猪γ干扰素基因真核重组表达质粒的构建及活性测定

以含有全长猪γ干扰素基因的质粒为模板,通过PCR方法扩增猪γ干扰素并引入酶切位点,酶切后与PCI真核表达载体连接在一起,测序后经比较,所扩增序列同Genebank报道的IFN-γ序列同源性为99·8%.将重组质粒转染PK细胞,检测所转染的pIFN-γ的抗病毒活性.试验结果表明,转染pIFN-γ试验组与正常细胞接毒对照组比较有显著差异,说明所构建的重组质粒转染后具有抗病毒作用.     

马γ-干扰素成熟蛋白基因在大肠埃希氏茵中表达及其多克隆抗体制备

采用RT-PCR从ConA刺激的马外周血单核细胞（PBMC）中扩增获得含信号肽的马γ-干扰素（Equine interferon-γ，EIFN-γ）基因，将其克隆至载体pCR2．1-TOPO中。通过PCR方法从重组质粒（pCR-EIFN-γ）中扩增马干扰素成熟蛋白（MatureEIFN-γ，mEIFN-γ）基因，并将其亚克隆至原核表达载体pET-28a（＋）。重组子经测序鉴定正确后转化至大肠杆菌BL21（DE3），经IPTG诱导，对其产物进行SDS-PAGE分析和Western blot鉴定，将纯化后的表达产物免疫新西兰白兔制备多克隆抗体。结果表明，mEIFN-γ基因全长为441b口，含一个开放阅读框，编码146个氨基酸的成熟蛋白；对其表达产物以可溶性和包涵体两种形式存在，重组蛋白相对分子量约为18Ku，且具有免疫反应活性。mEIFN-γ基因在大肠杆菌中高效表达并在非变性和变性条件下，采用Ni^＋亲和层析纯化方法均可获得高纯度的重组马γ-干扰素制备的多克隆抗体，为建立马γ-干扰素检测方法、监测机体免疫状态和研究机体免疫机制奠定了基础。     

Bovine NK cells acquire cytotoxic activity and produce IFN-gamma after stimulation by Mycobacterium bovis BCG- or Babesia bovis-exposed splenic dendritic cells

Early interactions of innate immune cell populations, such as dendritic cells (DC) and natural killer (NK) cells, can affect the ability of the acquired immune response to control infection of intracellular microorganisms. In this study, we investigated the activation of bovine NK cells by CD13(+) splenic DC stimulated with either Mycobacterium bovis BCG or Babesia bovis merozoites. Splenic DC were used either immediately after selection (cytokine(-)) or after exposure to GM-CSF, IL-4 and Flt3L for 72 h (cytokine(+)). Phenotypic analyses showed up-regulation of MHCII, CD80 and CD86 on cytokine(+) DC when compared to cytokine(-) DC. Purified NK cells (CD335(+)CD3(-)CD2(+/-)CD8alpha(+/-)) were co-cultured with microbial-exposed cytokine(-) DC or cytokine(+) DC in either transwell or cell-to-cell format and NK cell IFN-gamma production and cytotoxicity were assessed. NK cell IFN-gamma production was dependent on cell-to-cell contact. Microbial-stimulated cytokine(+) DC induced significantly more IFN-gamma production from NK cells than cytokine(-) cells. In contrast, cytotoxicity and perforin up-regulation were more pronounced in NK cells cultured with cytokine(-) DC than cytokine(+) DC. Therefore, activation of bovine NK cells by microbial-stimulated CD13(+) splenic DC is influenced by the maturation state of the DC suggesting different roles for the splenic DC during disease-induced maturation.     

Extent of Mycobacterium bovis infection in dairy cattle herds subject to partial culling as determined by an interferon-gamma assay

The interferon-gamma (IFN-γ) assay is employed as a complementary diagnostic test for bovine tuberculosis (BTB) in many countries. To simplify this assay, we established a 96-well plate format using the ESAT-6 and CFP-10 antigens and then employed it to determine the extent of Mycobacterium (M.) bovis infection in dairy herds with a history of BTB outbreaks in a country where only selective culling is practiced. The sensitivity and specificity of this IFN-γ assay were 85.9% and 100%, respectively, based on comparison with the conventional single intradermal tuberculin test (SIDT). The IFN-γ assay was also positive in 30.4% and 36.8% of SIDT-negative animals from herds with recent and remote BTB outbreaks, respectively. Of 14 SIDT-negative, IFN-γ positive cattle, five (35.7%) were culture positive and an additional six were positive based on a polymerase chain reaction-based test for M. bovis. Therefore, the IFN-γ assay has the potential to serve as a specific and sensitive test for M. bovis infection in dairy cattle. Further, the results indicated that a substantial portion of SIDT-negative animals in herds with previous BTB outbreaks were actually infected with M. bovis. Accordingly, the present selective-culling strategy may require modifications to include this more sensitive assay.     

鸡包涵体肝炎过程中IFN-γ的mRNA原位杂交

FAV-Ⅷ型禽腺病毒口服接种感染2日龄SPF雏鸡,攻毒后取不同日龄的肝脏于福尔马林溶液中固定,并进行石蜡包埋,石蜡切片进行原位杂交染色.结果表明,着染部位主要出现在坏死灶或血管周围的成团或散在的淋巴细胞的胞浆内,在病情严重时的5 dpi开始增多,至20 dpi表达量也很高,以后开始减少,直到30 dpi基本消失.说明鸡包涵体肝炎过程中肝脏IFN-γ的mRNA大量上调,可能与T淋巴细胞的增多有关;而且IFN-γ作为免疫增强剂与抗病毒感染和病症恢复也有关.     

马γ-干扰素成熟蛋白基因在大肠埃希氏菌中表达及其多克隆抗体制备

采用RT-PCR从ConA刺激的马外周血单核细胞(PBMC)中扩增获得含信号肽的马γ-干扰素(Equine interferon-γ,EIFN-γ)基因,将其克隆至载体pCR2.1-TOPO中。通过PCR方法从重组质粒(pCR-EIFN-γ)中扩增马干扰素成熟蛋白(Mature EIFN-γ,reEIFN-γ)基因,并将其亚克隆至原核表达载体pET-28a( )。重组子经测序鉴定正确后转化至大肠杆菌BL21(DE3),经IPTG诱导,对其产物进行SDS-PAGE分析和Western blot鉴定,将纯化后的表达产物免疫新西兰白兔制备多克隆抗体。结果表明,mEIFN-γ基因全长为441bD,含一个开放阅读框,编码146个氨基酸的成熟蛋白;对其表达产物以可溶性和包涵体两种形式存在,重组蛋白相对分子量约为18Ku,且具有免疫反应活性。mEIFN-γ基因在大肠杆菌中高效表达并在非变性和变性条件下,采用Ni~ 亲和层析纯化方法均可获得高纯度的重组马γ-干扰素制备的多克隆抗体,为建立马γ-干扰素检测方法、监测机体免疫状态和研究机体免疫机制奠定了基础。