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目的体外克隆表达汉滩病毒全长及部分核蛋白编码基因，筛选病毒核蛋白在人体体液应答中的主要抗原决定簇区，以期获得高质量核蛋白抗原，为研制新型肾综合征出血热（ＨＦＲＳ）基因工程诊断试剂奠定基础。方法利用聚合酶链反应（ＰＣＲ）和限制性内切酶酶切的方法分离汉滩病毒７６－１１８Ｓ基因全长及部分氨基端编码基因（Ｓ、ＳＡ、ＳＢ、ＳＣ和ＳＤ），分别将各编码基因克隆入Ｔ７原核表达载体，在大肠杆菌中表达；免疫印迹试验（Ｗｅｓｔｅｒｎｂｌｏｔａｎａｌｙｓｉｓ）分析重组抗原活性。结果完整和截短核蛋白皆得到有效表达，相对分子质量分别为５００００（ｒｅｃｏｍｂｉｎａｎｔＳｐｒｏｔｅｉｎ，ｒＳ）、４５００（ｒＳＡ）、２２０００（ｒＳＢ）、２５０００（ｒＳＣ）和２７０００（ｒＳＤ），Ｗｅｓｔｅｒｎｂｌｏｔ分析表明截短核蛋白（ｒＳＢ、ｒＳＣ和ｒＳＤ）具有较好的抗原活性；粗提抗原Ｄｏｔｂｌｏｔ方法检测ＨＦＲＳ患者血清结果与ＩＦＡ法一致；重组小片段抗原与抗汉滩病毒单克隆抗体５Ｈ５及Ｈ７可发生特异性反应。结论汉滩病毒核蛋白的氨基端含有一主要抗原决定簇区；原核表达的截短核蛋白在ＨＦＲＳ的血清学诊断中具有一定的价值。
Objective To clone and express the full-length Hantaan virus and some nucleoprotein genes in vitro and screen for the major antigenic determinants of human humoral response in order to obtain high-quality nuclear protein antigens. In order to develop new hemorrhagic fever with renal syndrome (HFRS) Gene engineering diagnostic reagents to lay the foundation. Methods The full-length and some amino-terminal coding genes (S, SA, SB, SC and SD) of Hantaan virus 76-118S gene were isolated by polymerase chain reaction (PCR) and restriction endonuclease digestion. The coding gene was cloned into the prokaryotic expression vector T7 and expressed in E. coli. Western blotting was used to analyze the recombinant antigen activity. Results The complete and truncated nuclear proteins were all expressed efficiently. The relative molecular masses were 50 000 (rS), 4500 (rSA), 22000 (rSB), 25000 (rSC) and 27000 (rSD) respectively. Shorter nuclear proteins (rSB, rSC and rSD) had good antigenic activity. The results of Dot-blot assay of crude extracts with HFRS were consistent with those of IFA. Recombinant small fragment antigens and anti-Hantaan virus monoclonal antibodies 5H5 and H7 were Specific reaction occurred. Conclusion The amino terminal of Hantaan virus nucleoprotein contains a major antigenic determinant. The prokaryotic expression of truncated nucleoprotein is of value in the serological diagnosis of HFRS.