【摘要】：目的:構(gòu)建含有乙肝表面抗原單鏈抗體基因的重組質(zhì)粒,使其能夠在真核細(xì)胞中表達(dá)出乙肝表面抗原的單鏈抗體,探討其是否具有活性以及能否與人乙肝表面抗原特異性結(jié)合。方法:1.構(gòu)建重組質(zhì)粒合成含有乙肝表面抗原單鏈抗體基因的質(zhì)粒,通過PCR獲得乙肝表面抗原單鏈抗體的基因,將其插入真核表達(dá)載體p Fuse-m Ig G2-Fc中,構(gòu)建重組質(zhì)粒。2.重組質(zhì)粒鑒定將重組質(zhì)粒進(jìn)行酶切反應(yīng)及測(cè)序。3.收集目的蛋白將測(cè)序正確的重組質(zhì)粒轉(zhuǎn)染293F細(xì)胞,目的蛋白得以表達(dá)并分泌到細(xì)胞外,收集細(xì)胞培養(yǎng)的上清液得到目的蛋白,然后利用Protein G瓊脂糖親和層析柱和超濾管分別對(duì)其進(jìn)行純化濃縮,得到純度及濃度較高的目的蛋白。4.目的蛋白純度檢測(cè)使用SDS-PAGE膠進(jìn)行考馬斯亮藍(lán)染色以及western blotting對(duì)其進(jìn)行檢測(cè)。5.檢測(cè)目的蛋白活性及特異性結(jié)合能力使用native-PAGE膠進(jìn)行考馬斯亮藍(lán)染色、ELISA、免疫組化、免疫熒光等方法檢測(cè)目的蛋白的活力及其對(duì)人乙肝表面抗原的特異性結(jié)合能力。結(jié)果:1.重組質(zhì)粒進(jìn)行酶切后經(jīng)瓊脂糖凝膠電泳出現(xiàn)2條理論大小的條帶,測(cè)序結(jié)果顯示插入的序列正確。2.得到相對(duì)分子量約為50KD的目的蛋白且純度較高。3.ELISA、免疫組化、免疫熒光等試驗(yàn)結(jié)果證實(shí)目的蛋白對(duì)人乙肝表面抗原有特異性結(jié)合能力。結(jié)論:1.成功的構(gòu)建了重組質(zhì)粒。2.成功地在真核細(xì)胞中表達(dá)出乙肝表面抗原的單鏈抗體。3.驗(yàn)證了乙肝表面抗原的單鏈抗體與乙肝表面抗原有特異性結(jié)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.
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R392

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本文編號(hào)：2225479