登革病毒特异性抗原片段的筛选鉴定及其ELISA方法的建立

Screening and identification of dengue virus-specific antigens and the establishment of ELISA detection method for dengue antibody

目的筛选、鉴定登革病毒共同及型特异性抗原，用纯化抗原建立检测登革病毒抗体的ELISA方法。方法分别利用DNAStar及ANTHEPROT软件对登革病毒1—4型、流行性乙型脑炎及黄热病毒M、E、NS1蛋白进行分析，预测可能的抗原表位。并根据表位位置和氨基酸序列的相似性，分析登革病毒的共有及型特异性抗原表位，并参考GenBank中的序列信息进行比对，分析其在不同登革病毒株中的保守性。然后选择预测得分值较高的表位，利用pE332a、pMAI-c2x原核表达系统进行原核表达，Western blot验证其免疫学反应特异性。Western blot检测阳性抗原片段经亲和纯化后，包被ELISA微孔板，并对ELISA反应条件进行系统优化。结果经系统的生物信息学分析，共预测获得登革病毒抗原表位18个，登革病毒型特异性抗原表位25个，并对其中得分值较高的5段进行了高效表达，经Western blot分析，获得登革1—4型（Den-Ag5），登革2、4型（Den-Ag3），登革1—3型（Den—Ag2）病毒共同抗原片段各一段，登革1、2、4型（Den-Ag1、Den-Ag4）共同抗原片段两段，与流行性乙型脑炎病毒、黄热病毒及所用甲病毒多克隆抗体均无交叉反应。选择Den-Ag5、Den-Ag1和Den-Ag2作为检测用抗原，建立了检测登革病毒抗体的ELISA方法，初步应用表明，所建立方法具备良好特异性，可检测50～200倍稀释的患者血清，S／N比值均在15以上。结论经系统筛选，获登革病毒特异性抗原片段5段，并建立了检测登革病毒抗体的ELISA方法。

Objective To screen and identify the dengue virus-specific antigens, then establish the ELISA detection method for dengue virus antibody. Methods Using bioinformatie software DNAStar and AN- THEPROT to analyze the hydrophilicity, flexibility, surface probability and antigenieity of dengue virus type 1- 4, Japanese encephalitis virus and Yellow fever virus M, E and NS1 protein amino acid sequence and also con- sider the influence of secondary structure. Then in accordance with epitopes location and amino acid sequence similarity, forecast the share and specific epitopes. Reference the sequence information of different dengue virus strains in GenBank to analyze the epitopes conservative. Based on the results of bioinformatic analysis, 5 specific epitopes were amplified and inserted into prokaryotic expression vector pMAL-C2x or pET32a. Then the vectors was transferred into E. coli Rosetta（ DE3 ）. Isopropyl-β-D-thiogalactoside（IPTG） was used to induce the expression of gene segments. SDS-PAGE were used to identify the expression proteins, and the antigenicity were tested using Western blot. Using the antigen selected by Western blot, ELISA method for dengue virus antibody detection was established. Results Eighty shared epitopes and 25 specific epitopes were forecasted, and 5 antigenie fragments encloude analyzed epitopes from dengue virus type 2 and 3 were expressed in E. coli successfully. One dengue virus type 1-4 shared antigens （ Den-Ag5 ）, one dengue virus type 2 and 4 shared antigens （ Den- Ag3 ）, one dengue virus type 1-3 shared antigens （Den-Ag2） and two dengue virus type 1,2 and 4 shared antigens（ Den-Ag1, Den-Ag4）were conformed using Western blot. Using antigens Den-Ag5, Den-Ag1 and Den- Ag2, the ELISA method for dengue virus antibody detection were established. Conclusion Based on the bioinformatic analysis and Western blot verification, 5 dengue virus specific antigen were conformed, and the ELISA detection method for dengue virus antibody were established.