抗CD20嵌合抗体片段F(ab′)2突变体在大肠杆菌中的高效分泌表达

High Level Expressiong of Chimeric Antibody Fragment F(ab′)_2 Directed Against CD20 in Escherichia coli

构建抗CD2 0嵌合抗体片段F(ab′) 2 突变体 ,研究其在大肠杆菌中的高效表达及其表达产物的生物学活性。采用PCR法构建抗CD2 0嵌合抗体片段F(ab′) 2 突变体 ,并用双脱氧终止法测定DNA序列 ;采用 19L发酵罐高密度发酵抗CD2 0嵌合抗体片段F(ab′) 2 突变体 ,采用亲和色谱和分子筛色谱法纯化表达产物 ,并用SDS PAGE和薄层激光扫描鉴定纯化产物 ;采用活细胞间接免疫荧光法测定纯化产物与靶细胞的结合活性 ;MTT法测定纯化产物对Raji细胞的生长抑制作用 ,并研究其作用机理。DNA序列测定结果表明 ,抗CD2 0嵌合抗体片段F(ab′) 2 突变体已成功构建 ,表达可溶性产物的产量达 36 0mg L ,具有与Raji细胞 (CD2 0 + )结合的活性 ,并抑制Raji细胞的生长 ,其作用机理为诱导Raji细胞凋亡。

The use of tumor antigen specific antibody for the delivery of therapeutic agents offers the possibility of targeting therapy with reduced toxicity to normal tissues compared to conventional treatments. In previous work, the human mouse chimeric antibody fragment Fab′ directed against CD20 was constructed from the new anti CD20 antibody HI47 (a mouse IgG3, κ).The chimeric antibody fragment Fab′ could reduce its antigenicity, but the yield, quality and affinity of chimeric antibody fragment Fab′ restrict its use. To improve affinity of chimeric antibody fragment Fab′, a new phasmid pYZcpp3, which expresses chimeric antibody fragment F(ab′) 2, was constructed by adding a sequence encoding a small peptide, (CPP) 3, to C terminus of heavy chain constant region of chimeric antibody fragment Fab′. Using the pYZcpp3 to transform E.coli . 16c9, the genetically engineered bacteria 10916# was obtained. 10916# can secret the soluble chimeric antibody fragment Fab′ and F(ab′) 2 into periplasmic. The yield was up to 360 mg/L with the percent of F(ab′) 2 up to 45% in 19L fermentor by the high density fermentation technology. Without denaturation and renaturation, the F(ab′) 2 has possessed the native three dimensional structure. The purity of F(ab′) 2 was more than 90% after the purification of protein G affinity chromatography and S200 size exclusion chromatography. The F(ab′) 2 could distinguish and bind to Raji cells (CD20 +) by FACS. F(ab′) 2 could inhibit the proliferation of Raji cells in vitro by MTT, IC50 was 22 8 μg/mL. HI47 and its chimeric fragments F(ab′) 2 induced a significant level of apoptosis (23 5%, 20 8%, respectively), independent of any cross linking agents, in Raji cells after 24 h incubation. The chimeric antibody fragment F(ab′) 2 directed against CD20 is possible to apply to tumor therapy in clinic in the future.