猪a-干扰素单克隆抗体的制备及其特性鉴定

发布时间：2018-05-27 12:00

本文选题：重组猪IFN-α + 重组蛋白　；参考：《甘肃农业大学》2009年硕士论文

【摘要】： IFN-α(interferon alpha,IFN-α)是在特定诱生剂的作用下由细胞产生的一种具有生物活性的糖蛋白,可诱导同种动物细胞产生广谱的抗病毒活性。目前已发现近百种疫病能通过动物传染给人,其中很多为病毒性传染病,有些目前尚未发现合适的疫苗和药物来预防和治疗,有些对病毒的生物学特性仍未研究清楚。IFN-α是抗病毒能力最强的干扰素,但无论是医学还是兽医学领域对IFN-α临床应用仍然极为有限,因此开展IFN-α单克隆抗体的研制,为评价机体抗病毒水平以及IFN-α作用机制研究奠定基础,对推动IFN-α在临床上广泛应用具有重要作用。本研究运用杂交瘤细胞技术,以重组猪IFN-α蛋白免疫BALB/c小鼠,无菌条件下制备小鼠免疫脾细胞,与骨髓瘤细胞SP2/0细胞融合,通过通过GST和GST-pIFN-α双重筛选和多次克隆化培养,成功获得了3株能分泌抗IFN-α的单克隆杂交瘤稳定细胞株。用单克隆抗体亚型鉴定试剂盒鉴定其亚型,确定1株单克隆抗体为IgG2a,2株单克隆抗体为IgG2b。将分泌单克隆抗体的杂交瘤细胞株的染色体计数,发现杂交瘤细胞株染色体平均数为92条,而骨髓瘤细胞株染色体平均数仅为66条,从遗传角度证明确实获得了杂交瘤细胞株;杂交瘤细胞腹水和细胞上清经ELISA检测,腹水中3株杂交瘤分泌单抗的效价均可达1:105,而细胞上清的效价可达到1:104;单抗的抗原结合位点分析表明,3株单抗的抗原结合位点基本一致;SDS-PAGE结果显示,3株单抗的重链分子量均在60ku以上,轻链分子量在30ku以上;在杂交瘤细胞的稳定性试验中,3株细胞经3次克隆化培养,细胞上清中的抗体效价均在1:104～1:105之间,说明该3株杂交瘤细胞分泌单抗的能力稳定,具有较好的遗传稳定性。
[Abstract]:IFN- 伪 interferon alpha-IFN- 伪) is a bioactive glycoprotein produced by cells under the action of a specific inducer, which can induce a broad spectrum of antiviral activity in the same species of animal cells. At present, it has been found that nearly 100 kinds of epidemic diseases can be transmitted to people through animals, many of which are viral infectious diseases. Some of them have not yet found suitable vaccines and drugs to prevent and treat them. Some of the biological characteristics of the virus have not been studied clearly. IFN- 伪 is the most potent antiviral interferon, but the clinical application of IFN- 伪 in both medicine and veterinary medicine is still very limited. Therefore, the development of monoclonal antibodies against IFN- 伪 has been carried out. It lays a foundation for evaluating the level of anti-virus and the mechanism of IFN- 伪, and plays an important role in promoting the wide application of IFN- 伪 in clinic. In this study, BALB/c mice were immunized with recombinant porcine IFN- 伪 protein by hybridoma cell technique. Spleen cells were prepared under sterile conditions and fused with myeloma cell line SP2/0 cells. GST and GST-pIFN- 伪 double screening and multiple cloning culture were used. Three monoclonal hybridoma stable cell lines secreting anti IFN- 伪 were successfully obtained. The subtypes were identified by using monoclonal antibody subtype identification kit, and one monoclonal antibody was identified as IgG2a2 and two monoclonal antibodies were IgG2b. The chromosome count of hybridoma cell lines secreting monoclonal antibodies showed that the average chromosome number of hybridoma cell lines was 92, while the average chromosome number of myeloma cell lines was 66. From the genetic point of view, hybridoma cell lines were obtained. Ascites and supernatants of hybridoma cells were detected by ELISA. The titers of McAbs secreted by three strains of hybridoma in ascites were 1: 105, while the titers of supernatants reached 1: 104.The antigen-binding sites of McAbs were basically consistent with those of SDS-PAGE. The molecular weight of the chain is above 60ku. The molecular weight of light chain was above 30ku, and in the stability test of hybridoma cells, the antibody titers in the supernatant were between 1: 104 and 1: 105, which indicated that the monoclonal antibody secreted by the three hybridoma cells was stable, and the antibody titers in the supernatant were between 1: 104 and 1: 105, which indicated that the three hybridoma cells had stable ability to secrete monoclonal antibodies. It has good genetic stability.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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