【作者】 韩春来；

【导师】 汪明；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2005， 博士

【摘要】 干扰素是一种具有抗病毒、抑制细胞增殖和免疫调节活性的细胞因子,干扰素与干扰素受体结合后启动信号传导和干扰素基因的转录。本文克隆并表达了惠阳胡须鸡IFN-α、IFN-β、IFNAR1、IFNAR2基因;首次克隆了鸡IFNGR2全长cDNA;分析了干扰素受体在外周血淋巴细胞和各器官组织中的分布;并对IFN-α、IFN-β与其受体在体外的相互作用和重组蛋白的二级结构进行了研究。 以肝脏DNA为模板,克隆了惠刚胡须鸡IFN-α基因,与Genbank上IFN-α的同源性为96.9%～97.9%,是一个新的亚型;克隆了惠阳胡须鸡IFN-β和AA肉鸡IFN-β基因,与白色来亨鸡IFN-β的核苷酸序列同源性分别为99.84%、100%,目前鸡IFN-β尚未发现亚型的存在。构建了鸡IFN-α/pGEX-6P-1和IFN-β/pGEX-6P-1重组质粒,在BL21中诱导表达,可溶性蛋白通过亲和层析得到纯化并采用Western-blot得到了鉴定。IFN-α、IFN-β重组蛋白具有较高的生物学活性,在CEF/VSV系统中的抗病毒活性为7.9×10~5U/mg和6.4×10~4U/mg。 从惠阳胡须鸡肝脏总RNA中扩增了Ⅰ型干扰素受体的两个亚基:IFNAR1、IFNAR2。惠阳胡须鸡IFNAR1、IFNAR2分别与红色原鸡的IFNAR1、IFNAR2的氨基酸序列同源性为99.47%和98.82%,均为新的亚型。在IFNAR1氨基酸序列的35-133、134-237、240-319和341-447处有四个典型的干扰素受体家族的保守性motif:Fibronectin Ⅲ型分子,123-129和302-321处有两个典型的linker;在IFNAR2氨基酸序列的37-93处和129-240处有两个Fibronectin Ⅲ型分子,在94-105和157-166处有两个典型的linker。构建了鸡IFNAR1EC/pGEX-6P-1、IFNAR2EC/pGEX-6P-1重组质粒并转化到BL21中诱导表达,可溶性蛋白通过亲和层析得到纯化并采用Western-blot得到了鉴定。 以惠阳胡须鸡脾脏总RNA为模板,采用5′RACE和3′RACE的方法首次克隆了IFN-γ受体β链全长cDNA序列,共2221bp。这一基因编码334个氨基酸,与小鼠、大鼠和人IFNGR2氨基酸的同源性分别为29%、28%和30%;在氨基酸序列的31-119和126-217处含有两个Fibronectin Ⅲ型分子,在96-115和115-131处有两个linker。将采用RACE克隆的这一基因命名为惠阳胡须鸡IFNGR2(HuiYang chicken IFNGR2)。Genome BLAST的结果显示IFNGR2与IFNAR1、IFNAR2、IL-10R2串联排列于鸡的1号染色体上。 采用Northern杂交和RT-PCR的方法,分析了鸡IFN-α、IFN-β、IFNAR1EC、IFNAR2EC和IFNGR2EC基因在脾脏、胸腺、法式囊、胸肌、心肌、肾脏、肝脏、盲肠扁桃体、未刺激外周血淋巴细胞(PBL)和ConA刺激PBL中的表达水平。IFN-α、IFN-β基因在组织和未刺激的PBL中呈关闭状态,在ConA刺激的PBL中表达水平很高。未刺激的PBL中IFNAR1、IFNGR2的表达水平很高,IFNAR2的表达水平较低,ConA刺激后IFNAR1、IFNAR2和IFNGR2的表达水平没有明显上升。在脾脏、胸腺、法氏囊和盲肠扁桃体中均可检测到IFNAR1、IFNAR2和IFNGR2的表达;IFNGR2在胸肌、心肌、肝脏和肾脏中表达水平也较高。RT-PCR的结果与Northern杂交的结果基本一致。鸡IFNAR1、IFNAR2和IFNGR2在免疫器官中的表达量较高,表明其参与机体的抗病毒和免疫调节过程。更多还原

【Abstract】 Interferon exert the biological activities such as antivirus activity, antiproliferative actions and immune response through binding to their receptors. Interferon receptor play a critical role in the signal transmission and activation of transcription of interferon genes. This paper cloned,expressed IFN-α , IFN- β ,IFNAR1,IFNAR2 genes, firstly cloned IFNGR2 gene of HuiYang chicken and detected their transcript levels in peripheral blood leukocyte and tissues; secondary structures and interactions of IFN- α / β and their receptors were analysed.IFN- α gene was cloned from liver genomic DNA of HuiYang chicken.The homologies were from 96.9% to 97.9% between HuiYang IFN- α and IFN- α s on Genbank, indicating this IFN- α gene was a new subtype.The genes of IFN- β were cloned from liver genomic DNA of HuiYang chicken and Arbor Acres respectively,exhibiting 99.84% and 100% identity to gene of IFN- P of white leghorn.IFN- β was a single copy gene without subtype until now. IFN- α ,IFN- β genes were subcloned into pGEX-6P-1, the recombinant plasmids were transformed into BL21 and expressed.The GST fusion proteins in the supernatant were purified using Glutathione Separose 4B column and examined using western-blot.Recombinant IFN- α ,IFN- β expressed in E.coli exert high antiviral activities as 7.9 × 10~5U/mg and 6.4 × 10~4 U/mg respectively.IFNARl and IFNAR2 genes of HuiYang chicken were cloned from liver total RNA.Comparison of HuiYang IFNARl with gallus gallus IFNAR1,the homology of amino acids sequences was 99.47%; there was 99.35% homology between amino acid sequence of Huiyang IFNAR2 and that of gallus.gallus. HuiYang IFNARl, IFNAR2 were new subtypes.The extracellular part of HuiYang IFNAR1 was composed of four tipical conserved motif of interferon receptor family(Fibronectin Ⅲ molecule ) on 35-133、 134-237、 240-319 and 341-447 of amino acid sequence,which connected by two linkers on 123-129 and 302-321.There were two Fibronectin Ⅲ molecules on 37-93 and 129-240 of IFNAR2 amino acid sequences,which connected by two linkers on 94-105 and 157-166. IFNAR1EC and IFNAR2EC genes were subcloned into pGEX-6P-l, the recombinant plasmids were transformed into BL21 and expressed.The soluble proteins were purified using Glutathione Separose 4B column and examined using western-blot.The full length cDNA of IFNGR2 was firstly cloned from HuiYang chicken spleen total RNA using 5’ RACE and 3’ RACE. This 2221bp gene encoded 334 aminoacids ,with 28, 29%, 30% identity with amino acids of rat, mouse, human IFNGR2 respectively and given name as HuiYang IFNGR2. Extracellular part of HuiYang IFNGR2 contained two FNⅢ domains on 31-119 ,126-21 of amino acid sequences and two linkers on 96-115,115-131. HuiYang IFNGR2 gene was localized on the chromosome1q26 of chicken in tandem with IFNARl, IL-10R2 and IFNAR2 through Genome BLAST.To analyse the expression of IFN- α , IFN- β ,IFNAR1,IFNAR2 and IFNGR2 in different organs and peripheral blood leukocyte (PBL) using Northern blot and RT-PCR, the total RNAs were extracted from spleen,thymus, muscle ,caecum tonsil,cardiac muscle,cloacal bursa,liver,kidney ,unstimulated PBL and stimulated PBL with ConA .IFN- α and IFN- β were detectable when PBL stimulated with ConA,but not detectable in the organs and unstimuted PBL IFNAR1 and IFNGR2 was highly expressed in unstimulated PBL,but IFNAR2 was lowly expressed.Expression of of IFNAR1,IFNAR2 and IFNGR2 did not increased after ConA activation.IFNARl,IFNAR2 and IFNGR2 transcripts were detectable in spleen, thymus, cloacal bursa and caecum tonsil and IFNGR2 was also also detected in muscle,cardiac muscle,cloacal bursa,liver,kidney.RT-PCR results consisted with the results of Nothern blot.The high expression of interferon receptor in immune organs indicated interferon receptor may play important role in antivirus and immue response.The Gel-filtration and GST interacting trials confirmed stalbe formation of binary complex of IFN- α -IFNAR2EC and IFN- β -IFNAR2EC in vitro;IFNARlEC did not bind to IFN- β , IFN- β or IFNAR2EC. IFN-α更多还原