A型口蹄疫合成肽疫苗的免疫效力评价

【目的】研制A型口蹄疫(foot-and-mouth disease,FMD)新型合成肽疫苗,为畜牧业减少经济损失。【方法】应用两种含有口蹄疫病毒(foot-and-mouth disease virus,FMDV)AF/72株VP1[131-159]、VP4[20-35]、3A[21-35]和3B[29-42]4个抗原表位的合成肽联合CpG寡聚脱氧核苷酸(5’-TCGCGAACGTTCGCCCGATCGTCGGTA-3’)在豚鼠上进行了免疫效力评价。【结果】2.5μg/只的合成肽364能保护4/5的豚鼠免受FMDV AF/72株的攻击,灭活苗组获得完全保护,其他组的保护效力仅为3/5;保护效力最高的两组的外周血CD4+T淋巴细胞含量高达33.7%和36.6%,而其他组不超过27.7%,PBS组仅为18.1%。【结论】本研究筛选出一组免疫效力较好的口蹄疫A型合成肽疫苗,可以作为候选疫苗进行进一步评价。     

稳定表达鼠源整联蛋白αvβ6的CHO-677细胞系的构建

口蹄疫是由口蹄疫病毒(FMDV)引起的一种高度接触性传染病,主要侵害偶蹄动物。乳鼠常作为一种重要的实验动物模型用于FMDV的研究;整联蛋白αvβ6是FMDV的重要受体之一。为深入研究整联蛋白αvβ6在FMDV感染乳鼠中所发挥的作用,克隆了乳鼠整联蛋白αvβ6的两个亚基,并将其导入中国仓鼠卵巢细胞(Chinese hamster ovary,CHO-677)基因组中,构建了稳定表达乳鼠整联蛋白αv和β6亚基的细胞系CHO-677-mαvβ6,并分别选用两种不同血清型的野生型FMDV毒株Asia1/HN/CHA/06和O/BY/CHA/2010感染细胞系来分析细胞系对FMDV的易感性。首先通过PCR和间接免疫荧光试验证明了细胞系中整联蛋白αvβ6在基因水平成功导入,在蛋白水平成功表达。然后,通过实时荧光定量RT-PCR检测病毒RNA拷贝数,并结合TCID50试验测定了代表毒株在两个细胞上的生长曲线。结果表明,与亲本细胞CHO-677相比,细胞系CHO-677-mαvβ6对FMDV更易感,从αvβ6的功能性上进一步验证了细胞系被成功构建。     

表达O型口蹄疫病毒 VP1基因的重组病毒BHV-1的构建与鉴定

摘要：【目的】为了构建表达口蹄疫病毒（O/China/99）VP1基因的牛疱疹病毒1型,将人工合成的口蹄疫病毒VP1基因插入到巨细胞病毒（CMV）启动子之下构建gE基因缺失转移载体。【方法】利用磷酸钙介导转染法将该转移载体与亲本病毒BHV-1/gE-/LacZ+的基因组DNA共转染牛鼻甲细胞后收获增殖的病毒。通过筛选白色病毒蚀斑,得到重组病毒BHV-1/gE-/VP1。【结果】PCR检测结果表明VP1基因已经插入到了重组病毒BHV-1/gE-的基因组中,间接免疫荧光试验和Western blot证实了BHV-1/gE-/VP1中的VP1基因在感染的细胞中获得了表达。【结论】本研究成功的构建了表达口蹄疫病毒VP1基因的重组病毒BHV-1/gE-/VP1,为研制口蹄疫及其他重要牛传染病的BHV-1病毒载体疫苗奠定了基础。     

颗粒溶素和穿孔素真核共表达及其对A549细胞的影响

目的：克隆人颗粒溶素和穿孔素基因并构建真核表达载体pGNLY-2A-PFP,观察其在人肺癌A549细胞中的表达情况。方法：体外分离培养外周血单个核细胞并抽提总RNA,RT-PCR扩增人颗粒溶素和穿孔素的基因片段,并将其分别插入pMD18-T进行测序,鉴定正确后构建pIRES-GNLY、pIRES-PFP及pGNLY-2A-PFP并转染人肺癌A549细胞,RT-PCR和间接免疫荧光检测目的蛋白表达,流式细胞Annexin V/PI检测转染后细胞凋亡情况。结果：成功获取了人颗粒溶素和穿孔素cDNA,构建了真核表达载体pIRES-GNLY、pIRES-PFP、pGNLY-2A-PFP,转染A549细胞后检测出了目的蛋白的表达,pGNLY-2A-PFP转染组细胞死亡率高于其他对照组（P<0.05）,死亡细胞以凋亡为主。结论：人颗粒溶素和穿孔素基因可以在人肺癌A549细胞中表达,二者共表达能够促进细胞凋亡,这将有助于颗粒溶素和穿孔素在肿瘤治疗中应用的后续研究。     

ECMS对O-Asia Ⅰ型FMD疫苗免疫功能增强的初探

目的证实ECMS增强O-Asia I型FMDV疫苗免疫功能的有效性,选择ECMS使用的有效浓度及诱导抗体的维持时间。方法本文用ELISA法测定了免疫后3、6、14周O型FMDV VP1及AsiaI型FMDV抗体的滴度。结果与结论与疫苗组和阳性组对照,100μgECMS诱导O-Asia I型FMDV疫苗产生两种抗体的效价较高,维持时间较长。相对来讲,ECMS诱导O型FMDV疫苗的抗体含量较高,但下降较快;诱导Asia I型FMDV疫苗的抗体较低,但下降非常缓慢。100μgECMS的效果优于公认的皂树皂甙（QA）佐剂。     

Specific interference between two unrelated internal ribosome entry site elements impairs translation efficiency

Internal ribosome entry site (IRES) elements allow simultaneous synthesis of multiple proteins in eukaryotic cells. Here, two unrelated IRESs that perform efficiently in bicistronic constructs, the picornavirus foot-and-mouth disease virus (FMDV) and the cellular immunoglobulin heavy chain binding protein (BiP) IRES, were used to generate a tricistronic vector. Functional analysis of the tricistronic RNA evidenced that the efficiency of protein synthesis under the control of BiP IRES was lower than that of the FMDV IRES, relative to the efficiency measured in bicistronic vectors. A specific competition between these elements was verified using two separate mono- or bicistronic constructs in vivo and in vitro. In contrast, no interference was detected with the hepatitis C virus (HCV) IRES. The interference effect of FMDV IRES was observed in cis and trans, in support of competition for common transacting factors different than those used in cap- and HCV-dependent initiation.     

Foot-and-mouth disease virus capsid protein VP2 activates the cellular EIF2S1-ATF4 pathway and induces autophagy via HSPB1

Foot-and-mouth disease virus (FMDV) can result in economical destruction of cloven-hoofed animals. FMDV infection has been reported to induce macroautophagy/autophagy; however, the precise molecular mechanisms of autophagy induction and effect of FMDV capsid protein on autophagy remain unknown. In the present study, we report that FMDV infection induced a complete autophagy process in the natural host cells of FMDV, and inhibition of autophagy significantly decreased FMDV production, suggesting that FMDV-induced autophagy facilitates viral replication. We found that the EIF2S1-ATF4 pathway was activated and the AKT-MTOR signaling pathway was inhibited by FMDV infection. We also observed that ultraviolet (UV)-inactivated FMDV can induce autophagy. Importantly, our work provides the first piece of evidence that expression of FMDV capsid protein VP2 can induce autophagy through the EIF2S1-ATF4-AKT-MTOR cascade, and we found that VP2 interacted with HSPB1 (heat shock protein family B [small] member 1) and activated the EIF2S1-ATF4 pathway, resulting in autophagy and enhanced FMDV replication. In addition, we show that VP2 induced autophagy in a variety of mammalian cell lines and decreased aggregates of a model mutant HTT (huntingtin) polyglutamine expansion protein (HTT103Q). Overall, our results demonstrate that FMDV capsid protein VP2 induces autophagy through interaction with HSPB1 and activation of the EIF2S1-ATF4 pathway.     

The quasispecies (extremely heterogeneous) nature of viral RNA genome populations: biological relevance--a review

We review evidence that cloned (or uncloned) populations of most RNA viruses do not consist of a single genome species of defined sequence, but rather of heterogeneous mixtures of related genomes (quasispecies). Due to very high mutation rates, genomes of a quasispecies virus population share a consensus sequence but differ from each other and from the consensus sequence by one, several, or many mutations. Viral genome analyses by sequencing, fingerprinting, cDNA cloning etc. indicate that most viral RNA populations (quasispecies) contain all possible single and double genomic site mutations and varying proportions of triple, quadruple, etc. site mutations. This quasispecies structure of RNA virus populations has many important theoretical and practical implications because mutations at only one or a few sites may alter the phenotype of an RNA virus.