IgG Fab-BPI融合蛋白真核表达载体的构建及其在CHO细胞中的表达

The cDNA cloning and expression of Fab-BPI fusion protein in CHO cell line

目的构建抗抗LPSFab-BPI真核表达载体,并在CHO细胞中表达。方法通过RT-PCR,获得广谱抗LPSmAbC3A2IgG的Fd和完整轻链LCcDNA片段。在分别构建了pcDNA3-Fd和pcDNA3-LC表达载体的基础上,将包括BPIN端抗LPS活性中心的180bpDNA片段,通过一段长16个氨基酸的linker连接于上述Fab表达载体中Fd基因的下游,构建成Fab-BPI融合蛋白FB真核表达载体。将pcDNA3-LC质粒中包括hCMV启动子、LC和BGHpolyA等序列在内的片段,插入到pcDNA3-Fd中hCMV启动子上游,从而构建成单质粒的Fab-BPI表达载体pcD-NA3-FB,转染真核细胞。结果序列测定表明,重组质粒构建正确。pcDNA3-FB转染CHO细胞后,经ELISA、免疫荧光和mR-NA鉴定表达成功,免疫印迹试验显示,与抗κ轻链和抗BPI抗体反应的蛋白区带的Mr均为60000左右。交叉反应试验证明,Fab和FB与多种革兰氏阴性菌的LPS有交叉反应性。结论成功地在CHO细胞中表达出了抗LPSFab-BPIFB融合蛋白。FB作为融合蛋白,集广泛交叉反应性和强大中和活性于一身,有可能成为更适于临床应用的新的抗LPS免疫制剂。

Aim To construct eukaryotic expression vector of Fab -BPI,and express it in CHO cell line.Methods Fd and light chain cDNA were obtained by RT -PCR from mRNA of C3A2cells.On the basis of constructing recombinant vector pcDNA3-Fd and pcDNA3-LC,an180bp DNA fragment containing bioactive center of BPI was cloned and linked to the downstream of Fd sequen ce in vector pcDNA3-Fd.Then a 1.7kb fragment was amplified from pcDNA3-LC,containing hCMV promoter,LC and BGH polyA sequence,and inserted into upstream of hCMV promoter in vector pcDNA3-Fd to construct Fab -BPI fusion pretion expression vector pcDNA3-F B.pcDNA3-Fab was transfected into CHO cell line,and screened with G418,and then identified byELISA and immunofluorescent staini ng.Results ELISA and im-munofluorescence staining reveale d that Fab -BPI fusion protein(FB)were expressed in the culture medium and cell cytoplasm,which pre-sented as a 60kd protein in western -b lot,and all mRNA of Fd,LC and BPI could be detected by RT -PCR.Cross -reactivity of Fab and FB were identified by ELISA with R -LPSs and S -LPSs.Conclusion The successful expression of Fab -BPI fu sion protein(FB)laid the foundaion for study of sepsis treatment.With t he broad cross -reactivity and stron g neutralization of LPS,FB protein ma y become a new immunetherpy a-gent for clinical application.