草鱼出血病的免疫学研究进展

由于草鱼病毒性出血病发病率高,且尚未找到有效防治的药物,至今一直成为草鱼养殖发展中"瓶颈"问题的根本原因。因此,采取免疫学途径防治草鱼出血病是解决此问题的关键之所在。综述了采用草鱼出血病疫苗、干扰素及免疫增强剂进行草鱼出血病防治的研究现状;提出口服型免疫增强剂是生产上防治草鱼出血病最为有效的途径之一。     

猪α-干扰素的原核表达

重组质粒pMDIFN-α中的猪α-干扰素基因经双酶切,亚克隆到原核表达载体pET-．28a,构建了重组表达质粒pETIFN-α,经过SDS-PAGE蛋白质电泳,测得表达蛋白的相对分子质量约为26500．表达蛋白的Western-blotting分析显示：改进后的半干胶转印免疫印迹法灵敏度高、特异性强、重复性好;获得的重组融合蛋白具有良好的反应原性．     

草鱼干扰素的分离纯化及某些理化和生物学特性

通过ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ阴离子交换层析、Ｓｅｐｈａｃｒｙｌ Ｓ－２００凝胶层析、高效液相层析和梯度聚丙烯酰张胶电泳等技术,对病毒诱生的草鱼血清干扰素进行了分离纯化。纯化的草鱼干扰素在ＳＤＳ－聚丙烯酰胺凝胶电泳中呈现单一组份,分子量为３８ｋＤ,等电点为５．２５,过碘酸－Ｓｃｈｉｆｆ试剂反应表现为典型的糖蛋白染色特征。理化和生物学特性研究表明,草鱼干扰素具有１０００００ｇ（２ｈ）离心不沉降、耐     

程序性细胞死亡因子10抑制Ⅰ型干扰素表达并促进FMDV复制

旨在探究宿主蛋白程序性细胞死亡因子10(programmed cell death factor 10,PDCD10)通过抑制Ⅰ型干扰素表达进而促进口蹄疫病毒(foot-and-mouth disease virus,FMDV)的复制.首先,本研究验证了过表达和沉默PD-CD10对FMDV复制的影响,接着利用双荧光素酶...     

鸡IFN-γ重组鸡痘病毒对鸡传染性腔上囊炎MB43弱毒疫苗免疫鸡免疫应答的影响

将重组鸡痘病毒（rFPV—IFN-γ）与IBDMB43弱毒疫苗联合接种SPF鸡，研究重组鸡IFN-γ对IBD疫苗免疫效果的影响。结果显示，rFPV—IFN-γ和MB43弱毒疫苗联合免疫组（MB43＋rFPV-IFN-）＂）及rlFN-γ和MB43弱毒疫苗联合免疫组（MB43＋rIFN-γ）在免疫后第2周即可检出ELISA抗体，比MB43疫苗单独免疫组早1周。用IBDVGx株攻击后，MB43＋rFPV-IFN-γ组和MB43＋rIFN-γ组免疫鸡的脾只有个别淋巴细胞核浓缩、核崩解，少数滤泡萎缩变性坏死；胸腺损伤程度轻于MB43疫苗单独免疫组和非免疫对照组。免疫后1周，3个免疫组外周血CD8^＋T淋巴细胞含量均显著高于非免疫对照组，CD4^＋T淋巴细胞含量变化不明显；攻毒后第2周，3个疫苗免疫组CD4^＋、CD8^＋T淋巴细胞含量均显著升高，各组之间CD4^＋、CD8^＋T淋巴细胞含量无明显差异。证实，rFPV—IFN-γ和rIFN-γ可加强疫苗的细胞免疫和体液免疫应答。     

Kinetics of Interferon-Gamma Production and its Comparison with Anti-Listeriolysin O Detection in Experimental Bovine Listeriosis

The kinetics of the production of interferon gamma (IFN-) in whole blood culture and its comparison with anti-listeriolysin O (ALLO) detection by ELISA were studied during oral infection of calves with Listeria monocytogenes. Culture filtrate antigen (CFA), listeriolysin O (LLO), and sonicated antigen (SA) were used to prime the peripheral blood mononuclear cells (PBMCs) and the plasma from orally infected calves. IFN- and ALLO appeared as early as day 7 of an oral infection. IFN- was detected earlier with LLO than with SA. The Max50 interleukin (IL-2) activity and IFN- estimated in the culture supernatant from PBMCs primed in vitro with different antigens of L. monocytogenes revealed high induction of IL-2 and IFN- by CFA, LLO and live antigen. IFN- assay and ALLO detection were used for testing cases of repeat breeding in dairy cattle. It appeared that detection of IFN- employing LLO can be used to diagnose listerial infections.     

PK-15细胞的ISG15基因敲除促进PRV的复制

本试验旨在研究干扰素刺激基因15（interferon-stimulated gene 15,ISG15）敲除对猪伪狂犬病病毒（PRV）复制的影响。通过CRISPR/Cas9技术构建猪ISG15基因敲除猪肾上皮（PK-15）细胞系,利用CCK-8试剂盒检测PK-15敲除ISG15基因对细胞活力的影响,采用间接免疫荧光技术检测PK-15以及PK15-ISG15^-/-细胞感染PRV的增殖差异,通过RT-qPCR检测PRV-EP0、PRV-gE、PRV-VP16和IFN-β的转录水平,Western blot检测PRV-gE和ISG15的蛋白表达水平,以及通过病毒噬斑检测对子代病毒感染力的影响。结果表明,sgRNA1和sgRNA2均成功敲除ISG15基因;CCK-8试剂盒检测细胞活力结果表明,敲除ISG15基因对PK-15细胞活力无影响;间接免疫荧光检测结果表明,PRV感染后,PK15-ISG15^-/-细胞中的荧光强度明显高于PK-15细胞;RT-qPCR和Western blot结果表明,敲除ISG15可以促进PRV的转录和蛋白表达;病毒噬斑试验进一步显示,敲除ISG15可以促进PRV的复制。另外,RT-qPCR结果显示,敲除ISG15可以抑制PRV感染引起的IFN-β转录上调。本研究成功构建了PK15-ISG15^-/-细胞系,并通过PRV感染试验证实ISG15基因可以抑制PRV在PK-15细胞中的增殖,并推测这种抑制作用可能与IFN通路有关。     

Genetic characterization of bovine viral diarrhea virus strains in Beijing,China and innate immune responses of peripheral blood mononuclear cells in persistently infected dairy cattle

To acquire epidemiological data on the bovine viral diarrhea virus (BVDV) and identify cattle persistently infected (PI) with this virus, 4,327 samples from Holstein dairy cows were screened over a four-year period in Beijing, China. Eighteen BVD viruses were isolated, 12 from PI cattle. Based on genetic analysis of their 5''-untranslated region (5''-UTR), the 18 isolates were assigned to subgenotype BVDV-1m, 1a, 1d, 1q, and 1b. To investigate the innate immune responses in the peripheral-blood mononuclear cells of PI cattle, the expression of Toll-like receptors (TLRs), RIG-I-like receptors, interferon-α (IFN-α), IFN-β, myxovirus (influenza virus) resistance 1 (MX1), and interferon stimulatory gene 15 (ISG15) was assessed by qPCR. When compared with healthy cattle, the expression of TLR-7, IFN-α, and IFN-β mRNA was downregulated, but the expression of MX1 and ISG-15 mRNA was upregulated in PI cattle. Immunoblotting analysis revealed that the expression of interferon regulatory factor 3 (IRF-3) and IRF-7 was lower in PI cattle than in healthy cattle. Thus, BVDV-1m and 1a are the predominant subgenotypes in the Beijing region, and the strains are highly divergent. Our findings also suggest that the TLR-7/IRF-7 signaling pathway plays a role in evasion of host restriction by BVDV.     

Research Progress on Cytokine Adjuvants

As an adjuvant, cytokine plays a key role in regulating the innate and adaptive immunity and enhancing the vaccine specific immune responses. The researches on the interleukin, interferon, granulocyte macrophage colony stimulating factors, tumor necrosis factors, and chemotactic factor have made considerable progress. Now we reviewed the cytokine adjuvant application in order to provide reference for the future application.