为研究波形蛋白(vimentin)对H9N2亚型禽流感病毒(AIV)复制的影响,构建了真核表达载体FLAG-vimentin,并采用Western blot技术验证FLAG-vimentin重组载体和病毒NP蛋白的表达情况,用荧光定量PCR技术检测病毒HA基因水平.同时,设计合成siRNA,敲低内源性vimentin,检测其对病毒HA基因表达水平的影响.构建原核表达载pCold I-vimentin,蛋白纯化后孵育细胞,采用Western blot、荧光定量PCR技术检测病毒的蛋白表达和基因水平的变化.结果证实,H9N2亚型AIV感染FLAG-vimentin重组载体转染的Hela细胞12h时,病毒NP蛋白表达减少;而在感染24和36h时,病毒NP蛋白表达上升.荧光定量PCR结果亦显示,在AIV感染后12h时,HA基因表达水平明显降低.siRNA敲低Hela细胞内源性vimentin后,H9N2亚型AIV的HA基因表达水平在病毒感染12 h时明显升高,24h时差异较小,36h时有所升高.纯化后的vimentin重组蛋白孵育细胞,在病毒感染36 h时,H9N2亚型AIV的NP蛋白表达显著被抑制.荧光定量PCR结果亦证实病毒HA基因在AIV感染后36 h时表达水平明显降低.上述结果表明,H9N2亚型AIV在人源Hela细胞上的早期复制中,vimentin能起到一定程度上的抑制作用,且其重组表达蛋白孵育细胞能够阻断H9N2亚型AIV对细胞的感染,为深入研究vimentin抗病毒复制的机制提供了重要的试验依据,同时为开展抗H9N2亚型AIV的药物研发提供了参考.

The bursa of Fabricius (BF) is a vital central humoral immune organ unique to birds. The bioactive peptide BP7 derived from bursa is reported to promote the vaccine immune response and antibody production. However, the regulatory effect on antigen presentation and B cell differentiation has been infrequently reported. In this paper, chicken macrophage HD11 cells were used for the cell model, and the cellular molecular expressions were determined by the fluorescent quantitative PCR (qPCR) after BP7 treatment. Then, the miRNA expression profile was analyzed by high-throughput sequencing. In addition, BALB/C mice were used as the animal model to detect B cell subtype with flow cytometry (FCM). The results showed that the expressions of four immune active molecules, IL-1β, IL-6, iNOS, and IFN-α, in HD11 cells were significantly increased with 100 ng/mL BP7 treatment. Compared with the control group, there were 58 up-regulated and 61 down-regulated miRNAs in HD11 cells with BP7 treatment. The gene ontology (GO) function analysis found that BP7 mainly affected the various biological processes, molecular function, and MHC protein complex. Pathway analysis showed that 100 ng/mL BP7 stimulated various physiological metabolic pathways and signal transduction pathways, including the intestinal immune network producing IgA in HD11 cells. Furthermore, it was found that BP7 in vitro stimulated B cell populations, as well as plasma cells in spleen cells from the immunized mice. Additionally, B cell activation subpopulations were increased in mice immunized with the AIV vaccine and BP7. These results proved that BP7 stimulated various differentially expressed genes in chicken macrophage HD11 cells, and induced B cell differentiation in the immunized mice, which suggested that BP7 might participate in the antigen presentation process, thereby promoting the differentiation of B cells. These results provide an important basis for the mechanism of bursal-derived peptide on B cell development, and offer the experimental basis for the development of adjuvants.

CpG oligodeoxynucleotides (CpG ODN) present adjuvant activities for antigen proteins, which can induce humoral and cellular immune responses to antigens. However, the immunomodulatory functions of CpG ODNs with different sequences are very different. In this paper, six CpG ODNs with different sequences were designed based on CpG2007 as a template. Through the screening of CEF cells in vitro, the stimulating activity of CpG ODNs was determined. Then, two selected CpG ODN sequence backbones were modified by substituting the oxygen with sulfur (S-CpG) and verifying the immune activity. Next, to prove the feasibility of S-CpG as an immune potentiator, two immune models with or without white oil adjuvant were prepared in 20-day-old chicken vaccinations. The screening experiment in vitro showed that the inducing roles of CpG ODN 4 and 5 could strongly stimulate various immune-related molecular expressions. Additionally, CpG ODN 4 and 5 with sulfation modification significantly induced various cytokines' expressions. Furthermore, CpG ODN 4 and 5 induced the strongly humoral and cellular immune responses during vaccination, in which white oil, as an adjuvant, could significantly improve the immune effect of CpG ODN. These results provide an important experimental basis for exploring the structural characteristics and vaccine immunity of CpG ODN.

Avian influenza caused by H9N2 subtype avian influenza virus (AIV) poses a great threat to the healthy development of the poultry industry. Vimentin is closely related to intracellular lipid metabolism, which plays an important role during the viral infection process. However, the function of lipid metabolism and vimentin on H9N2 AIV replication is unclear. In this paper, the cholesterol level and 3-hydroxy-3-methylglutaryl coenzyme a reductase (HMGCR) phosphorylation were investigated in vimentin knockout (KO) and human cervical carcinoma cells (HeLa) cell with or without AIV infection. The results showed that compared to the control group without infected with H9N2 subtype AIV, the cholesterol contents were significantly increased, while HMGCR phosphorylation level was reduced in both KO and HeLa cell after virus infection. Furthermore, viral replication was significantly inhibited in the cells treated with the cholesterol inhibitor lovastatin. Compared with the control group, adenylate activated protein kinase (AMPK), a kinase regulating HMGCR enzymatic activity was inhibited in both KO and HeLa cells in the infected virus group, and AMPK phosphorylation levels were significantly lower in KO HeLa cell than that of HeLa cells. Additionally, after MβCD treatment, viral hemagglutinin (HA) gene level was significantly decreased in HeLa cells, while it was significantly increased in KO HeLa cells. In addition, vimentin expression was significantly increased in MβCD-treated HeLa cells with the viral infection and returned to normal levels after exogenous cholesterol to backfill the MβCD-treated cells. Therefore, the disruption of lipid rafts during the binding phase of viral invasion of cells significantly reduced viral infection. These studies indicated that the lipid rafts and cholesterol levels might be critical for H9N2 subtype AIV infection of human-derived cells and that vimentin might play an important role in the regulation of lipids on viral replication, which provided an important antiviral target against influenza virus.

为研究Cbl蛋白(Casitas b-lineage lymphoma)对H9N2亚型禽流感病毒(AIV)复制的影响,采用PCR技术扩增Cbl基因片段并连接到FLAG-N载体中,Western blot技术验证FLAG-Cbl重组载体表达及病毒NP蛋白的表达情况,并采用荧光定量PCR技术检测病毒的血凝素(HA)、核蛋白(NP)和非结构蛋白(NS)基因水平;采用RNAi技术检测Cbl对病毒HA基因表达水平的影响.结果 显示:EcoR Ⅰ、HindⅢ双酶切鉴定及测序分析发现,成功构建真核表达重组载体FLAG-Cbl;Western blot结果证实H9N2亚型AIV感染FLAG-Cbl重组载体转染的A549细胞6h时,病毒NP蛋白表达降低;荧光定量PCR结果证实病毒NP、NS及HA基因在AIV感染FLAG-cbl重组载体转染的A549细胞的6和12 h时表达水平均明显降低;RNAi敲低A549细胞内源性Cbl蛋白后,H9N2亚型AIV的HA基因表达水平在病毒感染12、24和36 h明显升高.结果 表明:Cbl蛋白能在早期抑制H9N2亚型AIV在A549细胞上的复制,为深入研究Cbl蛋白抗病毒复制的机制提供了重要的试验依据,同时为开展抗AIV的药物研发提供了参考.

试验旨在探索法氏囊活性肽BP7调节鸡未成熟B细胞的分子基础.利用BP7刺激禽前B淋巴细胞DT40细胞,采用荧光定量PCR(qPCR)检测IgM的mRNA水平,并采用基因芯片分析基因表达谱及其生物学功能.结果 显示,BP7刺激的DT40细胞产生IgM的mRNA水平明显升高.基因芯片分析发现,BP7处理的DT40细胞中共有1345个差异表达基因.通路分析发现,BP7诱导DT40细胞的差异表达基因涉及17条通路,包括受体互作、信号通路、代谢和蛋白质分解相关通路等.通路网络分析发现,细胞因子-细胞因子受体互作是BP7刺激后DT40细胞相关途径中的关键通路.基因本体论(Go)功能分析发现,BP7刺激DT40细胞中涉及的免疫相关功能主要包括免疫应答、免疫应答信号、Th1型免疫应答、细胞因子的产生和调节及其受体活性等方面.该研究阐述了法氏囊活性肽BP7调节禽未成熟B细胞的分子基础,为进一步研究法氏囊活性肽调控B细胞分化的分子机制提供了新的数据.