乙肝表面抗原单链抗体的构建、表达及其活性的检测

发布时间：2018-09-05 21:13

【摘要】：目的:构建含有乙肝表面抗原单链抗体基因的重组质粒,使其能够在真核细胞中表达出乙肝表面抗原的单链抗体,探讨其是否具有活性以及能否与人乙肝表面抗原特异性结合。方法:1.构建重组质粒合成含有乙肝表面抗原单链抗体基因的质粒,通过PCR获得乙肝表面抗原单链抗体的基因,将其插入真核表达载体p Fuse-m Ig G2-Fc中,构建重组质粒。2.重组质粒鉴定将重组质粒进行酶切反应及测序。3.收集目的蛋白将测序正确的重组质粒转染293F细胞,目的蛋白得以表达并分泌到细胞外,收集细胞培养的上清液得到目的蛋白,然后利用Protein G琼脂糖亲和层析柱和超滤管分别对其进行纯化浓缩,得到纯度及浓度较高的目的蛋白。4.目的蛋白纯度检测使用SDS-PAGE胶进行考马斯亮蓝染色以及western blotting对其进行检测。5.检测目的蛋白活性及特异性结合能力使用native-PAGE胶进行考马斯亮蓝染色、ELISA、免疫组化、免疫荧光等方法检测目的蛋白的活力及其对人乙肝表面抗原的特异性结合能力。结果:1.重组质粒进行酶切后经琼脂糖凝胶电泳出现2条理论大小的条带,测序结果显示插入的序列正确。2.得到相对分子量约为50KD的目的蛋白且纯度较高。3.ELISA、免疫组化、免疫荧光等试验结果证实目的蛋白对人乙肝表面抗原有特异性结合能力。结论:1.成功的构建了重组质粒。2.成功地在真核细胞中表达出乙肝表面抗原的单链抗体。3.验证了乙肝表面抗原的单链抗体与乙肝表面抗原有特异性结合能力。
[Abstract]:Aim: to construct a recombinant plasmid containing hepatitis B surface antigen (HBsAg) scFv gene to express HBV SFAV in eukaryotic cells, and to investigate whether it has activity and whether it can bind specifically with human HBsAg. Method 1: 1. The recombinant plasmid containing hepatitis B surface antigen single chain antibody gene was synthesized. The gene of hepatitis B surface antigen single chain antibody was obtained by PCR and inserted into the eukaryotic expression vector p Fuse-m Ig G2-Fc to construct the recombinant plasmid. 2. The recombinant plasmid was digested by enzyme digestion and sequenced. 3. The recombinant plasmid was transfected into 293F cells. The target protein was expressed and secreted out of the cell. The supernatant of cell culture was collected to obtain the target protein. Then Protein G agarose affinity chromatography column and ultrafiltration tube were used to purify and concentrate it to obtain the target protein with high purity and concentration. Objective to detect the purity of protein using SDS-PAGE gel for Coomassie brilliant blue staining and western blotting for its detection. The activity and specific binding ability of the target protein were detected by using native-PAGE glue for Coomassie brilliant blue staining ELISA.Immunohistochemical and immunofluorescence methods were used to detect the activity of the target protein and its specific binding ability to human hepatitis B surface antigen. The result is 1: 1. The recombinant plasmid was digested by agarose gel electrophoresis with two theoretical size bands. The sequencing results showed that the inserted sequence was correct. 2. The target protein with relative molecular weight of about 50KD was obtained and its purity was high. 3. The results of immunohistochemistry and immunofluorescence showed that the target protein had specific binding ability to human hepatitis B surface antigen. Conclusion 1. The recombinant plasmid. 2. 2 was successfully constructed. The single chain antibody of hepatitis B surface antigen was successfully expressed in eukaryotic cells. The specific binding ability of single chain antibody of hepatitis B surface antigen with hepatitis B surface antigen was verified.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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