Alloferon and Zanamivir Show Effective Antiviral Activity against Influenza A Virus (H1N1) Infection In Vitro and In Vivo

The use of vaccines is the most effective and reliable method for the prevention of viral infections. However, research on evaluation of effective therapeutic agents for use in treatment after infection is necessary. Zanamivir was administered through inhalation for treatment of pandemic influenza A/H1N1 in 2009. However, the emergence of drug-resistant strains can occur rapidly. Alloferon, an immunomodulatory drug developed as an NK cell activator, exerts antiviral effects against various viruses, particularly influenza viruses. Therefore, alloferon and zanamivir were administered in combination in an effort to improve the antiviral effect of zanamivir by reducing H1N1 resistance. First, we confirmed that administration of combined treatment would result in effective inhibition of viral proliferation in MDCK and A549 cells infected with H1N1. Production of IL-6 and MIP-1α in these cells and the activity of p38 MAPK and c-Jun that are increased by H1N1 were inhibited by combined treatment. Mice were then infected intranasally with H1N1, and examination of the antiviral efficacy of the alloferon/zanamivir combination was performed. The results showed that combined treatment after infection with H1N1 prevented weight loss, increased the survival rate, and improved lung fibrosis. Combined treatment also resulted in reduced infiltration of neutrophils and macrophages into the lungs. Combined treatment effectively inhibited the activity of p38 MAPK and c-Jun in lung tissue, which was increased by infection with H1N1. Therefore, the combination of alloferon/zanamivir effectively prevents the development of H1N1-mediated inflammation in the lungs by inhibiting the production of inflammatory mediators and migration of inflammatory cells into lung tissue.     

国产甲型H1N1流感病毒裂解疫苗的安全性及免疫原性

目的观察国产甲型H1N1流感病毒裂解疫苗的安全性及免疫原性。方法按照随机、对照、盲法的原则,0、21d程序选择老年组、少年组和少儿组各220人,按1︰1比例随机接种15μg、30μg甲型H1N1流感疫苗;成年组330人,按1︰1︰1比例随机接种15μg、30μg甲型H1N1流感疫苗和安慰剂对照(PBS);0、28d程序只选择成年组220人,按1︰1比例随机接种15μg、30μg甲型H1N1流感疫苗。观察各组接种后的总体不良反应率、全身和局部不良反应率以及免疫后HI抗体阳转率、保护率、GMT水平和平均增长倍数。结果1210名观察对象总体不良反应发生率为21.82%,均以1级不良反应为主,未观察到3级及3级以上不良反应、其他异常反应和严重不良事件。30μg剂量组免疫后HI抗体阳转率和保护率与15μg剂量组比较,差异无统计学意义。两接种程序同一剂量组内第1针免疫后HI抗体阳转率、保护率、免疫后GMT及增长倍数各指标比较,差异均无统计学意义。结论国产甲型H1N1流感病毒裂解疫苗具有良好的安全性和免疫原性,按照0、21d程序各接种1针15μg甲型H1N1流感疫苗,即可在12～60岁人群中产生良好的免疫效果。     

甲型H1N1流感疫苗免疫安全性观察

目的观察国产甲型H1N1流感疫苗的免疫安全性。方法对3～65岁的人群接种国产甲型H1N1流感疫苗和接种季节性流感疫苗进行接种副反应观察。结果 78972人接种甲型H1N1流感疫苗者,有91人次局部轻微红肿,另有6人出现发热,1人出现过敏,接种反应率为0.124%。季节性流感疫苗接种24899人次,局部轻微红肿26人次,发热2人次,接种反应率为0.112%,结论国产甲型H1N1流感疫苗副反应少,免疫安全性可靠,群众可放心接种。     

38例甲型H1N1流感患者病毒核酸阴转时限的观察

目的观察甲型H1N1流感患者病毒核酸阴转时限。方法选择2009年9月16～27日我院收治的甲型H1N1流感确诊病例38例,经同一名医生进行咽拭子采集,采用RT-PCR方法检测甲型通用、甲1通用、季节性流感、甲型H1N1亚型4个病毒亚型,以甲型通用、甲1通用、甲型H1N1亚型均阴转作为判定病毒核酸阴转的标准。结果 38例甲型H1N1流感患者病毒核酸阴转的时限最短1 d,最长14 d,平均4.5 d;病毒核酸阴转时间主要集中在第3、4、5天,第5天与第4天相比,甲型通用、甲1通用和甲型H1N1的阴转比例均明显增加(P<0.05);发病36 h内接受抗病毒治疗者,病毒核酸阴转平均时间为4.1 d;37～72 h接受抗病毒治疗者,病毒核酸阴转平均时间为5.2 d;有3例甲型通用、甲1通用、甲型H1N1亚型病毒核酸未同时阴转。结论甲型H1N1流感抗病毒治疗1周,大部分患者病毒核酸阴转,不具有传染性;尽早(36 h内)接受抗病毒治疗,可以缩短病毒核酸阴转时间;甲型通用、甲1通用、甲型H1N1亚型具有较好的一致性,对三者未同时阴转的情况,应注意是否同时合并其他甲型流感病毒亚型感染。     

人甲型流感病毒H1N1亚型NS1蛋白的原核表达及纯化

目的表达并纯化人甲型流感病毒H1N1亚型NS1蛋白。方法用RT-PCR法从人甲型流感病毒株A/PR/8/34(H1N1)中扩增NS1基因,克隆入原核表达载体pTXB1,构建重组原核表达质粒pTXB1/NS1,经酶切鉴定后,转化大肠杆菌BL21(DE3),IPTG诱导表达,SDS-PAGE分析表达形式和表达量。经几丁质柱亲和层析纯化表达蛋白,串联飞行时间质谱仪检测其相对分子质量。结果所构建的重组表达质粒pTXB1/NS1序列完整,插入的基因片段全长690bp。以1.0mmol/LIPTG37℃诱导4h,重组蛋白表达量最高,占菌体总蛋白的50%以上。破菌上清及沉淀中均有目的蛋白表达。纯化的NS1蛋白纯度达95%以上,相对分子质量约为26000。结论已成功表达并纯化了人甲型流感病毒H1N1亚型NS1蛋白,为其进一步的研究奠定了基础。     

Transgenic Chicks Expressing Interferon-Inducible Transmembrane Protein 1 (IFITM1) Restrict Highly Pathogenic H5N1 Influenza Viruses

Mammalian cells utilize a wide spectrum of pathways to antagonize the viral replication. These pathways are typically regulated by antiviral proteins and can be constitutively expressed but also exacerbated by interferon induction. A myriad of interferon-stimulated genes (ISGs) have been identified in mounting broad-spectrum antiviral responses. Members of the interferon-induced transmembrane (IFITM) family of proteins are unique among these ISGs due to their ability to prevent virus entry through the lipid bilayer into the cell. In the current study, we generated transgenic chickens that constitutively and stably expressed chicken IFITM1 (chIFITM1) using the avian sarcoma-leukosis virus (RCAS)-based gene transfer system. The challenged transgenic chicks with clinical dose 10⁴ egg infective dose 50 (EID₅₀) of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 (clade 2.2.1.2) showed 100% protection and significant infection tolerance. Although challenged transgenic chicks displayed 60% protection against challenge with the sub-lethal dose (EID₅₀ 10⁵), the transgenic chicks showed delayed clinical symptoms, reduced virus shedding, and reduced histopathologic alterations compared to non-transgenic challenged control chickens. These finding indicate that the sterile defense against H5N1 HPAIV offered by the stable expression of chIFITM1 is inadequate; however, the clinical outcome can be substantially ameliorated. In conclusion, chIFITM proteins can inhibit influenza virus replication that can infect various host species and could be a crucial barrier against zoonotic infections.     

表达甲型H1N1流感病毒神经氨酸酶的重组腺病毒的构建及其免疫原性

目的构建表达甲型H1N1流感病毒神经氨酸酶(Neuraminidase,NA)的重组腺病毒,并检测其免疫原性。方法从质粒pMD19T-simple-NA中扩增NA基因,克隆至穿梭质粒pShuttleCMV中,经同源重组获得重组腺病毒质粒,转染Ad-293细胞,包装出重组腺病毒Ad-NA,RT-PCR和免疫荧光法检测NA基因在Vero细胞中的转录和表达。CsCl密度梯度离心纯化重组腺病毒,免疫小鼠,ELISA法检测免疫小鼠血清中抗NA抗体滴度。结果重组腺病毒质粒经PacⅠ酶切鉴定表明带有目的基因的穿梭质粒已整合到腺病毒基因组中;NA基因在Vero细胞中成功转录和表达;重组腺病毒可刺激小鼠产生抗NA抗体,初免后4周,抗体水平达最高,为1∶100 000。结论成功构建了表达甲型H1N1流感病毒NA蛋白的重组腺病毒,其可刺激小鼠产生有效的免疫应答,为甲型H1N1流感病毒基因工程疫苗的研发奠定了基础。     

以重配技术制备H1N1流感病毒Vero细胞适应株

目的以重配技术制备H1N1流感病毒Vero细胞适应株,为以Vero细胞为基质生产流感疫苗奠定基础。方法以流感病毒Vero细胞适应株A/Yunnan/1/2005Va(H3N2)为母本株,与WHO推荐的2007～2008年度北半球流感疫苗生产用毒株A/Caledonia/20/99(H1N1)共同感染SPF鸡胚和Vero细胞,用抗A/Yunnan/1/2005Va(H3N2)抗体筛选重配病毒,在Vero细胞连续传12代,采用血凝抑制试验和RT-PCR鉴定病毒型别。将重配病毒经Vero细胞大量培养,重配前的病毒经鸡胚大量培养,分别经甲醛灭活,制备灭活疫苗,免疫ICR小鼠,检测其免疫原性。结果获得了1株Vero细胞适应的高产H1N1流感病毒株,连续传9代后,病毒血凝滴度维持在512,免疫原性与重配前的毒株相比,差异无统计学意义。结论通过流感病毒Vero细胞适应株与流行株的重配和抗体筛选,可以获得H1N1流感病毒Vero细胞适应株。     

H5N1亚型禽流感病毒免疫血清精制工艺的建立

目的建立马抗H5N1亚型禽流感病毒免疫血清的精制工艺。方法以硫酸铵盐析法提取免疫马血浆中的IgG抗体,以8～256μg/mgIgG胃蛋白酶(活性单位1:3000)进行消化,以阳离子交换层析柱纯化F(ab’)2抗体,并参照《中国药典》三部(2005版)要求,对试制的样品进行检定。结果经一步50%硫酸铵和多步33%硫酸铵盐析,获得了较为纯净的IgG抗体。8μg胃蛋白酶用量可完全消化1mgIgG抗体分子,阳离子交换方法分离F(ab’)2抗体纯度可达90%以上,高于常规工艺制备的抗体纯度。以此工艺试制的样品,各项质量指标均符合《中国药典》三部(2005版)质量标准。结论已初步建立了马抗H5N1亚型禽流感病毒免疫血清的精制工艺。     

静注甲型H1N1流感人免疫球蛋白的研制

目的研制高效价静注甲型H1N1流感人免疫球蛋白。方法用甲型H1N1流感疫苗(简称甲流)对固定供血浆者按疫苗说明书免疫后2周开始采集原料血浆,采用血凝抑制法检测原料血浆和制品的甲流抗体效价;采用柱层析法从高效价甲流免疫血浆中提取富含IgM免疫球蛋白的样品,模拟低温乙醇蛋白分离法工艺从小量混合血浆分离IgG样品,测定血浆和提取样品的甲流抗体效价,分析甲流中和抗体效价与IgG、IgM含量的对应关系,判断甲流中和抗体的免疫球蛋白类型;按本公司静脉注射人免疫球蛋白(Intravenous immunoglobulin,IVIG)生产工艺制备静注甲型H1N1流感人免疫球蛋白,并与普通IVIG及相应合并血浆的甲流抗体效价进行比较。结果筛选到甲流抗体效价≥320 HU/ml的合格血浆9 866袋,合格率达33.5%,其中抗体效价≥640 HU/ml的血浆占合格血浆的49%,血浆质量符合《中国药典》三部(2010版)"血液制品生产用人血浆规程"要求;甲流抗体效价与IgG含量呈相应比例关系,而与IgM含量无关,甲流中和抗体的免疫球蛋白类型以IgG为主;制备的甲流人免疫球蛋白制品的甲流中和抗体效价达2 560 HU/ml,为普通IVIG的197倍,其他质量指标符合《中国药典》三部(2010版)"静注人免疫球蛋白制检规程"要求。结论成功研制了静注甲型H1N1流感人免疫球蛋白,在甲流疫情得到有效控制的情况下,仍具有战略储备意义。     

甲型H1N1流感病毒实时荧光PCR诊断试剂盒的制备

目的制备甲型H1N1流感病毒实时荧光PCR诊断试剂盒,并进行验证。方法采用磁珠法从甲型H1N1流感疑似患者咽拭子样品中提取病毒RNA,逆转录合成cDNA。根据NCBI最新公布的大流行甲型H1N1流感病毒(2009)基因序列,设计针对编码基质蛋白M基因的引物和探针,检测甲型流感病毒;设计针对血凝素(HA)基因和神经氨酸酶(NA)基因特异性的引物和探针,检测甲型H1N1流感病毒;同时针对人的RNaseP基因设计用于内部控制的引物和探针。所有探针均为Taqman探针,5'端标记FAM,3'端标记BHQ1。对最佳荧光PCR反应条件进行优化,在此基础上组装成甲型H1N1流感病毒实时荧光PCR诊断试剂盒,对其特异性、灵敏度、精密性和稳定性进行验证。与市售试剂盒的检测结果进行对比,并对63份临床甲型H1N1流感疑似患者咽拭子样品进行检测。结果设计的PCR引物及探针能对甲型H1N1流感病毒进行准确检测,与流感病毒的其他型和亚型无交叉反应;试剂盒的灵敏度为0.004个血凝素单位;试验内变异系数小于2.5%,批间变异系数小于5%;试剂盒放置-20℃保存,稳定性良好;检测20份甲型H1N1流感疑似患者咽拭子样品的结果与市售试剂盒一致;检测63份流感疑似患者咽拭子样品,其中大流行甲型H1N1流感病毒阳性36份,普通甲型流感病毒阳性5份。结论所制备的甲型H1N1流感病毒实时荧光PCR诊断试剂盒具有较高的灵敏度、特异性、精密性和稳定性,可用于目前流行的甲型H1N1流感病毒的快速检测。     

Identification of Important N-Linked Glycosylation Sites in the Hemagglutinin Protein and Their Functional Impact on DC-SIGN Mediated Avian Influenza H5N1 Infection

DC-SIGN, a C-type lectin mainly expressed in dendritic cells (DCs), has been reported to mediate several viral infections. We previously reported that DC-SIGN mediated H5N1 influenza A virus (AIVs) infection, however, the important DC-SIGN interaction with N-glycosylation sites remain unknown. This study aims to identify the optimal DC-SIGN interacting N-glycosylation sites in HA proteins of H5N1-AIVs. Results from NetNGlyc program analyzed the H5 hemagglutinin sequences of isolates during 2004–2020, revealing that seven and two conserved N-glycosylation sites were detected in HA1 and HA2 domain, respectively. A lentivirus pseudotyped A/Vietnam/1203/04 H5N1 envelope (H5N1-PVs) was generated which displayed an abundance of HA5 proteins on the virions via immuno-electron microscope observation. Further, H5N1-PVs or reverse-genetics (H5N1-RG) strains carrying a serial N-glycosylated mutation was generated by site-directed mutagenesis assay. Human recombinant DC-SIGN (rDC-SIGN) coated ELISA showed that H5N1-PVs bound to DC-SIGN, however, mutation on the N27Q, N39Q, and N181Q significantly reduced this binding (p < 0.05). Infectivity and capture assay demonstrated that N27Q and N39Q mutations significantly ameliorated DC-SIGN mediated H5N1 infection. Furthermore, combined mutations (N27Q&N39Q) significantly waned the interaction on either H5N1-PVs or -RG infection in cis and in trans (p < 0.01). This study concludes that N27 and N39 are two essential N-glycosylation contributing to DC-SIGN mediating H5N1 infection.     

人用H5N1禽流感裂解疫苗的制备工艺

目的设计3种工艺,制备不同裂解程度的禽流感疫苗,并观察其免疫效果。方法用禽流感毒种R1203株制备3种禽流感疫苗(全病毒、裂解-1和裂解-2),并分别以不同剂量免疫大鼠和家兔,肌肉接种2针(间隔14d),初免后14d和28d静脉采血,检测动物血清中血凝抑制抗体和中和抗体。结果两种动物在疫苗接种1针后14d,血抑抗体和中和抗体滴度均较低;接种2针后14d,均显著高于1针;裂解-2疫苗接种两种动物后的抗体反应均高于其他两种疫苗,且家兔的中和抗体量-效反应明显。结论裂解-2疫苗的工艺优于其他两种疫苗,其中和抗体能正确反映疫苗的质量。     

The Fight against the Influenza A Virus H1N1: Synthesis,Molecular Modeling,and Biological Evaluation of Benzofurazan Derivatives as Viral RNA Polymerase Inhibitors

The influenza RNA polymerase complex, which consists of the three subunits PA, PB1, and PB2, is a promising target for the development of new antiviral drugs. A large library of benzofurazan compounds was synthesized and assayed against influenza virus A/WSN/33 (H1N1). Most of the new derivatives were found to act by inhibiting the viral RNA polymerase complex through disruption of the complex formed between subunits PA and PB1. Docking studies were also performed to elucidate the binding mode of benzofurazans within the PB1 binding site in PA and to identify amino acids involved in their mechanism of action. The predicted binding pose is fully consistent with the biological data and lays the foundation for the rational development of more effective PA–PB1 inhibitors.     

Combining Molecular Docking and Molecular Dynamics to Predict the Binding Modes of Flavonoid Derivatives with the Neuraminidase of the 2009 H1N1 Influenza A Virus

Control of flavonoid derivatives inhibitors release through the inhibition of neuraminidase has been identified as a potential target for the treatment of H1N1 influenza disease. We have employed molecular dynamics simulation techniques to optimize the 2009 H1N1 influenza neuraminidase X-ray crystal structure. Molecular docking of the compounds revealed the possible binding mode. Our molecular dynamics simulations combined with the solvated interaction energies technique was applied to predict the docking models of the inhibitors in the binding pocket of the H1N1 influenza neuraminidase. In the simulations, the correlation of the predicted and experimental binding free energies of all 20 flavonoid derivatives inhibitors is satisfactory, as indicated by R(2) = 0.75.     

H5N1流感病毒疫苗株(NIBRG-14)在MDCK-G1细胞上的遗传稳定性

目的分析H5N1流感病毒疫苗株(NIBRG-14)在MDCK-G1细胞上的遗传稳定性。方法将H5N1种子毒株在MDCK-G1细胞上传代,收获病毒液作为P2病毒,继续传至P15,每隔5代[即P1(原代)、P5、P10、P15]进行测序。分别以含不同浓度(1、2、4μg/mL)TPCK-trypsin的培养基及不同病毒接种MOI(1、0. 1、0. 01、0. 001、0. 000 1、0. 000 01)在MDCK-G1细胞上培养P1 H5N1病毒,检测血凝滴度,计算CCID50,确定最适MOI及TPCK-trypsin浓度。将P1和P15 H5N1病毒接种鸡胚,收获尿囊液,经Sepharose 4 Fast Flow凝胶柱层析法纯化病毒后,进行电镜观察;采用培养法和指示细胞培养法(DNA染色法)检测支原体。结果 H5N1流感病毒疫苗株(NIBRG-14)在MDCKG1细胞传代过程中血凝滴度增加,从1∶128升至1∶1 024,P1、P5、P10和P15 H5N1种子病毒8条基因序列(PB1、PB2、PA、HA、NP、NA、M、NS)经DNAMAN比对结果一致。H5N1种子病毒株在MDCK-G1细胞传代的最适TPCKtrypsin浓度为4μg/mL,最适MOI为0. 001。两种方法检测P1、P15 H5N1种子病毒株均未被支原体感染。结论H5N1种子病毒株在MDCK细胞中传至P15时,病毒滴度增加但基因序列未发生改变,表明H5N1种子病毒株在MDCK-G1细胞中传代具有一定的遗传稳定性。     

H5N1高致病性人禽流感DNA疫苗表达质粒的构建及其免疫原性

目的构建H5N1高致病性人禽流感DNA疫苗表达质粒,并观察其免疫原性。方法分析H5N1人禽流感病毒HA基因的进化关系,人工合成人禽流感病毒HA基因(包含多数H5N1人禽流感病毒共有序列),构建含细胞毒性T淋巴细胞抗原4(CTLA4)和HA融合基因的真核表达质粒pVAX-CLHA及HA基因的真核表达质粒pVAX-HA,经酶切及测序鉴定正确后,转染293-T细胞,经RT-PCR法检测转染细胞中HA基因mRNA的转录水平,间接免疫荧光法(IFA)检测其表达;分别以质粒pVAX-CLHA及pVAX-HA免疫BALB/c小鼠,测定血清HI抗体效价。结果重组DNA疫苗表达质粒pVAX-CLHA和pVAX-HA经酶切及测序证明构建正确,转染的293-T细胞可检测到目的基因的转录及蛋白的表达;3次免疫后均能诱导BALB/c小鼠产生HI抗体,pVAX-CLHA诱导的HI抗体高于pVAX-HA。结论已成功构建了含H5N1HA及CTLA4融合基因的DNA疫苗表达质粒,其对小鼠具有良好的免疫效果,为H5N1高致病性人禽流感DNA疫苗的开发奠定了基础。     

A novel sequence-based antigenic distance measure for H1N1, with application to vaccine effectiveness and the selection of vaccine strains

H1N1 influenza causes substantial seasonal illness and was the subtype of the 2009 influenza pandemic. Precise measures of antigenic distance between the vaccine and circulating virus strains help researchers design influenza vaccines with high vaccine effectiveness. We here introduce a sequence-based method to predict vaccine effectiveness in humans. Historical epidemiological data show that this sequence-based method is as predictive of vaccine effectiveness as hemagglutination inhibition assay data from ferret animal model studies. Interestingly, the expected vaccine effectiveness is greater against H1N1 than H3N2, suggesting a stronger immune response against H1N1 than H3N2. The evolution rate of hemagglutinin in H1N1 is also shown to be greater than that in H3N2, presumably due to greater immune selection pressure.     

无血清微载体培养Vero细胞和H1N1流感病毒条件的优化

目的优化无血清微载体培养Vero细胞和H1N1流感病毒的条件,为Vero细胞流感疫苗的开发奠定基础。方法在搅拌瓶中采用不同的细胞接种量和不同的微载体浓度培养Vero细胞,制备流感病毒,并检测不同的pH值、TPCK-胰酶含量、病毒接种量及病毒收获时间对流感病毒血凝滴度的影响。结果以(1.0～5.0)×105个/ml的Vero细胞接种至浓度为3mg/ml的无血清微载体中,病毒培养液pH值为7.2～7.4,TPCK-胰酶含量为1.0μg/ml,病毒接种量为1.0MOI,并在感染48h后补加TPCK-胰酶,培养72h后收获上清与细胞内病毒,血凝滴度可达1:512。结论已获得了无血清微载体培养Vero细胞和H1N1流感病毒的适宜条件,为应用生物反应器大规模制备流感病毒奠定了基础。     

H5N1亚型禽流感病毒样颗粒的构建及鉴定

目的构建同时含有禽流感H5N1疫苗株NIBRG-14的血凝素(Hemagglutinin,HA)、神经氨酸酶(Neurami-nidase,NA)及基质蛋白(Matrix protein,M1)3种基因的重组杆状病毒,并研究其表达产物的生物学特性。方法利用RT-PCR法从禽流感H5N1疫苗株NIBRG-14中扩增HA、NA及M1基因片段,并同时亚克隆至杆状病毒穿梭质粒pFastBacDual中,构建重组杆状病毒穿梭质粒,转染Sf9昆虫细胞,包装重组杆状病毒Bacmid-HA-NA-M1a。Westernblot法和血凝试验检测HA、NA及M1蛋白在Sf9细胞中的表达;表达的重组蛋白经超速离心浓缩及蔗糖密度梯度离心纯化后,透射电镜观察病毒样颗粒(Virus-like particles,VLPs)的形态结构,并通过单向免疫扩散(Single radialimmunodiffusion,SRID)试验检测HA的含量。结果已成功构建了重组杆状病毒,该病毒可同时表达HA、NA及M1蛋白,3种蛋白可自行组装成VLPs,并分泌至细胞培养上清中,血凝效价可达1∶64;重组杆状病毒表达的蛋白形成的VLPs与NIBRG-14标准抗血清形成沉淀环,浓缩及纯化后的重组蛋白HA含量分别为36和27μg/ml;透射电镜观察可见直径约100 nm的典型流感VLPs。结论构建的重组杆状病毒可表达VLPs,为禽流感新型疫苗的研制奠定了基础。