AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

Molecular characterization of VP4,VP6,VP7 and NSP4 genes of group B rotavirus strains from outbreaks of gastroenteritis

Objective:To characterize VP4,VP6,VP7 and NSP4 genes of representative GBR strains(NIV- 005625.MV-04622 and NIV-094456) delected as the major eliolngic agenl in the outbreaks of gastroenteritis in western India.Methods:Fecal specimens collected during the outbreaks of gastroenteritis were processed for RNA isolation.RT-PCR using GBR VP4.VP6.VP7 and NSP4 gene specific primers,nucleotide sequencing of the amplicons and phylogenetic analysis of the sequences.Results:Phylogenetic analysis of all of the VP4.VP6.VP7 and NSP4 gene sequences revealed clustering of GBR strains in Indian-Bangladeshi lineage of genotype G2 with 95.8%- 99.4%nucleotide and 97.3%-100.0%amino acid identities.However,all three strains showed the presence of unique amino acid substitutions in the VP4 protein suggesting alteration in the antigenicity of outbreak strains of GBR.The VP8* and VP5* regions of VP4 proteins showed respectively 0.5%-6.3%and 0.2%-1.1%amino acid divergence from human GBR strains of Indian-Bangladeshi lineage.Conclusions:These data confirm the reported variability of VP8* region and suggest the possible role of this region in the perpetuation of GBR infections in the environment.This is the first study to document the phylogenetic relationship of VP4,VP6.VP7 and NSP4 genes of GBR strains detected in the outbreaks of gastroenteritis from India with the CBR strains from other parts of world.

PRRSV Nsp4基因突变株对β2M表达的影响

[目的]本试验旨在探究猪繁殖与呼吸综合征病毒(PRRSV)Nsp4基因是否在抑制细胞β2M表达方面发挥关键性作用,以进一步研究PRRSV对抗原递呈的免疫抑制机制。[方法]建立PRRSV的反向遗传平台并成功拯救出PRRSV野生毒株SH2020。根据之前对Nsp4基因不同结构域的研究以及氨基酸位点功能预测,在其DomainⅡ和DomainⅢ分别筛选出3个和5个不同的氨基酸位点,并在病毒感染性克隆质粒上对这些位点进行不同的氨基酸替换突变,再将构建好的感染性克隆质粒转染Marc-145细胞以拯救各个Nsp4突变PRRSV病毒,将拯救的PRRSV病毒以MOI=0.02感染PAM细胞,并通过RT-qPCR和Western blot验证PRRSV Nsp4基因是否能够在PAM细胞β2M表达方面发挥作用。[结果]成功拯救出PRRSV D2-5(Nsp4-A80L)突变毒株,并且在Marc-145细胞上的生长动力学特性与野生毒株SH2020相似;而Nsp4基因其余位点突变则会导致PRRSV生长受到显著抑制从而无法进行试验。Western blot和RT-qPCR试验结果证实PRRSV D2-5毒株能够显著促进β2M在Marc-145和PAM细胞的表达,而野生毒株SH2020则表现出了对β2M表达的抑制作用。[结论]Nsp4基因在PRRSV抑制细胞β2M表达方面发挥重要作用,并且Nsp4-A80L突变能够显著促进β2M的表达。

PRRSV非结构蛋白NSP4及其3个主要结构域过表达慢病毒载体的构建及鉴定

旨在构建猪繁殖与呼吸综合征病毒(PRRSV)NSP4蛋白及其3个主要结构域的过表达慢病毒载体,包装慢病毒并检测4种慢病毒在人单核细胞白血病细胞THP-1和猪肺泡巨噬细胞PAM中的表达。试验以质粒pXJ41-HA-NSP4为模板,分别扩增NSP4全长及其3个结构域NSP4-DⅠ、NSP4-DⅡ、NSP4-DⅢ片段,连接到pCD513B慢病毒表达载体中,构建重组质粒pCD513B-NSP4、pCD513B-NSP4-DⅠ、pCD513B-NSP4-DⅡ、pCD513B-NSP4-DⅢ;将慢病毒重组质粒与包装质粒pLP1、pLP2、pLP/VSVG共转染HEK-293T细胞,包装获得4种重组慢病毒rLV-NSP4、rLV-NSP4-DⅠ、rLV-NSP4-DⅡ、rLV-NSP4-DⅢ,并感染HEK-293T细胞测定病毒滴度;分别将4种重组慢病毒感染THP-1细胞、PAM细胞,荧光显微镜观察目的蛋白表达,荧光定量PCR(qPCR)检测目的基因转录水平,Western blot检测不同感染时间目的蛋白表达水平。结果:4种慢病毒rLV-NSP4、rLV-NSP4-DⅠ、rLV-NSP4-DⅡ、rLV-NSP4-DⅢ感染HEK-293T细胞的病毒滴度分别为2.2×10^(6)、2.8×10^(6)、2.5×10^(6)和2.5×10^(6) TU/mL;荧光显微镜观察显示4种慢病毒感染THP-1细胞和PAM细胞后阳性细胞数均随时间的增加而增多;qPCR结果表明目的基因转录水平在慢病毒感染后60 h内随着感染时间增加而升高;Western blot表明4种慢病毒能够成功感染THP-1细胞和PAM细胞并稳定表达相应的目的蛋白,且蛋白表达水平随着感染时间增加而升高。综上,本研究成功包装PRRSV NSP4及其3个主要结构域重组过表达慢病毒,为进一步研究PRRSV NSP4及其3个结构域的功能奠定了基础。

PbrARF4 contributes to calyx shedding of fruitlets in ‘Dangshan Suli’ pear by partly regulating the expression of abscission genes

Fruitlet calyx shedding in pear plants is apparently regulated via numerous pathways that involve both environmental triggers and phytohormones cues such as auxin. In this study, we found at 10 days after full bloom (DAFB) higher levels of indoleacetic acid (IAA) and tryptophan (Trp) in calyx persistence fruitlet (CPF) than calyx shedding fruitlet (CSF) ofDanshan Suli’ pear (Pyrus bretschneideri Rhed.). Consisting with this, the activity of indolealdehyde oxidase (IAAIdO), which promotes IAA synthesis, was remarkably increased, and that of peroxidase(POD), which degrades IAA, dropped markedly in CPF but not in CSF. Further, qRT-PCR results revealed that most of 31 PbrARFs (encoding auxin response factors) in Pyrus bretschneideri were highly expressed in CPF, whereas PbrARF4, PbrARF24 and PbrARF26 were significantly downregulated in CPF vis-a-vis CSF. Phylogenetic analysis revealed that 6 PbrARFs clustered in the group III, where PbrARF4 showed the closest affinity with AtARF1 that promotes organ abscission, indicating a putative role of PbrARF4 in mediating the process of calyx shedding in pear. In fact, the ectopic overexpression of PbrARF4 in Solanum lycopersicum resulted in an earlier-formed and deeper abscission layer (AL) in the transgenic plants, whose calyxes were more prone to wilt at the mature red stage (MR) compared with the control plants (wild-type). More importantly, expression levels of the abscission genes SILS and Sl Cel2 in transgenic plants overexpressing PbrARF4 were significantly upregulated in comparation with the WT, whereas those of Sl BI and Sl TAPG2 were considerably inhibited. Further, PbrJOINTLESS and PbrIDA,the two genes related to calyx shedding in pear, were up-regulated more in CSF than CPF. The findings contribute to a better understanding of PbrARFs involved in fruitlet calyx shedding of pear, which could prove beneficial to improving the quality of pear fruit.

Quantitative trait loci identification reveals zinc finger protein CONSTANS-LIKE 4 as the key candidate gene of stigma color in watermelon(Citrullus lanatus)

Stigma color is a critical agronomic trait in watermelon that plays an important role in pollination.However,there are few reports on the regulation of stigma color in watermelon.In this study,a genetic analysis of the F2 population derived from ZXG1553(P1,with orange stigma)and W1-17(P2,with yellow stigma)indicated that stigma color is a quantitative trait and the orange stigma is recessive compared with the yellow stigma.Bulk segregant analysis sequencing(BSA-seq)revealed a 3.75 Mb segment on chromosome 6 that is related to stigma color.Also,a major stable effective QTL Clqsc6.1(QTL stigma color)was detected in two years between cleaved amplified polymorphic sequencing(CAPS)markers Chr06_8338913 and Chr06_9344593 spanning a~1.01 Mb interval that harbors 51 annotated genes.Cla97C06G117020(annotated as zinc finger protein CONSTANS-LIKE 4)was identified as the best candidate gene for the stigma color trait through RNA-seq,quantitative real-time PCR(qRT-PCR),and gene structure alignment analysis among the natural watermelon panel.The expression level of Cla97C06G117020 in the orange stigma accession was lower than in the yellow stigma accessions with a significant difference.A nonsynonymous SNP site of the Cla97C06G117020 coding region that causes amino acid variation was related to the stigma color variation among nine watermelon accessions according to their re-sequencing data.Stigma color formation is often related to carotenoids,and we also found that the expression trend of ClCHYB(annotated asβ-carotene hydroxylase)in the carotenoid metabolic pathway was consistent with Cla97C06G117020,and it was expressed in low amounts in the orange stigma accession.These data indicated that Cla97C06G117020 and ClCHYB may interact to form the stigma color.This study provides a theoretical basis for gene fine mapping and mechanisms for the regulation of stigma color in watermelon.

Cellular Senescence and SENEX Gene on the Peripheral CD4+CD25+ Treg Cells Enhancement in Elderly

Cellular senescence is a signal transduction process which maintained genomic stability and stopped mammalian cell growth. Furthermore, cellular senescence induces a protective response to a variety of DNA damage. However, this process is also associated with apoptosis, upregulated secretion of inflammatory cytokine, and promoted surrounding tissue damage. When cellular senescence accumulates to a certain extent, it triggers geriatric diseases, such as chronic inflammation, immune senescence-associated tumors and incontrollable infections. Cellular senescence gene SENEX, which was cloned in 2004, has been demonstrated to play a unique gatekeeper function in human endothelial cells when stress-induced pre-mature senescence and apoptosis occurr. The phenomenon that CD4+CD25+ Treg cells accumulated in the aged population has been well studied in recent years. Now Treg accumulation related to immune-pathology has attracted more interest. CD4+CD25+ Treg did not decline and age, but accumulated and suppressed immunoreaction. The enhanced Treg number and function may be associated with stress-induced premature senescence-mediated unique cellular senescence protection mechanisms, and SENEX may play a critical role in this process. In this article, we summarize the cellular senescence and SENEX gene in the accumulation and functional activity of CD4+CD25+ Treg in the elderly.

The Influence of Aerial Exposure on Sea Anemones Aulactinia veratra Mucin Genes Expression Using the RNA Sequencing

Mucin genes are the main component of mucus. The sea anemone species, Aulactinia veratra (Phylum Cnidaria) contains different types of mucin genes. In the intertidal zone, A. veratra is found to be exposed to air during the low tide and produces large quantities of mucus as an external covering. The relation between low tide and mucus secretion is still unclear, and what is the role of mucin during arial exposure is not yet investigated. This study hypothesised that the mucin genes in A. veratra would have significantly high expression in response to aerial exposure. Therefore, the aim of current study was to examine and analyses the response of A. veratra mucins in response to an experiment involving three hours of aerial exposure. To achieve this, aim the RNA-sequencing and bioinformatics analyses were used to examine the expression profile of A. veratra mucin genes in response to aerial exposure. The generated results have shown that, Mucin4-like and mucin5B-like were up-regulated in response to the three hours of aerial exposure in A. veratra. This finding shows a significant role of mucin5B-like and mucin4-like genes in response to air stress at low tide. The data generated from this study could be used in conjunction with future mucin gene studies of sea anemones and other cnidarians to compare A. veratra mucin gene expression results across time, and to extend our understanding of mucin stress response in this phylum.

G5型人A组轮状病毒LL36755株的VP4、VP6和NSP4编码基因的分子特征

最近在亚洲首次发现并报道了感染人的G5型人A组轮状病毒LL36755株,为进一步探讨其进化来源,克隆了G5型人A组轮状病毒LL36755株的VP4、VP6、NSP4编码基因,并分析其基因序列的分子特征。结果发现卢龙株LL36755为罕见的G5P[6]型,其VP6的亚群为SGⅡ型,NSP4的基因型为B型。系统进化树分析表明,卢龙株LL36755的VP7、VP4编码基因与猪来源的毒株关系密切,而VP6、NSP4编码基因与人来源的毒株紧密相联系。可以推断新的人腹泻A组轮状病毒LL36755株是猪的VP7,VP4编码基因与人的VP6,NSP4编码基因的自然重组;而且该毒株不是G5的原型,很可能是人类轮状病毒与猪轮状病毒毒株的自然重组后逐步进化而来。

NSP4基因突变的重组牛轮状病毒的拯救及其鉴定

【目的】通过反向遗传技术结合RNAi筛选和蚀斑克隆方法,拯救出毒力减弱的轮状病毒(rotavirus,RV)。【方法】以野生型轮状病毒CHLY基因组RNA为模板,通过重叠PCR方法在NSP4基因93—104 nt引入5个沉默突变核苷酸,并将毒力位点区135aa、136aa和138aa对应的核苷酸进行定向突变,构建了含有突变位点的转录质粒△pT7-NSP4/89/M。将该质粒与携带RNA聚合酶基因的重组质粒pcDNA3.1/T7-RNAP共转染已接种野生型RV病毒的MA104细胞单层,继续培养24 h,获得了携带NSP4变异基因的RV病毒粒子和野生型RV病毒粒子的混合病毒。将获得的混合病毒接种MA104细胞,通过RNA干扰和蚀斑克隆方法逐步筛选纯化以获得拯救RV。【结果】成功拯救出了NSP4基因变异的RV,该病毒在MA104细胞上的病变和致乳鼠腹泻效果较野生型RV明显减弱。【结论】通过反向遗传技术结合RNAi筛选和蚀斑克隆方法成功拯救出毒力减弱的RV;NSP4毒力位点135aa、136aa和138aa的变异对RV毒力改变和腹泻严重程度有影响。

人轮状病毒NSP4基因变异与功能关系的初步研究

在比较我国人A组轮状病毒一般腹泻患者分离株和重症患者分离株非结构蛋白 (NSP) 4cDNA序列时发现 ,两者在可能与致病性有关的区域 (aa131～ 14 6 )内存在着显著的差异。为进一步探讨这种变异是否与毒力改变有关 ,利用杆状病毒表达载体在昆虫细胞Sf9中表达两种毒株的NSP4 ,通过激光扫描共聚焦显微镜初步观察了它对细胞内钙离子浓度的影响。结果表明 :两种来源的NSP4均可使细胞内钙离子浓度明显升高 ,在 4 8h时大致升高3.1～ 3.4倍 ,96h时升高 5 .6～ 5 .8倍 ,但两种毒株之间的差别并不明显。研究证实 ,人轮状病毒NSP4与以往报道的动物轮状病毒NSP4一样 ,可以引起细胞内钙离子增高 ,即可能与病毒的致病性有关。但重症腹泻毒株SZ1NSP4第 131～ 14 6位氨基酸位点出现的变异并未提高其毒力。轮状病毒的毒力改变可能与其它因素有关。

Impact of Copy Number on Transgene Expression In Tobacco

对农杆菌介导法获得的转 β_葡糖醛酸酶 (β_glucuronidase ,GUS)基因 (uidA)烟草 (NicotianatabacumL .)进行GUS表达分析 ,发现部分转基因植株无GUS活性。进一步Southern杂交结果发现 ,GUS基因失活植株的基因组中整合了多个uidA拷贝 ,而GUS活性高的转基因植株多为uidA单拷贝整合 ,表明uidA基因失活与基因多拷贝整合有关。Northern杂交结果显示 ,失活植株无特异uidARNA杂交带 ,而GUS活性高的植株可检测到明显的杂交信号 ,说明多拷贝引起的基因失活发生在RNA水平。

猪繁殖与呼吸综合征病毒NSP4切割IKKα抑制β干扰素产生的研究

IKK复合体由IKKα、IKKβ和IKKγ3个亚基组成。早期研究结果表明催化亚基IKKα在调节β干扰素(IFNβ)产生方面发挥重要作用。猪繁殖与呼吸综合征病毒(PRRSV)感染及其非结构蛋白4(NSP4)表达能够显著抑制宿主的天然免疫反应。本研究结果显示PRRSV NSP4可以与IKKα结合,并能够切割IKKα。而且,丝氨酸蛋白酶活性是PRRSV NSP4切割IKKα所必需的,其中NSP4中的3个蛋白酶活性位点(His^(39)、Asp^(64)和Ser^(118))中任何一个发生突变均无法切割IKKα。进一步研究证明,NSP4通过切割IKKα抑制了NF-κB和IFNβ启动子的活性。本研究结果表明,PRRSV NSP4除了通过切割NEMO和MAVS抑制NF-κB和IFNβ启动子活性以外,也可以通过切割IKKα抑制机体NF-κB的激活和IFNβ的表达。这些研究结果为阐明PRRSV逃逸宿主天然免疫提供了一个新的证据,为深入研究PRRSV的致病机理奠定了基础。

轮状病毒NSP4基因在非复制型腺病毒载体中的表达及免疫活性

目的:研究以非复制型重组腺病毒表达人轮状病毒NSP4基因作为新型轮状病毒基因疫苗的可行性．方法:用AdEasy系统构建表达NSP4蛋白的重组腺病毒,并通过形态学观察、PCR、RT- PCR、Southern blot和Western blot等对重组腺病毒进行鉴定．通过滴鼻途径免疫♀小鼠,对免疫后母鼠生产的乳鼠(4日龄)用SA11株轮状病毒灌胃攻击,观察腹泻的产生情况并进行腹泻评级．结果:获得了重组腺病毒rvAdEasyNSP4． Southern blot表明,在重组腺病毒中确有特异性的NSP4基因整合,Western blot证明,目的蛋白得到表达．初次免疫小鼠后血清IgG抗体的滴度可达1:1 000,但阳转率仅为28．5％;再次免疫后,血清抗体阳转率达到了85．7％;第3次免疫后,血清抗体阳转率达到100%．血清IgA 阳转率可以达到71．4％．实验组乳鼠攻毒后腹泻的百分率和腹泻评分(反应腹泻严重程度) 均较对照组低．结论:腺病毒载体可有效表达轮状病毒非结构蛋白NSP4并可有效诱导小鼠免疫反应,这些结果的取得为新型轮状病毒疫苗的研制提供了依据．

Genetic Polymorphism of TLR4 Gene and Correlation with Mastitis in Cattle

Toll-like receptor 4 （TLR4） recognizes pathogen ligands and mediates signaling to initiate innate and adaptive immune responses. In this experiment, a 316 bp and 382 bp fragments of TLR4 gene named T4CRBR1 and T4CRBR2, of Chinese Holstein, Sanhe cattle, and Chinese Simmental was amplified by polymerase chain reaction （PCR）, respectively. The genetic polymorphisms in the three populations were detected by Single-Strand Conformational Polymorphism （SSCP） in the first locus and by digesting the fragments with restriction endonuclease Alu I in the second one. Results showed that both alleles （A and B） of two loci were found in all the three populations and the value of polymorphism information content （PIC） indicated that these were a moderate polymorphism. Statistical results of X^2 test indicated that two polymorphism sites in the three populations fitted with Hardy-Weinberg equilibrium （P 〉 0.05）. After sequencing, A-G single nucleotide polymorphism （SNP） was identified at nucleotide 4,525 in intron 1 of TLR4 gene and C-T SNP was identified at nucleotide 1,397 in exon 3 of TLR4 gene. Meanwhile, the effect of polymorphism of TLR4 gene on somatic cell score （SCS） was analyzed, the results indicated that the cattle with allele A in T4CRBR1 showed lower somatic cell score than that of allele B （P 〈 0.05）. In short, the allele A might play an important role in mastiffs resistance in bovine.

轮状病毒LLR疫苗株非结构蛋白NSP4基因稳定性研究

目的研究轮状病毒(RV)LLR疫苗株NSP4基因的遗传特征及基因稳定性,为疫苗的质量控制提供依据。方法将LLR株轮状病毒毒种LLR38代在原代牛肾细胞上连续传至49代,提取第38、43、44、49代病毒RNA。通过RT-PCR扩增NSP4基因片段,将其克隆入质粒pGEM-T中,进行序列测定与分析。结果 LLR株NSP4基因全长751 bp,含编码175个氨基酸的单一的开放读码框架(ORF)。各代次病毒的NSP4基因核苷酸与推导的氨基酸序列完全一致,与GenBank中LLR参考株(AY219873)进行比较,核苷酸与推导的氨基酸同源性分别为99.1%和99.7%,各关键功能区均未发生改变。与27株A～F不同基因群RV代表株进行比较,LLR株与A基因群RV的NSP4同源性较高,核苷酸推导的氨基酸同源性为89.2%～95.5%;与B、C、D、E、F基因群RV核苷酸推导的氨基酸同源性分别为83.5%～87.5%、85.2%～87.5%、65.9%～67.8%、27.6%～30.8%、77.8%;系统发生树显示LLR株与A基因群RV在一个分支内。结论轮状病毒LLR疫苗株在原代牛肾细胞上连续传代NSP4基因遗传特性极其稳定,从分子水平证明LLR疫苗株毒种及其生产的疫苗是安全的。

人轮状病毒NSP4蛋白的表达及其致小鼠腹泻作用的初步研究

研究人轮状病毒非结构蛋白NSP4在轮状病毒致病性中的作用。分离得到我国人轮状病毒97SZ8株,以谷胱甘肽S-转移酶融合蛋白的形式在大肠杆菌BL-21中表达NSP4蛋白C端86-175氨基酸并用GlutathioneSepharose^(TM) 4B亲和纯化。将纯化蛋白分别以0.4nmol和1.5nmol的剂量腹腔注射新生Balb/C乳鼠,记录腹泻发生和体重变化情况。当注射0.4nmol GST-NSP4_T重组蛋白时,有1只小鼠发生一过性腹泻(1/6),给予1.5nmol重组蛋白时,实验组所有乳鼠都先后出现了腹泻,存在一定的剂量依赖性。本研究初步在新生小鼠建立了一种人轮状病毒腹泻动物模型,该模型有望在人轮状病毒的致腹泻机理、治疗和预防研究中发挥重要作用。

高致病性PRRSV HuN4株Nsp4的原核表达及其抗体水平检测

为进一步研究高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)Nsp4的免疫学特性,本研究根据PRRSV HuN4株序列,设计并合成用于扩增PRRSV HuN4株Nsp4基因的引物。将扩增产物与原核表达载体pGEX-6P-1连接,转化到大肠杆菌BL21(DE3)中。Western blot试验显示,Nsp4在大肠杆菌中获得较高水平的表达,并且具有良好的反应活性。利用该蛋白建立ELISA方法,检测PRRSV HuN4株感染仔猪的不同时间内的血清,并比较抗Nsp4,囊膜蛋白和M蛋白,N蛋白的抗体水平,结果表明Nsp4抗体升高的时间晚于囊膜蛋白和M蛋白、N蛋白出现的时间,同时抗体水平也低于其他蛋白。

猪繁殖与呼吸综合征病毒NSP4切割天然免疫分子NEMO抑制I型干扰素的产生

猪繁殖与呼吸综合征病毒(PRRSV)侵入宿主细胞后能够强烈抑制宿主天然免疫反应。早期研究表明,PRRSV 4个非结构蛋白NSP1、NSP2、NSP4和NSP11均可以抑制I型干扰素(IFN-I)的产生,但NSP4抑制IFN-I的机理尚无相关报道。本研究显示PRRSV感染猪肺泡巨噬细胞(PAMs)导致宿主蛋白NEMO(NF-κB essential modulator)被切割,产生一条约40 ku的蛋白条带。通过检测PRRSV编码蛋白酶的切割功能,进一步表明其NSP4是切割NEMO的关键蛋白酶。免疫共沉淀试验表明NSP4与NEMO具有特异性相互作用;激光共聚焦试验结果表明两者在细胞浆中共定位。通过双荧光素酶报告基因试验显示,NSP4过表达可以显著抑制仙台病毒(SeV)和NEMO诱导的IFNβ启动子的激活,导致IFNβ表达量显著下降(p<0.01)。本研究结果表明,PRRSV NSP4可以切割宿主蛋白NEMO,从而显著抑制IFNβ启动子的激活,从分子水平解释了PRRSV NSP4抑制IFNβ产生的机理。

猪轮状病毒NSP4蛋白135、136和138位点氨基酸突变对毒力影响的研究

在比较我国猪轮状病毒(PRV)强弱毒株非结构蛋白NSP4 cDNA序列时发现,两者在可能与致病性有关的区域(aa112～aa175)内存在显著差异。为进一步研究这种变异是否与毒力改变有关,本研究从我国流行弱毒株JL94中扩增Nsp4基因并突变其135、136和138氨基酸位点,以谷胱甘肽S-转移酶GST重组蛋白的形式在大肠杆菌Rosseta中表达突变和未突变两种Nsp4蛋白C端编码aa 112～aa 175的截短蛋白并用Glutathione SepharoseTM4B亲和纯化。将纯化的两种蛋白均以40μg的剂量腹腔接种新生BALB/c乳鼠。结果显示,接种GST-Nsp4突变重组蛋白组,有大部分小鼠先后发生腹泻(7/12),接种未突变GST-Nsp4重组蛋白组,只有少部分小鼠发生先后腹泻(3/12),而注射PBS的对照组,均未发生腹泻。本研究证明PRV Nsp4第135位、136位和138位氨基酸位点的变异影响蛋白毒性的改变,其致腹泻活性具有明显差异(p<0.05)。为进一步研究PRV的致腹泻机理和防制及其疫苗的研制奠定基础。