【摘要】 目的:构建表达人干燥综合征抗原A(SSA)的毕赤酵母分泌型表达载体。方法:以人白血病淋巴细胞HL-60株cDNA为模板,PCR扩增目的基因SSA,纯化,双酶切,DNA浓缩回收,插入酵母分泌型表达载体pPIC9k,热冲击法转化感受态大肠杆菌JM109,阳性克隆提取质粒,双酶切鉴定并测序。结果:PCR产物大小符合要求,重组质粒pPIC9k-SSA双酶切鉴定与预期一致,测序结果正确。结论:成功构建SSA毕赤酵母分泌型表达载体,为下一步取代从动物胸腺中人工提取低纯度、低产量的SSA抗原,研制新型抗SSA诊断试剂盒打下基础。更多还原

【Abstract】 Objective To construct a secreted expression vector on human Sjogren′ s syndrome antigen A (SSA) in Pichia pastoris. Mothods Gene SSA was amplified with cDNA template from HL- 60 cell line by PCR. The PCR product was purified, digested by two restriction enzymes, and then inserted into yeast secretion expression vector pPIC9k dealt with the same way. The recombined plasmid was transformed into competent E.coli JM109 by heat treatment. The positive transformant plasmid was digested by two restriction enzymes again and identified. Results The product of PCR was found no error. The recombinant plasmid pPIC9k- SSA clone was identified by two restriction enzymes and sequencing was in accordance with our prediction. Conclusion The successful construction of secreted expression vector on SSA in Pichia pastoris provides a basic support for further work to replace the SSA in low purity and yield from the animal thymus, and will help to develop a new diagnostic test kit for detecting anti- SSA.更多还原