汉滩病毒截短核蛋白在大肠杆菌中表达及其抗原分析

EXPRESSION OF TRUNCATED REGIONS OF THE S GENOME SEGMENT OF HANTAAN VIRUS IN E.COLI AND THE ANTIGEN OF THE RECOMBINANT PROTEINS

目的 体外克隆表达肾综合征出血热病毒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－ １１８ Ｓ片段部分编码基因（ＳＡ、ＳＢ和ＳＣ），并分别克隆入Ｔ７ 原核表达载体，在大肠杆菌中表达；免疫印迹试验和酶联免疫吸附反应分析重组抗原活性。结果 截短核蛋白得到有效表达，分子量分别约为４．５ＫＤ（ｒＳＡ）、２５ｋＤ（ｒＳＢ）和１５．５ｋＤ（ｒＳＣ），Ｗｅｓｔｅｒｎ－ ｂｌｏｔ分析表明２５ｋＤ截短核蛋白具有较好的抗原活性；粗提抗原Ｄｏｔ－ＥＬＩＳＡ 方法检测ＨＦＲＳ患者血清结果与ＩＦＡ 法一致；２５ｋＤ重组小片段抗原与抗ＨＦＲＳＶ单克隆抗体５Ｈ５、Ｂ１１ 及Ｈ７可发生特异性反应。结论 汉滩病毒核蛋白的主要抗原决定簇主要集中在氨基端；

Aim\ In order to obtain a major antigenic domain for the human humoral response to hantann virus nucleocapsid protein and to use this information to develop the new genetic engineering diagnostic reagents,several truncated coding regions of the small(S) genome segment of hantaan virus in E.coli were constructed and expressed.Methods\ Fragments of truncated conserve gene(SA、SB、SC) were cloned into poly-histidine fusion protein expression vector PET-28c by PCR and restriction digest methods,and the recombinant proteins were examined by immunoblots and ELISA with polyclonal and monoclonal antibodies.Results\ The molecular weights of SA,SB and SC were 4500,25000 and 15000 respectively.Western-blot analyses identified recombinant SB protein presented the specific HFRSV antigenicity as entiere Sprotein and was used for the detection of specific IgG and IgM against HFRSV in patients serum by Dot-ELISA after purification.The specificity and stability of the test were equal to IFA.Conclusion\ A dominating antigenic region of Hataan virus nucleocapsid protein located at the amino-terminus.These recombinant truncated proteins are useful in the serodiagnosis of HFRSV infection.