汉坦病毒核壳蛋白重组抗原的制备和基因分型研究

摘要

目的研制新的汉坦病毒核衣壳工程抗原，建立肾综合征出血热病毒检测和基因分型方法。rn方法以一组引物克隆全长S基因片段和N端的部分S基因片段，并使它们在T7系统进行融合表达和非rn融合表达。用另组引物建立了逆转录-聚合酶链式反应(RT-PCR)，检测我国不同地区由8种主要宿主分rn离的37个汉坦毒株，2个阳性标准对照毒株和5个阴性对照标本。对其中20个毒株的PCR扩增产物先rn后用Rsa Ⅰ和HindⅢ作二级酶切，建立了逆转录-聚合酶链式反应-限制性片段长度多态性分析(RT-PCR-rnRFLP)分型法。rn结果非融合表达产量虽不及融合表达高，但以非融合表达的两个S基因片段产物作间接ELISA的包被rn抗原，其工作浓度均达1：10000，显示出良好的生物活性。RT-PCR检测结果表明，所有的毒株扩增出汉坦rn特异性核酸组份(299 bp或577 bp)。用RT-PCR-RFLP法分型，上述毒株被定为汉滩型的9株，汉城型的rn8株，余3株未能定型。rn结论非融合表达的小分子抗原生物活性较高，有望替代天然抗原用于HV抗原抗体检测。RT-PCR法与rncELISA法比较，二者阳性检出率分别为100％和84.6％，符合率为84.6％，但前者比后者敏感性高15.4％。rnRT-PCR-RFLP分型法与血清学分型法所得结果具有很高的符合率，但RFLP法的分型率为85％(17/20)，rn血清法的分型率为55％(11/20)，前者比后者高30％。%Objective To identify new recombinant antigens with potential for diagnosis of hemorrhagic fever with renal rnsyndrom (HFRS) and establish reverse transcription-polymerase chain reaction-restriction fragments length rnpolymorphism (RT-PCR-RFLP) for genotyping of hantavirus.rnMethods One group of primers was used to clone the full-length S genome segment and the partial S rngenorme segment of the N-terminal. The two cloned genes were both fusionally expressed and nonrnfusionally expressed in the T7 system. The other group of primers was used to establish a RT-PCR method rnto detect RNAs in 37 virus isolates, 2 positive standard virus strains of hantavirus and 5 negative controls.rnThe method for typing RFLP was set up by digesting the PCR products of 20 virus isolates with Ras Ⅰ and rnHind Ⅲ.rnResults The non-fusionally expressed products with a working concentration of 1:10 000 by chapping rnenzyme-linked immunosorbent assay (cELISA), presented good biological activity though yields were lower rnthan that of the fusionally expressed products.The specific component of the hantavirus genome (299 bp or rn577 bp) wes seen in all viral samples. The genotyping of hantavirus showed that 9 out of the total were rnhantann (HTN) viruses, 8 were seol (SEO) viruses and 3 were not determined.rnConclusions The good working titrer of expressed recombinant antigen showed that it has the potential to rnreplace the natural antigen for detecting hantavirus antibodies. On comparison with cELISA, the detection rnrates for these two methods were 100% and 84.6%, and the coincidence rate was 84.6%. The former rnhad a 15.4% higher sensitivity than the latter. The typing efficiency of RT-PCR-RFLP and sero-typing rnmethod was 85% (17/20) and 55% (11/20), respectively, showing that the former was 30% higher than rnthe latter, while their results were highly consistant.