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目的 构建抗ＨＢｓＡｇ人源单链抗体与人干扰素α的融合分子－抗ＨＢｓＡｇ单链抗体靶向干扰素，并在大肠杆菌中表达出活性融合蛋白。方法 利用限制性内切酶分别从含有抗ＨＢｓＡｇ人源单链抗体与人干扰素α的质粒中切出目的基因，连接到ｐＥＴ２２ｂ质粒中，构建成单链抗体靶向干扰素表达载体。利用ＳＤＳ—ＰＡＧＥ电泳、竞争性抑制实验与抗病毒实验对表达产物进行分析鉴定。结果 构建出含有抗体靶向干扰素融合基因的表达质粒，诱导１２ｈ后，在ＳＤＳ—ＰＡＧＥ上可以观察到相对分子质量约４５×１０４的目的蛋白，竞争性抑制实验与抗病毒实验表明表达的融合蛋白保留了单链抗体的ＨＢｓＡｇ特异结合活性与干扰素的抗病毒活性。结论 抗ＨＢｓＡｇ单链靶抗体靶向干扰素融合分子的构建及在大肠杆菌中的表达是成功的。
Objective To construct a fusion molecule of anti-HBsAg human single-chain antibody and human interferon-alpha targeting anti-HBsAg single-chain antibody targeting interferon, and express the active fusion protein in E. coli. METHODS: Restriction endonucleases were used to excise the target gene from plasmids containing human anti-HBsAg scFv and human interferon alpha, and ligated into pET22b plasmid to construct a single-chain antibody targeted interferon expression vector. The SDS-PAGE electrophoresis, competitive inhibition experiments and anti-virus experiments were used to analyze and identify the expression products. RESULTS: An expression vector containing the antibody-targeting interferon fusion gene was constructed. After induction for 12 hours, the target protein with a relative molecular mass of about 45.times.104 was observed on SDS-PAGE. The competitive inhibition experiment and the antiviral experiment showed fusion of the expression. The protein retains the HBsAg-specific binding activity of the single-chain antibody and the antiviral activity of the interferon. Conclusion The construction of an anti-HBsAg single-stranded target antibody-targeting interferon fusion molecule and its expression in E. coli are successful.