【作者】 滕巧泱；

【导师】 周继勇；

【作者基本信息】 浙江大学 ， 预防兽医学， 2008， 博士

【摘要】 1、chIL-2R基因的克隆与体外重组表达依据鸡白细胞介素-2受体亚基α链(chCD25)、β(chCD122)、γ(chCD132)的cDNA序列设计特异性引物,通过RT-PCR分别扩增和克隆了chCD25、chCD132 cDNA全长序列及chCD122部分cDNA序列。chCD25和chCD132 cDNA的长度分别为1027 bp和1047 bp,各自编码由211和348个氨基酸(aa)残基所组成的前体蛋白。chCD122 cDNA片段长度为660bp,编码该蛋白的部分胞外区序列。chCD25成熟蛋白由胞外区(172aa)、跨膜区(15aa)和胞浆区(4aa)三部分组成。chCD132成熟蛋白由由胞外区(210aa)、跨膜区(23aa)和胞浆区(94aa)三部分组成。基因定位分析显示,chCD25基因位于鸡染色体1,由五个外显子和四个内含子组成。chCD122基因也位于鸡染色体1,由12个外显子和11个内含子组成。chCD132基因位于鸡染色体4,由8个外显子和7个内含子组成；chCD25和chCD122在基因结构上与人和鼠的相应基因差异较大,而chCD132的则与人、鼠相似。这些结果说明,鸡白细胞介素-2受体(chIL-2R)基因具有明显的种属特异性。将编码chCD25、chCD132的胞外区序列及chCD122部分胞外区序列,分别插入pET32a或pET28a原核表达载体。经酶切及测序鉴定后,转化大肠杆菌BL21(DE3),在1 mM IPTG的诱导下表达4h。SDS-PAGE和Western blot鉴定的结果显示,chCD25、chCD122和chCD132蛋白被表达,它们的分子量大小分别为38KDα、30KDα、28 KDα。通过Ni柱变性纯化的方法,获得纯度较高的chCD25、chCD122和chCD132原核蛋白。2、chIL-2R单克隆抗体制备与功能验证以原核表达的chCD25、chCD122和chCD132重组蛋白为免疫原,免疫BALB/c小鼠,分别获得了7株(4H5、6C9、6A1、5E1、1G1、6C2、1E1)能稳定分泌抗chCD25单克隆抗体(mAbs)的细胞、4株(1G11,5A5,5B12,2C11)稳定分泌抗chCD122单克隆抗体的细胞、6株(1A1、1G11、1B9、3G11、F8、C10)稳定分泌抗chCD132单克隆抗体的细胞。Western blot检测结果显示,所有抗体细胞分泌的细胞上清均能与相应的重组蛋白发生反应。chIL-2R基因转染真核细胞的免疫细胞化学或荧光检测证实,除chCD122的mAb 5B12和chCD132的mAb 1A1外,所有mAb均能真核细胞表达的chIL-2R蛋白反应。用Con A诱导的体外淋巴细胞增殖试验证实,制备的chCD25 mAbs (5E1和6C9)、chCD122mAbs(5A5、1G11、2C11)及chCD132mAb(C10)能结合淋巴细胞表面的蛋白分子。体外淋巴细胞增殖抑制试验的研究表明,chCD25 mAb 6C9能有效地抑制鸡白细胞介素-2 (chIL-2)激活T淋巴细胞的增殖作用,并呈浓度依赖性。此提示,本文获得的chCD25基因是特异性针对chIL-2的。3、IBDV感染条件下的chIL-2R调节功能分析以IBDV感染鸡胚成纤维细胞,感染后不同时间点,在mRNA和蛋白水平分析chIL-2R及chCD132关联细胞因子的变化。结果显示,IBDV感染能激活CEF中chCD25、chCD122、chIL-2、鸡白细胞介素-4(chIL-4)、鸡白细胞介素-7(chIL-7)、鸡白细胞介素-9(chIL-9)、鸡白细胞介素-15(chIL-15)的mRNA转录显著上调,但CD132的mRNA转录处于抑制状态。与mRNA检测相对应,在IBDV体外感染48小时的CEF可检测到chCD25和chCD122蛋白的表达,不能检测到chCD132蛋白的表达。以致病性IBDV感染SPF鸡,在mRNA和蛋白水平分析chIL-2R及chCD132关联细胞因子的变化,结果显示：胸腺组织中chCD25、chCD122、chCD132、chIL-2、chIL-4、chIL-7、chIL-9、chIL-15的mRNA转录被显著抑制；与胸腺组织相反,法氏囊和脾脏组织中chCD25、chCD122的mRNA转录显著上调,chCD132的mRNA转录上调维持在较低水平,以法氏囊最不明显。与mRNA检测相对应,在IBDV感染的法氏囊及脾脏淋巴细胞表面可检测到chCD25和chCD122的表达,不能检测到chCD132蛋白的表达。这些数据显示,IBDV体内感染显著抑制了胸腺细胞chCD25、chCD122和chCD132亚基的表达,明显激活了法氏囊细胞chCD25和chCD122亚基的表达,但法氏囊细胞chCD132的mRNA基本没有明显变化。因此,我们可以确信,IBDV感染过程中chIL-2R对细胞免疫中枢和体液免疫中枢的调控机制是不同的。在非免疫组织中,IBDV感染后,脑、十二指肠、空肠组织中的chCD25. chCD122、chCD132在感染后表达下调；腺胃、盲肠、肾、肺及胰组织组织中的chCD25、chCD122、chCD132在感染后表达逐渐上调；肌胃组织中的chCD25、chCD122、chCD132处于初期的下调、中期上调和末期下调；肝组织中chCD122和chCD132在感染后表达上调,而chCD25处于初期上调,中末期下调；心组织中chCD132在感染后表达上调,而chCD25处于初期上调、中期下调和末期上调,chCD122处于初期下调,中末期上调。与chIL-2R关联的chIL-4 mRNA转录在脑抑制、在肾上调,chIL-7 mRNA转录在肌胃上调、在腺胃抑制,chIL-2、chIL-7、chIL-9 mRNAs的转录在十二指肠抑制、在盲肠上调,chIL-15mRNA转录在十二指肠上调、在空、盲肠抑制。chIL-2、chIL-7、chIL-9、chIL-15 mRNAs的转录在空肠抑制和在盲肠、心脏上调。这些结果证明,IBDV感染后chIL-2R在不同的非免疫组织的调控机制也是不同的。4、H5N1感染条件下的chIL-2R调节功能分析以H5N1感染CEF,感染后不同时间点,在mRNA和蛋白水平chIL-2R及chCD132关联细胞因子的变化。结果表明,H5N1病毒感染CEF后,能引起细胞内chIL-2R和chCD132细胞因子mRNAs表达上调。IL-2R蛋白水平的间接免疫荧光可检测到chCD25、chCD122和chCD132的表达。此结果显示,IBDV和H5N1感染过程中对chCD132调控是不同的。以H5N1感染SPF鸡,感染后不同时间在mRNA和蛋白水平分析chIL-2R及chCD132关联细胞因子的变化,结果表明,脾脏组织中chCD25、chCD122、chCD132的mRNA明显上调,法氏囊组织中仅出现chCD25和chCD132的mRNA上调,与此相反,胸腺组织中的chCD25、chCD122和chCD132的mRNA均显著下调。免疫组织化学检测也证实,脾脏组织中的chCD25+、chCD122+和chCD132+细胞的数量多于法氏囊组织,胸腺组织中不能检测到chCD25、chCD122+和chCD132+细胞。与之相对应的配体chIL-2、chIL-4在脾脏和胸腺中也出现了显著上调,在法氏囊中出现下调,说明H5N1病毒感染不仅导致了胸腺中chIL-2R+细胞从免疫中枢-胸腺向外周免疫器官的移动,同时启动了机体的TH1和TH2细胞免疫应答。就与chCD132相关联的其他细胞因子而言,chIL-15的mRNA在脾脏、胸腺、法氏囊组织中转录无不明显变化,说明chIL-15未能在机体抵抗H5N1的感染过程中发挥作用。chIL-7的mRNA在脾和法氏囊组织上明显上调,在胸腺中变化不明显,说明H5N1感染过程中chIL-7在维持外周T细胞的自我平衡过程发挥了重要作用。综合上述,H5N1病毒感染引起了免疫中枢chCD25、chCD122和chCD132分子的表达下调,外周免疫组织的上调,导致了chIL-15对chIL-2和chIL-4的调控能力破坏。更多还原

【Abstract】 Using a pair of specific primers designed from chicken interleukin-2 receptor a chain (chCD25),βchain (chCD122) and y chain (chCD132), the full cDNAs for chCD25, chCD132 and partial cDNA for chCD122 were amplified from a Chinese local breed Sanhuang broilers. The lengths of chCD25 and chCD132 cDNAs were 1027 bp and 1047 bp, respectively, whose precursor proteins comprised of 211 and 348 amino acids (aa), respectively. The predicted analysis of bioinformaitics revealed that the predicted mature chCD25 protein was composed of an extracellular domain (172 aa), a membrane-spanning region (15 aa) and a cytoplasmic domain (4 aa). The predicted mature chCD132 protein was composed of an extracellular domain (210 aa), a membrane-spanning region (23 aa) and a cytoplasmic domain (94 aa). The partial fragment of chCD122 was 660 bp in length, which encoded partial extracellular domain. In a further analysis, chCD25 gene was predicted to locus on the chromosome 1, which had five exons and four introns. chCD122 gene was also predicted to locus on the chromosome 1, which had 12 exons and 11 introns. chCD132 gene was predicted to locus on the chromosome 4, which had 8 exons and 7 introns. The gene structures of chCD25 and chCD122 were quite different from those of human and mouse, but not for chCD132. It suggests that chicken interleukin-2 receptor (chIL-2R) gene has obvious species specificity. In addition, the sequences encoding entire extracellular domains of chCD25 and chCD132 and partial extracellular domain of chCD122, were inserted into the plasmid pET32a, or pET28a vectors, respectively. Then, these recombinant plasmids were transformed E.coli BL21(DE3) and induced by 1 mM IPTG. The analysises of SDS-PAGE and Western blot showed that chCD25, chCD122 and chCD132 proteins were successfully expressed in the prokaryotic system, with molecular weights of 38 KD a,30 KD a and 28 KD a, respectively. The highly pure chCD25, chCD122 and chCD132 prokaryotic proteins were acquired.Using chCD25, chCD122 and chCD132 prokaryotic proteins as immunogen to immune BALB/c mosue, the stable cell lines secreting seven monoclonal antibodies (mAbs)to chCD25(4H5、6C9、6A1、5E1、1G1、6C2、1E1), four mAbs to chCD122 (1G11,5A5,5B12,2C11) and six mAbs to chCD132 (1A1、1G11、1B9、3G11、F8、C10) were acquired. The result of Western blot showed that all these antibodies could react to corresponding antigens. By immunocytochemistry or indirect immunofluorescence assay, the result demonstrated that all mAbs could react to chIL-2R eukaryotic proteins except of 5B12 mAb to chCD122 and 1A1 mAb to chCD132. The vitro proliferation experiments of Con A-stimulated T lymphocytes demonstrated that chCD25 mAbs (5E1 and 6C9), chCD122 mAbs (5A5,1G11,2C11) and chCD132 mAb (C10) had abilities to binding with proteins expressed on the surface of lymphocytes. The study on the inhibiton of lymphocytes proliferation in vitro indicated that mAb 6C9 to chCD25 could effectively inhibited chicken interleukin-2 (chIL-2) to stimulating the proliferation of T lymphocytes, which was dose-dependent. This hints that chCD25 gene here was specific to chIL-2.Chicken embryonic fibroblast (CEF) was inoculated with IBDV, and then the expression of chIL-2R and chCD132 cytokines mRNAs or proteins at different time point post-infection (p.i) was analyzed. The result demonstrated that the transcriptions of chCD25, chCD122, chCD132, chIL-2, chicken interleukin-4 (chIL-4), chicken interleukin-7 (chIL-7), chicken interleukin-9 (chIL-9), chicken interleukin-15 (chIL-15) up-regulated obviously in CEF, but chCD132 mRNA was inhibited. At 48h p.i, the expression of chCD25 and chCD122 proteins could be detected in IBDV-inoculated CEF, but not for chCD132, which was the same to that at mRNA level. Pathogenic IBDV was inoculated with SPF chicken, and then the expression of chIL-2R and chCD132 cytokines was analyzed at mRNA and amino acid levels. The result demonstrated that the transcriptions of chCD25, chCD122, chCD132, chIL-2, chIL-4, chIL-7, chIL-9, chIL-15 were inhibited obviously in thymus; In contrast, the expression of chCD25 and chCD122 proteins up-regulated markly in bursa of Fabricius and spleen, but chCD132 mRNA only weakly increased. The expression of chCD25 and chCD122 proteins could be detected in IBDV-inoculated bursa of Fabricius and spleen, but not for chCD132 protein, which was in accordance with that at mRNA level. These results suggest that the infection of IBDV obviously inhibited chCD25, chCD122 and chCD132 to express in thymus, enhanced chCD25 and chCD122 to express in cells from bursa of Fabricius, but nearly not affected chCD132 in bursa of Fabricius. Thus, we made sure that the immunoregulation mechanisms of IL-2R were different between cellular central immune and humoral centran immune during IBDV infection. In IBDV-inoculated non-immnue tissues, chCD25, chCD122 and chCD132 mRNAs down-regulated in brain, duodenum and jejunum, gradually up-regulated in glandular stomach, cecum, kidney, lung and pancreas; chCD25, chCD122 and chCD132 mRNAs expressed in gizard down-regulated in the initial stage, up-regulated in the intermediate stage and down-regulated in the end stage; in the liver, the expression of chCD122 and chCD132 mRNAs upregulated at the whole time of infection, but the expression of chCD25 mRNA initially up-regulated, then down-regulated; in heart, chCD132 mRNA up-regulated, differently, chCD25 mRNA up-regulated in the initial stage, down-regulated in the intermediate stage and up-regulated in the end stage, and chCD122 mRNA down-regulated in the initial stage and up-regulated finally. The transcriptional expression of chIL-4 was inhibited in the brain, and up-regulated in the kidney; The transcriptional expression of chIL-7 up-regulated in the gizard and was inhibited in the proventriculus; The transcriptional expression of chIL-2, chIL-7, chIL-9 was inhibited in the duodenum and up-regulated in the cecum; chIL-15 mRNA increased in the duodenum and was inhibited in the jejunum and cecum. ChIL-2, chIL-7, chIL-9, chIL-15 mRNAs were inhibited in the jejunum and up-regulated in the cecum and heart. These results confirmed that the immunoregulation mechanisms of IL-2R were different in each non-immune tissue.CEF was inoculated with H5N1, and then the expression of chIL-2R and chCD132 cytokines mRNAs or proteins at different time point post-infection (p.i) was analyzed. The result demonstrated that the infection of H5N1 vigorously increase the expression of chIL-2R and chCD132 cytokines mRNAs in CEF. Meanwhile, the chCD25, chCD122 and chCD132 proteins could be detected by the indirect immunofluorescence assay. This result suggests that immunoregulation of chCD132 happened in the infection of H5N1 was quite different from that of IBDV. On this basis, SPF chicken was inoculated with H5N1 in order to vivo analysis. The result demonstrated that the expression of chCD25, chCD122 and chCD132 mRNAs up-regulated obviously in the spleen. In bursa of Fabricius, chCD25 and chCD132 mRNAs increased. In contrast, chCD25, chCD122 and chCD132 mRNAs were obviously down-regulated in the thymus. The result of immunohistochemistry also confirmed that the quantities of chCD25+, chCD122+ and chCD132+cells were more than those of bursa of Fabricius, but no positive signals appeared in the thymus. ChIL-2 and chIL-4 mRNAs increased obviously in the spleen and thymus, but down-regulated in the bursa of Fabricius. This suggests that the infection of H5N1 causes that chIL-2R+cells migrated from central immune organ (thymus) to peripheral immune organ, as well as activation of TH1 and TH2 cellular immune responses. To other chCD132 cytokines, chIL-15 mRNA did not change obviously in the spleen, thymus and bursa of Fabricius. It hints that chIL-15 does not play a positive role in the process of H5N1 infection. ChIL-7 mRNA obviously increased in the spleen and bursa of Fabricius, but did not change in the thymus. This phenomenon indicated that chIL-7 played an important role in the maintaining balance of peripheral T cells. In conclusion, the infection of H5N1 caused that chCD25, chCD122 and chCD132 down-regulated in the central immune, and up-regulated in the peripheral immune organs, and chIL-15 demaged the immunoregulation functions of chIL-2 and chIL-4.更多还原