰式死亡曲线.     

IBDV血清Ⅱ型VP5蛋白原核表达及型特异性单克隆抗体的制备

本实验构建表达了传染性法氏囊病病毒(IBDV)血清Ⅱ型23/82株VP5蛋白,并制备出能够区分不同血清型IBDV的单克隆抗体(mAb).利用EcoR I和Xho I双酶切pUC57-23/82VP5质粒,获得23/82株IBDV VP5基因片段,连接到同样处理的原核表达质粒pET-28a,经酶切鉴定获得重组质粒pET-23/82VP5,转化到宿主茵BL21(DE3),在IPTG诱导下表达融合蛋白,SDS-PAGE分析表明该融合蛋白分子量大小约为23 ku.Ni-NTA柱纯化重组蛋白,复性后免疫8周龄BALB/c小鼠,3次免疫后,常规方法进行细胞融合,获得1株阳性杂交瘤细胞株(5D3),IFA和western blot分析表明该细胞株分泌的mAb仅与血清II型IBDV 23/82株VP5反应,不与血清I型IBDV Gt株VP5反应.腹水抗体间接ELISA效价为1×104.该mAb的制备为研究血清II型IBDV的VP5的功能和标记疫苗的研制奠定了基础.     

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。     

鸡传染性法氏囊病病毒VP3蛋白的原核表达及其B细胞抗原表位鉴定

为鉴定鸡传染性法氏囊病病毒(IBDV)VP3蛋白中的B细胞抗原表位,本研究将IBDV的VP3基因亚克隆于pET-28a中,构建了表达重组质粒pETVP3,经IPTG诱导在E.coli BL21(DE3)中表达了重组蛋白(rVP3).Western blot鉴定表明,rVP3能被IBDV抗血清特异性识别.同时,根据IBDV VP3的氨基酸序列,合成覆盖VP3全序列的重叠多肽,并与载体蛋白BSA藕联制备多肽人工结合抗原.Peptide-ELISA和Dot-ELISA检测结果表明VP3中有2个线性表位可以被已制备的单克隆抗体(MAb)识别,即~(728)PRDWDRLPYLNL~(739)和~(982)PKPKPKPNAPTQ~(993);Dot-ELISA结果显示,在VP3中还存在另外4个线性多克隆抗体识别位点:~(818)SLANAPQAGSKSQRA~(831),~(851)QREKD TIUSKKMETMGIYFATP~(872),~(876)ALNGHRGPSPGQLKYWQNTREI~(897)和~(961)QMKDLLLTAMEMK~(973).这些抗原表位的鉴定为开发IBD表位疫苗奠定了基础.     

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.