摘要
克隆人CD40 L基因功能性片段(E107-L261),并在CHO细胞中进行表达。从健康人扁桃体组织中提取总RNA,通过RT-PCR技术,扩增CD40 L胞外区cDNA功能性片段,经DNA测序证实后,将得到的片段基因插入pcDNA3.0表达载体,构建了pcDNA3.0-hCD40 L真核表达载体。将重组质粒pcDNA3.0-hCD40 L转染CHO细胞,经G418筛选出阳性细胞克隆,扩大培养,提取细胞总蛋白,用SDS-PAGE分离并western blot-ting鉴定其特异性,通过小鼠脾淋巴细胞增值实验检验其生物学活性。结果,CD40 L胞外区功能性片段(E107-L261)cDNA被正确克隆到真核表达载体pcDNA3.0中并在CHO中成功表达,具有免疫学和生物学活性。结论:真核表达载体pcDNA3.0-CD40 L的成功构建并在CHO中成功表达。
To clone the functional fragment of human CI）40 L （E107-L261） gene and to express it in CHO cells. RT-PCR was used to amplify the eDNA of the functional fragment of hCD40 L from the total RNA of activated human tonsillitis. After verification sequencing, the DNA fragment was cloned into expression plasmid pcDNA3. 0, and the eukaryotic expression vector pcDNA 3. 0-hCD40 L was constructe＆ The recombinant cloning vetor pcDNA3. 0-hCD40 L was transfected into CHO cells, and the consistently transfected cells were screened with G418. The total protein was extracted from positive clones, and seperated by SDS-PAGE. Westera-blotting was used to identify the immunological activity. Its biological activity was checked by enhancing lymphocytes of mice. Results The functional fragment in extracellular regin of hCD40 L was cloned correctly into eukaryotic expression vector pcDNA 3.0 and expressed successfully in CHO cells. It has immunological activity and Biological activity.
出处
《药物生物技术》
CAS
CSCD
2008年第2期115-117,共3页
Pharmaceutical Biotechnology
