摘要
根据HFRSV汉滩型（HTN）代表株76－118和汉城型（SEO）代表株R22基因资料，设计了两组引物，用电脑软件分析证明设计符合引物标准。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融合表达。非融合表达产量虽不及融合表达高，但生物活性好。以非融合表达的两个S基因片段产物作间接ELISA的包被抗原，其工作浓度均达1：100000用另一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种主要宿主分离的37个HFRSV毒株，2个阳性标准对照毒株和5个阴性对照标本，并与cELISA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％。对其中20个毒株的PCR扩增产物先后用RsaⅠ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－RFLP）分型法。被定为HTN型的9株，SEO型的8株，余3株未能定型。此20个毒株曾用血清学方法分型，仅11株分型成功．与RT-PCR－RFLP法结果符合。分型成功率RT-PCR－RFLP法比血清法高30％。
Two groups of primers were designed according to the gene backgrounds of prototypevirus of HTN and SEO serotypes and corrected by computer. One group of primers was used toclone entire S genome segment and partial S genome segment with respect to N-terminal. Thetwo cloned genes were fusionally expressed and non-fusionally expressed by T7 system. The nonfusionally expressed products whose working concentration were 1: 10000 presented a good biological activity though their yields were lower than the fusionally expressed products. The othergroup of primers was used to establish a method of RT-PCR to detect RNAs of 37 virus isolates ofHFRSV 2 positeve standard viruses and 5 negtive controls. on comparision with that ofcELISA, the detecting rates of two methods were 100 % and 84.6% respectively, the coincidental rate was 84. 6 % while the former had a 15. 4 % higher sensitivity than the latter. The typingmethod of RFLP was set up by digesting the PCR products of 20 virus isolates with Ras Ⅰ andHind Ⅲ resulting that 9 out of the total were HTN, 8 were SEO and 3 were not determined respectively. The same 20 viruses have been previoushy typed using serotyping method resultingthat only 11 could be typed successfully, showing a high consistency with that of RT-PCR-RFLPmethod and a 3o % lower typing efficiency than the latter.
出处
《中国病毒学》
CSCD
1999年第4期314-321,共8页
Virologica Sinica
基金
国家自然科学基金
