猪白细胞介素22克隆表达和活性研究及单克隆抗体的制备

发布时间：2018-11-10 20:14

【摘要】：白细胞介素22(IL-22),属于IL-10家族成员,主要由T淋巴细胞和固有淋巴细胞分泌,在体内通过与其受体(IL-22R1和IL-10R2)结合形成异源二聚体受体复合物并激活信号通路来发挥生物学功能。研究发现,人和鼠的IL-22在体内发挥抗炎和促炎的双重作用:一方面,它主要作用于黏膜上皮细胞,在黏膜损伤的修护,炎症反应及抗感染中发挥重要作用;另一方面,IL-22在组织中的长期过表达可导致宿主慢性炎症的发生。小肠作为宿主免疫的重要场所,是IL-22发挥生物效应的关键部位。近年来对IL-22的研究主要集中在人源和鼠源,鱼IL-22也曾有报道,但猪IL-22的研究尚未见报道。鉴于此,本研究旨在通过以下二方面的研究阐释猪IL-22能否发挥保护宿主抵御病原入侵、抵抗感染的作用,为猪IL-22的研究奠定基础。(1)猪白细胞介素22的原核表达和活性研究从猪外周血单核细胞衍生的DC细胞中克隆出大小为568 bp的猪IL-22基因序列和去信号肽的IL-22成熟序列。首先将该去信号肽基因片段克隆到原核表达载体PET-30a(Amp+)中,构建重组质粒PET-30a/IL-22,再将阳性重组质粒转化至大肠杆菌BL21(DE3)中诱导表达了大小为22 KD的重组蛋白His/IL-22,并利用Ni-NTA亲和层析法进行纯化。生物学活性检测结果显示:纯化的His/IL-22对猪IPEC-J2中IL-18、IFN-λ和BD-2均有上调表达的作用,当用终浓度为0.04μg/m L的重组蛋白处理IPEC-J2细胞时,随着His/IL-22对IPEC-J2细胞处理时间的延长,细胞中这三种细胞因子表达呈现逐渐增加的趋势,在48 h时,三种细胞因子的表达量均达到最大。此外,病毒增殖抑制试验发现His/IL-22具有抑制PEDV-CV777和Po RV在IPEC-J2中复制的作用。这一结果首次证明猪IL-22能够上调IPEC-J2中细胞因子的表达,并且帮助IPEC-J2抵抗病毒感染。(2)猪IL-22单克隆抗体的制备以前期试验获得的具有生物活性的His/IL-22蛋白作为抗原,腹腔免疫6-8周龄雌性BALB/C小鼠,取其脾细胞,通过细胞融合的方法通筛选出了4株能分泌抗猪IL-22抗体的阳性杂交瘤细胞,分别命名为2G10、2C7、6B11、7F3,这四株细胞分泌的抗体亚型均为Ig M K型。根据预实验中细胞培养上清抗体效价测定的结果选取了效价较高的6B11和7F3细胞株制备腹水,ELISA测定6B11细胞株培养上清及其腹水效价,分别为1∶50和1∶25 600,7F3细胞株的培养上清及其腹水效价分别为1∶100和1∶6 400。本次试验制备的猪IL-22单克隆抗体为建立准确快速检测猪IL-22的ELISA方法奠定了基础。
[Abstract]:Interleukin 22 (IL-22), a member of the IL-10 family, is secreted mainly by T lymphocytes and innate lymphocytes. In vivo biological functions are performed by binding to its receptors (IL-22R1 and IL-10R2) to form heterodimer receptor complexes and to activate signaling pathways. It has been found that IL-22 plays a dual role of anti-inflammatory and pro-inflammatory in vivo. On the one hand, it mainly acts on mucosal epithelial cells and plays an important role in the repair of mucosal injury, inflammatory response and anti-infection. On the other hand, long-term overexpression of IL-22 may lead to chronic inflammation of the host. Small intestine, as an important site of host immunity, is the key site of biological effect of IL-22. In recent years, the research on IL-22 is mainly focused on human and mouse sources, fish IL-22 has also been reported, but the study of porcine IL-22 has not been reported. In view of this, the purpose of this study is to elucidate whether porcine IL-22 can protect the host against pathogen invasion and infection through the following two aspects of research. (1) prokaryotic expression and activity of porcine interleukin-22; cloning of porcine IL-22 gene sequence and de-signal from porcine peripheral blood monocyte derived DC cells with the size of 568 bp IL-22 mature sequence of peptide. Firstly, the gene fragment was cloned into prokaryotic expression vector PET-30a (Amp), and the recombinant plasmid PET-30a/IL-22, was constructed. Then the positive recombinant plasmid was transformed into E. coli BL21 (DE3) to induce the expression of 22 KD recombinant protein His/IL-22, and purified by Ni-NTA affinity chromatography. The results of biological activity test showed that purified His/IL-22 could up-regulate the expression of IL-18,IFN- 位 and BD-2 in porcine IPEC-J2. When the recombinant protein with final concentration of 0.04 渭 g / mL was used to treat IPEC-J2 cells, With the prolongation of the treatment time of His/IL-22 on IPEC-J2 cells, the expression of these three cytokines increased gradually, and the expression of the three cytokines reached the maximum at 48 h. In addition, virus proliferation inhibition test found that His/IL-22 can inhibit the replication of PEDV-CV777 and Po RV in IPEC-J2. This result is the first to prove that porcine IL-22 can up-regulate the expression of cytokines in IPEC-J2. And to help IPEC-J2 resist virus infection. (2) the preparation of porcine IL-22 monoclonal antibody, using bioactive His/IL-22 protein obtained in previous tests as antigen, was intraperitoneally immunized with 6-8 week-old female BALB/C mice. Four positive hybridoma cells which secreted anti-porcine IL-22 antibody were selected by cell fusion method and named as 2G10C7C7B117F3. The antibody subtypes secreted by these four strains were all Ig M K type. Ascites were prepared from 6B11 and 7F3 cell lines with high titer, and supernatant and ascites titers of 6B11 cell lines were determined by ELISA according to the results of antibody titer test in cell culture supernatants. The supernatant and ascites titers of the cell lines at 1:50 and 1:25 were 1: 100 and 1:6 400, respectively. The monoclonal antibody to porcine IL-22 prepared in this study laid a foundation for the establishment of an accurate and rapid ELISA method for the detection of porcine IL-22.
【学位授予单位】：青海大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.4

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