CAJ | 학술논문

目的了解原核表达的登革病毒(Dengue virus,DV)1～4型融合的E蛋白结构域Ⅲ直接抑制登革病毒感染及其抗体的中和作用.方法通过连接肽将1～4型登革病毒包膜蛋白Ⅲ区串联的基因产物插入PET30a在大肠埃希菌中进行表达、纯化后,应用Western Blot及间接ELISA验证表达产物.将融合蛋白免疫新西兰大白兔制备免疫血清,应用间接免疫荧光检测多抗血清的活性.将融合蛋白及多抗血清分别进行阻断实验和中和实验,对抗原及抗体的功能进行研究.结果在大肠埃希菌中成功表达了串联的登革病毒1～4型E蛋白结构域Ⅲ融合蛋白,并得到兔抗免疫血清,分别对融合蛋白及兔抗免疫血清进行验证.融合蛋白能够阻断1～4型DV感染,兔抗免疫血清能中和1～4型DV,但中和抗体效价不同.结论串联表达的登革病毒包膜蛋白Ⅲ区可抑制登革病毒感染,串联rEⅢ蛋白免疫新西兰大白兔产生的针对DV1～4型包膜蛋白结构域Ⅲ区的抗体对登革病毒具有中和作用.
목적 료해원핵표체적등혁병독(Dengue virus,DV)1～4형융합적E단백결구역Ⅲ직접억제등혁병독감염급기항체적중화작용.방법 통과련접태장1～4형등혁병독포막단백Ⅲ구천련적기인산물삽입PET30a재대장애희균중진행표체、순화후,응용Western Blot급간접ELISA험증표체산물.장융합단백면역신서란대백토제비면역혈청,응용간접면역형광검측다항혈청적활성.장융합단백급다항혈청분별진행조단실험화중화실험,대항원급항체적공능진행연구.결과 재대장애희균중성공표체료천련적등혁병독1～4형E단백결구역Ⅲ융합단백,병득도토항면역혈청,분별대융합단백급토항면역혈청진행험증.융합단백능구조단1～4형DV감염,토항면역혈청능중화1～4형DV,단중화항체효개불동.결론 천련표체적등혁병독포막단백Ⅲ구가억제등혁병독감염,천련rEⅢ단백면역신서란대백토산생적침대DV1～4형포막단백결구역Ⅲ구적항체대등혁병독구유중화작용.
Objecfive To observe the ability of dengue virus type 1-4 envelope domain Ⅲ fusion protein to inhibit virus infection and analyze the neutralizing ability of polyclonal antibodies against rEⅢ.Methods After being connected by linker peptide.EⅢ protein of Dengue virus serotypes 1-4 were expressed in E coli BL21(DE3) then purified.Fusion proteins were verified by Western Blot and ELISA.Rabbits were immunized with fusion proteins to produce anti-rE Ⅲ serum.The activity of anti-rEⅢ serum were detected through indirect immunofluorescence assay test.Inhibition of dengue virus type 1 to 4 infection in BHK-21 cells by rEⅢ fusion protein were tested.Neutralizing activity of anti-rEⅢ serum was analyzed.Results Dengue virus type 1 to 4 envelope domain Ⅲ recombinant fusion protein was expressed in Ecoli BL21 and purified successfully.Then rEⅢ fusion protein and anti-rEⅢ serum were analyzed respectively and rEⅢ fusion protein can effectively inhibit dengue virus type 1 to 4 from infecting BHK cells.The anti-rE Ⅲ serums can neutralize dengue virus type 1 to 4 but with different neutralizing titer.Conclusion Dengue virus type 1-4 envelope domain Ⅲ fusion protein can directly inhibit DV infeetion.Antibodies induced by rE Ⅲ fusion proteins can neutralize dengue virus type 1-4.