布氏杆菌LPS抗原及其单克隆抗体的制备与纯化

发布时间：2018-09-02 05:40

【摘要】：布氏杆菌(Brucella Blues)是革兰氏阴性需氧杆菌，可引起人畜共患的布氏杆菌病，给世界各国畜牧业造成了严重的经济损失，同时也给人类健康造成极大的危害。该病目前尚不能完全根治，所以预防和检测显得尤为重要。本研究采用改良热酚法制备纯化布氏杆菌M5株的脂多糖（LPS），检测细菌LPS免疫原性；纯化的细菌LPS免疫BALB/c小鼠，，通过DOT-ELISA测定血清抗体效价。结果表明采用热酚法能获得较高纯度的细菌LPS，其免疫原性较高，DOT-ELISA检测的血清效价达到1:106，与全菌免疫原性接近。在此基础之上，将LPS免疫的BALB/c小鼠脾细胞和SP2/0细胞进行融合，制备杂交瘤细胞，采用间接ELISA法进行阳性筛选，经过三次筛选和3-4次细胞克隆化培养，获得四株稳定分泌抗体的杂交瘤细胞株，采用间接ELISA法分别测定其细胞上清抗体效价，分别达到1:104，1:105，1:105，1:105。对四株细胞的上清液进行Tricine-SDS-PAGE鉴定，上清中同时出现了细胞培养液中没有的蛋白，即为单克隆抗体目的蛋白。测定单克隆抗体的相对亲和力和稳定性，结果显示其相对亲和力分别为24.13μg/mL，24.65μg/mL，24.95μg/mL，23.16μg/mL，且具有良好的稳定性。对细胞上清采用硫酸铵盐析法初步纯化，再采用亲和层析法对单抗进一步纯化，纯化的蛋白进行效价测定和Tricine-SDS-PAGE鉴定，结果表明纯化后四株细胞的单抗效价略有降低，但仍可达1:104。电泳结果表明单抗的轻链分子量约为26ku，重链分子量约为37ku。本实验结果为布氏杆菌病检测方法的建立奠定了研究基础。
[Abstract]:Brucellosis (Brucella Blues) is a gram-negative aerobic bacillus, which can cause zoonotic brucellosis, which has caused serious economic loss to animal husbandry in the world, and also caused great harm to human health. At present, the disease can not be completely cured, so prevention and detection is particularly important. In this study, the lipopolysaccharide (LPS),) of purified brucellosis M5 strain was prepared by modified thermophenol method to detect the immunogenicity of bacterial LPS, and the purified bacterial LPS was used to immunize BALB/c mice. Serum antibody titers were determined by DOT-ELISA. The results showed that the high purity bacterial LPS, could be obtained by pyrophenol method. The immunogenicity of the serum detected by DOT-ELISA was 1: 106, which was close to the immunogenicity of the whole bacterium. On this basis, the spleen cells and SP2/0 cells of BALB/c mice immunized with LPS were fused and hybridoma cells were prepared. The hybridoma cells were screened by indirect ELISA method. Four hybridoma cell lines stably secreting antibodies were obtained and their supernatant antibody titers were determined by indirect ELISA assay. The titers of the supernatant antibodies reached 1: 104 and 1: 105 respectively. The supernatant of four cell lines was identified by Tricine-SDS-PAGE, and the protein which was not found in the cell culture medium was found in the supernatant, that is, the target protein of monoclonal antibody. The relative affinity and stability of monoclonal antibody were determined. The results showed that the relative affinity of McAb was 24.13 渭 g / mL ~ (-1) 24.65 渭 g / mL ~ (-1) ~ 24.95 渭 g / m ~ (-1) 路m ~ (-1) ~ (2) 渭 g / m ~ (-1), and it had good stability. The supernatant was purified by ammonium sulfate salting-out method, and further purified by affinity chromatography. The purified protein was determined by titer and Tricine-SDS-PAGE. The results showed that the titer of the McAb was slightly decreased, but it could reach 1: 104 after purification. The results showed that the molecular weight of light chain and heavy chain were about 26kuand 37ku. respectively. The results laid a foundation for the establishment of detection method for brucellosis.
【学位授予单位】：江苏科技大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R392

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