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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。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 2 0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。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的包被抗原 ,其工作浓度均达 1∶10 0 0 0 ,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特异性核酸组份(2 99bp或 5 77bp)。用RT PCR RFLP法分型 ,上述毒株被定为汉滩型的 9株 ,汉城型的 8株 ,余 3株未能定型。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cELISA法比较 ,二者阳性检出率分别为 10 0 %和 84 .6% ,符合率为 84 .6% ,但前者比后者敏感性高 15 .4 %。RT 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/ 2 0
Objective To develop a new Hantavirus nucleocapsid engineering antigen and to establish a method of detecting and genotyping hemorrhagic fever with renal syndrome. Methods A set of primers was used to clone the full-length S gene fragment and partial N-terminal S gene fragment, and to make them fused and non-fused in T7 system. Reverse transcriptase-polymerase chain reaction (RT-PCR) was established with the other primers to detect 37 strains of Han, 8 positive control strains and 5 negative control strains isolated from 8 main hosts in different areas in China. . The PCR products of 20 strains of them were digested with RsaI and Hind Ⅲ, and the RT PCR RFLP typing method was established. Results Although the yield of non-fusion expression was lower than that of fusion expression, both S gene fragment products expressed by non-fusion were used as antigen for indirect ELISA. Their working concentrations were all 1:10 0 0 0, showing good bioactivity. RT-PCR results showed that all strains amplified hantan-specific nucleic acid components (2 99bp or 5 77bp). By RT PCR RFLP method typing, the strains were designated as the Han beach type 9, Seoul type 8, more than 3 strains failed to stereotypes. Conclusion The non-fusion expression of small molecule antigen biological activity is high, is expected to replace the natural antigen for HV antigen antibody detection. RT-PCR and cELISA method, the positive rate of the two was 100% and 84.6%, respectively, with a coincidence rate of 84.6%, but the former was 15.4% more sensitive than the latter. RT PCR RFLP typing and serological typing results obtained with a high coincidence rate, but the RFLP method was 85% (17/2 0