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目的建立登革病毒2型(DENV-2)前膜蛋白(prM)的原核稳定表达系统,并制备prM多克隆抗体,研究登革病毒特异性prM抗体的中和作用及抗体依赖性感染增强作用(ADE)。方法应用大肠杆菌表达系统表达全长DENV-2 prM蛋白,经电洗脱法纯化重组蛋白,免疫家兔,制备兔抗prM蛋白多抗;应用空斑减数中和试验(PRNT)和流式细胞术(FCM)分别检测prM兔多克隆抗体的中和作用和ADE效应。结果重组prM蛋白在原核系统可获得高效表达,prM蛋白的表达量占大肠杆菌全菌蛋白的15%左右;其免疫血清经Western blotting法证实为登革病毒特异性prM多克隆抗体,用ELISA法检测效价为1:800 000。PRNT表明,prM抗体对成熟和不成熟DENV-2感染C6/36细胞中和作用有限,最大中和程度分别为49.84%和50.22%;FCM结果表明,prM抗体能在较大的抗体稀释度范围(10-2～10-6)增强成熟和不成熟DENV-2感染,且在10-3稀释度时达到最高峰。结论重组DENV-2 prM蛋白有很强的免疫原性,其诱生的prM抗体对登革病毒仅有较弱的中和活性,但却呈现出较强的ADE作用,揭示prM抗体是一种感染增强性抗体。
Objective To establish prokaryotic stable expression system of dengue virus type 2 (DENV-2) pro-membrane protein (prM) and to prepare prM polyclonal antibody to study the neutralization of dengue virus-specific prM antibody and the enhancement of antibody-dependent infection (ADE). Methods The full-length DENV-2 prM protein was expressed in Escherichia coli (E.coli) expression system. The recombinant protein was purified by electroelution method and immunized rabbits to prepare polyclonal anti-prM protein. The plaque reduction neutralization test (PRNT) and flow cytometry Cytometry (FCM) was used to detect the neutralization and ADE effects of the polyclonal antibody against prM. Results The recombinant prM protein was highly expressed in prokaryotic system. The expression level of prM protein was about 15% of that of E. coli. The immune serum was confirmed to be dengue virus specific prM polyclonal antibody by Western blotting. The test titer is 1: 800 000. PRNT showed that the prM antibody had a limited neutralization effect on C6 / 36 cells infected with mature and immature DENV-2, the maximum degree of neutralization was 49.84% and 50.22%, respectively. FCM results showed that the prM antibody could neutralize in a large range of antibody dilution (10-2 ~ 10-6) enhanced the maturation and immature DENV-2 infection and reached its peak at 10-3 dilution. Conclusions Recombinant DENV-2 prM protein has strong immunogenicity. The prM antibody induced by it has only weak neutralizing activity against dengue virus, but shows strong ADE effect. It reveals that prM antibody is a Infection enhances antibodies.