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目的 :选用高效表达载体分别高效表达人源抗HBsAg抗体的Fd段和L链 ,经包涵体纯化和变性复性使Fd段和L链之间形成二硫键 ,最终制备有活性、高产量的人源抗HBsAg基因工程抗体Fab。方法 :用PCR法从可溶性表达重组质粒抗HBsAgFadComb3扩增Fd段和L链后分别构建高效表达载体PQE32 Fd和PQE32 L ,并分别导入大肠杆菌M15中进行表达 ,用SDS PAGE筛选出高效表达克隆。结果 :SDS PAGE筛选的高效表达克隆M15 PQE32 Fd和M15 PQE32 L经IPTG诱导后以包涵体形式表达 ,其表达量很高。从高效表达克隆重新扩增Fd段和L链后进行测序鉴定发现所得的序列与已报道的抗HBsAg抗体Fab基因吻合率为 97%。结论 :虽然已有表达可溶性人源抗HBsAg基因工程抗体Fab的报道 ,但因其表达量低而不能实际应用。筛选出高效表达人源抗HBsAg抗体Fd段和L链的克隆 ,因为包涵体形式表达需经变性复性 ,虽然变性复性后其产量会受影响 ,但因表达量很高 ,所以还是有很高的实际应用价值。其包涵体变性复性条件仍需进一步探讨。
OBJECTIVE: To efficiently express Fd and L chains of human anti-HBsAg antibody by high expression vector respectively. After purification and denaturalization renaturation, disulfide bond was formed between Fd segment and L chain. Finally, an active and high yield Human anti - HBsAg genetically engineered antibody Fab. Methods: High expression plasmids PQE32 Fd and PQE32 L were constructed by PCR from recombinant soluble plasmid HBsAgFadComb3. The recombinant plasmids were introduced into E. coli M15 for expression respectively. Highly expressed clones were screened by SDS PAGE. Results: The highly expressed clones M15 PQE32 Fd and M15 PQE32 L screened by SDS PAGE were expressed in inclusion bodies after IPTG induction, and their expression levels were high. The sequences of Fd and L chains were amplified by high-performance cloning and sequenced. The results showed that the obtained sequence was 97% identical to the reported anti-HBsAg Fab gene. Conclusion: Although the expression of soluble human anti-HBsAg genetically engineered Fab has been reported, its low expression can not be applied in practice. The clones that efficiently expressed Fd segment and L chain of human anti-HBsAg antibody were screened out. Because the expression of the inclusion body needs to be denatured and refolded, although its yield will be affected after denaturation and refolding, the expression is still very high High practical value. Its inclusion body denaturation refolding conditions still need to be further explored.