摘要
目的 利用pBAd/gⅢA表达载体和大肠杆菌Top 10克隆和表达κ、λ轻链C区基因片段,探索生物工程技术制备小分子抗原的可行性。方法 经RT-PCR从正常人的外周血淋巴细胞mRNA中扩增出κ、λ轻链恒定区基因,限制性双酶切后分别克隆到同样酶切的pBAd/gⅢ A质粒中,转化大肠杆菌。酶切鉴定与PCR法筛选出阳性克隆菌株,经Arabinose诱导表达出κ、λ轻链片段。Ni^(2+)-NTA亲和层析纯化后,以150 mg/mL SDS-PACE与Western blot鉴定纯化产物,并测定蛋白的相对浓度。结果 SDS-PACE与Western blot结果显示,纯化后的产物在M_r 16000处呈致密的单一条带,与克隆基因表达产物的相对分子质量一致,该带经HRP标记的羊抗人IgG Fab抗体证实为抗体片段成分。结论 κ、λ轻链C区基因的pBAd/gⅢA表达在技术上是可行的,该原核系统表达的片段具抗原性,为后续的开发应用打下了基础。
Objective To construct and express human antibody κ,λ light chain constant gene. Methods The mRNA isolated from peripheral blood lymphocytes of four healthy donors were reverse transcribed and amplified for human immunoglobulin κ,λ light chain canstant region with specific primer. The light chain genes were subsequently inserted into the corresponding sites of pBAd/gⅢ A to generate a κ-pBAd/gⅢ A and λ-pBAd/gⅢ A plasmid. The plasmid transformed into E. coli. Top10, and clones with correct insert genes were selected. Induced by arabinose, the expression protein was found in the periplasmic by SDS-PAGE. Results After Ni-NTA purification. The molecular weigh of the purified protein was about M, 16 000 and the protein showed good immunoreactivity with the HRP-Fab antibody by SDS-PAGE and western blot. Conclusion Expression of human antibody κ,λ light chain constant gene was available in technology. It lays the foundatian for further applications.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2003年第5期394-396,共3页
Immunological Journal
