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目的克隆、真核表达严重急性呼吸综合征冠状病毒(SARS-CoV)受体ACE2基因,建立安全、可靠的SARS-CoV保护性体液免疫评价体系。方法采用Trizol一步法提取人右心衰心房组织总RNA,逆转录巢式聚合酶链反应(RT-nested-PCR)扩增ACE2全长基因,克隆人pcDNA4/HisMax-TOPO表达载体,重组质粒转染293T细胞进行瞬时表达,Western Blot检测其真核表达;建立细胞融合抑制实验以检测SARS-CoV中和抗体,并与SARS-CoV假病毒中和试验进行平行比较。结果重组质粒可在真核细胞中表达ACE2蛋白,其蛋白表达细胞可与S蛋白表达细胞产生细胞融合现象,并可用于中和抗体的检测;其结果与SARS-CoV假病毒中和试验较为一致。结论克隆和真核表达了SARS-CoV受体ACE-2基因;基于其蛋白真核表达的细胞融合抑制实验可用于SARS-CoV中和抗体的检测。
Objective To clone and express the ACE2 gene of severe acute respiratory syndrome coronavirus (SARS-CoV) receptor and establish a safe and reliable evaluation system of SARS-CoV protective humoral immunity. Methods Trizol was used to extract total RNA of human atrial tissue from human right heart failure. The full-length cDNA of ACE2 was amplified by RT-nested-PCR. The recombinant plasmid pcDNA4 / HisMax-TOPO was cloned. 293T cells were transiently transfected and eukaryotic expression was detected by Western Blot. Cell fusion inhibition assay was established to detect SARS-CoV neutralizing antibodies, and compared with the neutralization test of SARS-CoV pseudovirus. Results Recombinant plasmids could express ACE2 protein in eukaryotic cells. The expression of ACE2 protein in Eukaryotic cells resulted in cell fusion with S protein-expressing cells, and could be used to detect neutralizing antibodies. The results were in good agreement with the neutralization test of SARS-CoV pseudovirus . Conclusion The SARS-CoV receptor ACE-2 gene was cloned and eukaryotic expressed. The cell fusion inhibition assay based on the eukaryotic expression of SARS-CoV receptor could be used to detect SARS-CoV neutralizing antibody.