论文部分内容阅读
目的探讨如何在无药物抗性改变；无转化细胞生物指示系统改变的情况下准确快速地筛选出人源抗乙型肝炎病毒表面抗原（ＨＢｓＡｇ）抗体Ｆａｂ片段／ＩＦＮ－α融合蛋白原核表达载体。方法在 ＩＦＮ－α基因两端加上 ＸｂａⅠ酶切位点后用 ＰＣＲ法扩增，再重组入以抗体库技术筛选得到的人源抗ＨＢｓＡｇ－Ｆａｂ片段表达载体的相应位点上，构建 ａｎｔｉ－ＨＢｓＡｇ－Ｆａｂ／ＩＦＮ－α融合蛋白表达载体。取 ＰＣＲ扩增得到的 ＩＦＮ－α ＤＮＡ，用地高辛标记系统进行化学标记，制备 ＩＦＮ－α ＤＮＡ探针。取构建的融合蛋白表达载体转化受体菌．增菌后，以溶菌酶加煮沸法批量制备载体 ＤＮＡ，点样于硝酸纤维膜上， ８０℃烤２ｈ，预杂交液 ４２℃，２ｈ膜预杂交，标记探针 ４２℃， ４ｈ杂交，洗膜后用酶标抗地高辛抗体显色。同时设单纯人源抗 ＨＢｓＡｇ－Ｆａｂ片段表达载体为阴性对照， ＩＦＮ－α质粒为阳性对照。阳性克隆再以 ＳａｃＩ和 ＸｂａⅠ酶切，琼脂糖电泳鉴定。结果４０株转化细胞中筛选出７个阳性克隆，酶切鉴定显示两者符合率达１００％，成功地筛选出了ａｎｔｉ－ＨＢｓＡｇ－Ｆａｂ／ＩＦＮ－α融合蛋白表达载体。结论人源ＨＢｓＡｇ抗体Ｆａｂ片段／ＩＦＮ－α融合蛋白表达载?
Objective To explore how to screen the prokaryotic expression vector of human anti-hepatitis B virus surface antigen (HBsAg) Fab fragment / IFN-α fusion protein accurately and rapidly in the absence of change of drug resistance and in the absence of altered biological indicator system. Methods After the site of Xba Ⅰ restriction enzyme was added to both ends of IFN-α gene, it was amplified by PCR and recombined into the corresponding site of the human anti-HBsAg-Fab fragment screened by antibody library technology to construct anti- HBsAg-Fab / IFN-α fusion protein expression vector. The IFN-α DNA obtained by PCR amplification was chemically labeled with a digoxigenin labeling system to prepare an IFN-α DNA probe. The constructed fusion protein expression vector was transformed into recipient bacteria. After enrichment, lysozyme plus boiling method batch preparation of vector DNA, spotted on a nitrocellulose membrane, baked at 80 ℃ 2h, pre-hybridization solution 42 ℃, 2h membrane prehybridization, labeling probe 42 ℃, 4h hybridization, washing membrane After using enzyme-labeled anti-digoxin antibody color. At the same time, pure human anti-HBsAg-Fab fragment expression vector was used as a negative control, IFN-α plasmid as a positive control. The positive clones were digested with SacI and XbaI and identified by agarose gel electrophoresis. Results The positive clones were screened from 40 transformed cells. The coincidence rate of the two clones was 100%. The anti-HBsAg-Fab / IFN-α fusion protein expression vector was successfully screened. Conclusion The expression of human HBsAg Fab fragment / IFN-α fusion protein