摘要
目的:构建人源化的抗人肺癌单域抗体hu3D3VH基因,在大肠杆菌中表达,对其蛋白活性进行分析。方法:采用CDR移植技术对mAb3D3的重链可变区进行人源化,通过重叠PCR获得hu3D3VH的基因。构建pET22(b+)/hu3D3VH表达载体,并转化大肠杆菌BL21(DE3),在IPTG诱导下表达。表达产物通过Ni亲和层析柱纯化。采用间接ELISA和竞争抑制ELISA法进行活性分析。结果:通过重叠PCR获得序列正确的目的基因。目的蛋白以包涵体的形式表达,表达量占菌体总蛋白的30%以上。纯化后,目的蛋白的纯度达95%以上。hu3D3VH具有与亲本抗体相同的抗原反应性,并能抑制mAb3D3与L342细胞的结合。结论:获得的人源化单域抗体hu3D3VH,保留了与mAb3D3相同的反应性和特异性,为进一步临床应用奠定了基础。
AIM. To construct the gene of humanized single-domain antibody hu3D3V, against human lung cancer, to express it in E. coil and analyze its activity. METHODS： The mAb3D3VH was humanized by using CDRs grafting technique. The hu3D3VH gene was assembled by overlapping PCR. The expression vector pET22（b ＋ ）/hu3D3VH was constructed and transformed into E. coil BL21 （ DE3 ） and the hu3D3VH protein was expressed under IPTG induction. After purification by Ni2^＋ -affinity chromatographic column, the activity of hu3D3VH protein was analyzed by indirect ELISA and competitive inhibition ELISA. RESULTS： The target gene with correct sequence was obtained by overlapping PCR. The hu3D3V, protein was expressed as inclusion bodies with the yield of more than 30% of total bacterial proteins. After purification, the purity of hu3D3V, was more than 95%. The reactivity of purified protein was the same as parent antibody, and could inhibit the binding of mAb3D3 to L342 cells. CONCLUSION： The hu3D3V, still retains the reactivity and specificity of mAb3D3, which lays the foundation for its further clinical application.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2006年第2期193-195,199,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
福建省自然科学基金资助项目(No.C0410004)
关键词
人源化
抗人肺癌单域抗体
表达
活性分析
humanization
single-domain antibody agai-nst human lung cancer
expression
activityanalysis
