人源抗HBsAg抗体Fd段和L链高效表达菌株的筛选

The efficient expression of anti-HBsAg human Fd and L chain in E.coli

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:Choosing efficient expression vector express anti HBsAg Fd and L chain in E.coli to produce anti HBsAg human Fab.Purificating inclusion bodies to denaturating and refolding the protein.Methods:Expression vector of PQE32 Fd、PQE32 L was constructed and transformed into E.coli strain M15,efficient expression clone was screened by SDS PAGE.Results:After induced by IPTG,M15 PQE32 Fd、M15 PQE32 L expressed insoluble recombinant protein in inclusion bodies.The Fd、L chain gene sequence conform with that reported in NCBI BLAST.Conclusion:The M15 PQE32 expression system is stable and efficient for expressing anti HBs human Fd、L chain.The denaturation and protein refolding of the anti HBsAg Fab expressed in inclusion bodies need to be studied further.