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目的构建人源Fab抗体文库,筛选抗HBsAg抗体片段并进行初步鉴定。方法收集20份临床检验废弃的成人乙肝感染者淋巴细胞,抽提总RNA,逆转录成cDNA,构建抗乙肝病毒人源免疫型Fab抗体文库。以HBsAg包板进行4轮循环的吸附-洗脱-扩增,挑单克隆用Phage-ELISA、DNA测序筛选阳性克隆,对阳性克隆进行可溶性表达,并用ELISA对其特异性进行鉴定。结果构建的人源Fab型抗体文库的库容为2.0×108,并具有良好的多样性。经过4轮筛选,成功获得4株能与HBsAg结合的人源抗体克隆,分别命名为hFabHB1、hFabHB2、hFabHB3和hFabHB4。对其中的hFabHB1进行可溶性表达,ELISA鉴定阳性。结论成功构建了抗乙肝病毒人源免疫型Fab抗体文库,从中筛选获得的4株人源Fab抗体片段有望在乙型肝炎的预防和治疗上发挥作用。
Objective To construct human Fab antibody library and screen anti-HBsAg antibody for preliminary identification. Methods Totally 20 lymphocytes were isolated from clinical samples from adult patients with hepatitis B virus infection. Total RNA was extracted and reverse transcribed into cDNA to construct a library of anti-hepatitis B virus human immunotype Fab antibody. After 4 rounds of adsorption-elution-amplification with HBsAg on the plate, positive clones were screened by Phage-ELISA and DNA sequencing. The positive clones were expressed in soluble form and their specificities were identified by ELISA. Results The constructed human Fab antibody library had a capacity of 2.0 × 108 with good diversity. After 4 rounds of screening, four human antibody clones capable of binding to HBsAg were successfully obtained and named hFabHB1, hFabHB2, hFabHB3 and hFabHB4, respectively. The soluble expression of hFabHB1 was confirmed by ELISA. Conclusion The library of anti-hepatitis B virus human immunotype Fab antibody was successfully constructed. The four human Fab antibody fragments screened from it are expected to play a role in the prevention and treatment of hepatitis B.