慢病毒载体介导的西尼罗河病毒prME蛋白的表达

将西尼罗河病毒(WNV)的主要免疫原性基因prME插入HIV-1慢病毒载体pHR中,构建了转移质粒pHR-WNV/prME。在脂质体介导下,将该质粒与p△8.2、pVSV/G共转染293T细胞,48h后收获细胞上清。将上清转导正常的293T细胞,通过报告基因荧光观察、间接免疫荧光检测。结果表明,利用HIV-1慢病毒载体系统包装得到了假型病毒,可以高效转导293T细胞,并实现了WNVprME基因在被转导细胞中的表达,从而为基于慢病毒载体的WNV新型疫苗的研制奠定基础。

毕赤酵母分泌表达JEV prME蛋白及其形成病毒样颗粒的免疫原性鉴定

应用毕赤酵母分泌表达日本脑炎病毒(Japanese encephalitis virus,JEV)prME蛋白,鉴定其表达效果与免疫原性,以期为JEV亚单位疫苗的研制奠定基础。RT-PCR扩增JEV SA14-14-2株prME基因,将其连接到毕赤酵母表达载体pPICZa-A,分别获得pPICZa-prME和携带JEV Cap蛋白C末端19个Aa信号肽的pPICZa-SprME质粒。表达载体用PmeⅠ酶切线性化,通过电转化转入毕赤酵母X33并诱导发酵培养。利用SDS-PAGE和Western blotting鉴定酵母发酵上清中目的蛋白的表达情况。利用GE蛋白层析纯化柱纯化目的蛋白,利用电镜观察纯化前后的目的蛋白,将不同剂量纯化后的prME蛋白与弗氏佐剂混合以及定量纯化后的prME蛋白与不同剂量的核酸佐剂混合分别免疫4周龄小鼠,定期采血,ELISA检测被免小鼠血清的抗体水平,空斑减少试验测定抗体中和效价。SDS-PAGE结果表明毕赤酵母可以分泌表达完整的prME蛋白,目的蛋白在70–100 kDa之间;Western blotting结果显示分泌表达的prME蛋白具有良好的反应原性,进一步证明prME蛋白在酵母X33中以整体的形式分泌表达,没有发生水解切割。纯化目的蛋白,根据洗脱时间和体积表明其分子量大于1×10~6 Da,因此推断prME蛋白可能形成多聚化的颗粒。电镜观察发现直径30–50 nm的病毒样颗粒(Virus like particles,VLPs)。免疫试验结果表明,纯化后的重组蛋白10–15μg/只接种小鼠在3周后抗体达到高峰值,之后逐渐下降,免疫7周后小鼠血清仍可检测到JEV抗体。将prME VLPs以10μg/只的剂量与不同剂量的核酸佐剂配伍后接种小鼠,ELISA检测结果表明核酸佐剂可明显增强JEV prME VLPs免疫应答,免疫4周后小鼠血清的中和抗体效价为1∶80–1∶160。上述结果表明毕赤酵母表达JEV prME虽不能发生水解切割,但仍可形成VLP并诱导免疫小鼠产生较高水平中和抗体。

Study on DNA Immunization by Recombinants Encoding Japanese Encephalitis Virus prME and E Proteins

To study the expression characteristic of Japanese encephalitis virus (JEV) prME and E proteins and the efficacy of DNA immunization by different recombinant plasmids containing JEV prME (2001 bp) and E (1500 bp) genes, two recombinants (pJME and pJE) containing JEV prME and E genes fused with FLAG were constructed and then transfected into HepG2 and COS-1 cells by liposome fusion. The expression feature of FLAG-prME (about 72 kDa) and FLAG-E (about 54 kDa) proteins in transfected cells were analyzed by Western blot and two antibody systems (anti-FLAG and anti-E). BALB/c mice were immunized with 100 μg of two kinds of recombinants by intramuscular injection, and JEV JaGAr-01 strains (10 5 PFU/100 μl)were given to BALB/c mice by intraperioneal injection 3 wk after twice DNA immunization by a lethal virus challenge. BALB/c mice were observed for 21 days after challenge. 80% plaque reduction neutralization test was performed to titrate neutralization antibody before and after viral challenge. It was found that the expression of proteins associated with pJME and pJE was determined in transfected cells with anti-FLAG and a new protein of 11 kDa was detected in HepG2 and COS-1 cells transfected with pJME. Only E (53 kDa) protein was identified as transfected with pJME using anti-E. Higher level of neutralization antibodies and the efficacy of protective immunity were induced with pJME immunization, and were similar to those induced by inactivated Japanese encephalitis vaccine, but were better than those induced with pJE. It concludes that the expression level from prM to E proteins of JEV is different in vitro, and the in vitro expression efficiency of pJME was better than that of pJE. FLAG-prME protein expressed by pJME could be cleaved by peptidase from host. The efficacy of DNA immunization is correlated to the expression characterization of related proteins expressed in vitro.

狂犬病毒载体的构建与鉴定

目的构建狂犬病毒载体,为新型疫苗研究提供依据。方法将狂犬病毒减毒株SAE基因组分段扩增,然后经逐步拼接得到全基因组克隆,并在其中引入转录终止-起始元件和多克隆位点获得重组质粒pSAETOPO。将绿色荧光蛋白(EGFP)基因和西尼罗病毒prME基因分别克隆至pSAETOPO,然后将带有这两个基因的狂犬病毒全长cDNA切下并克隆至pcDNA3.1(+)载体。将获得的重组质粒pcSAE-GFP和pcSAE-ME转染细胞,分别观察辅助病毒对外源基因表达的影响。结果获得狂犬病毒载体质粒pcSAE-GFP和pcSAE-ME,经酶切分析和序列测定表明所构建克隆是正确的。pcSAE-GFP及pcSAE-ME转染细胞后,在辅助病毒存在下可表达相应外源基因。结论构建的狂犬病毒载体质粒可表达外源基因,为进一步获得表达外源基因的重组狂犬病毒奠定了基础。

乙型脑炎病毒样颗粒疫苗的制备及其免疫保护效率的初步评价

将乙型脑炎病毒prME及其信号肽基因片段克隆到穿梭载体AcBacmid中,构建重组穿梭载体AcBac-prME。Lipofectine介导其转染Sf9细胞,获得重组杆状病毒Ac-prME。Western-blotting、间接免疫荧光试验及电镜观察,证实Ac-prME介导prME在Sf9细胞中高效表达,且形成了病毒样颗粒。小鼠免疫和攻毒试验表明,该病毒样颗粒免疫可以使小鼠获得有效抵抗乙型脑炎病毒强毒株攻击的能力,保护率高达100%,从而为研制基于乙型脑炎病毒样颗粒的亚单位疫苗奠定了基础。