重组腺病毒表达牛纽布病毒VP1蛋白及免疫效果评价

Expression of VP1 protein of bovine nebovirus by recombinant adenovirus and evaluation of its immune effect

旨在构建一种表达牛纽布病毒(bovine nebovirus,BNeV)VP1蛋白的人5型复制缺陷型重组腺病毒rAd5-BNeV_VP1,并评价其免疫效果。以优化合成的BNeV VP1基因序列构建重组穿梭质粒pDC316-VP1,利用AdMax腺病毒包装系统包装重组腺病毒rAd5-BNeV_VP1,并在小鼠中评价其免疫效果。结果显示,通过比对国内BNeV VP1氨基酸序列,根据HEK293细胞密码子偏嗜性优化合成国内流行毒株Bo/LN-13/18/CH株的BNeV VP1序列,序列大小为1650 bp。将pDC316-VP1与骨架载体pBHGIox_E1,3Cre共转染入HEK293细胞包装出重组腺病毒rAd5-BNeV_VP1。RT-PCR扩增出1650 bp的BNeV VP1基因条带,Western blot及间接免疫荧光反应证实rAd5-BNeV_VP1能够成功表达VP1蛋白,蛋白大小约为57.5 kDa,表明成功包装出rAd5-BNeV_VP1且VP1基因能在重组腺病毒中稳定表达,其TCID_(50)为10-5.34/0.1 mL。通过肌肉注射和滴鼻2种途径免疫小鼠均能产生较高的特异性抗体水平,二免7 d后肌肉注射组抗体效价最高可达1∶105,滴鼻组抗体效价最高可达1∶104;滴鼻组可产生高于PBS组2倍的INF-γ和IL-2(P<0.05)。结果表明,本研究构建表达BNeV VP1蛋白的重组腺病毒rAd5-BNeV_VP1具有良好的免疫原性,可刺激小鼠快速产生针对BNeV VP1的特异性抗体,为今后BNeV疫苗的研发奠定了基础。

The aim of this study was to construct the recombinant human adenovirus rAd5-BNeV_VP1 that expresses the VP1 protein of bovine nebovirus(BNeV)and evaluate its immune effect.The recombination shuttle plasmid pDC316-VP1 was constructed using the optimized gene sequence of BNeV VP1.The recombinant adenovirus rAd5-BNeV_VP1 was packaged by AdMax adenovirus packaging system.The immune effect was evaluated in mice.BNeV VP1 sequence with a size of 1650 bp of Bo/LN-13/18/CH strain was optimized and synthesized according to codon preference of HEK293 cells by comparing the amino acid sequence of BNeV VP1 in China.Shuttle plasmid pDC316-VP1 and backbone vector pBHGIox_E1,3Cre were co-transfected into HEK293cells to package recombinant adenovirus rAd5-BNeV_VP1.BNeV VP1 gene band with a size of about 1650 bp was amplified by RT-PCR.Western blot and indirect immunofluorescence assay confirmed that rAd5-BNeV_VP1 successfully expressed VP1 protein with a size of about 57.5 kDa.The results showed that rAd5-BNeV_VP1 was successfully packaged and VP1 gene was stably expressed in the recombinant adenovirus.TCI_(50)was 10~(-5.34)/0.1 mL.A high level of specific antibody was detected in mice after immunized by intramuscular and intranasal routes.The highest antibody titer was 1:10~5 in the group of intramuscular and 1:10~4 in the group of intranasal on 7 d after the second immunization.The group of intranasal can produce two times of INF-γand IL-2 than the group of PBS(P<0.05).The recombinant adenovirus rAd5-BNeV_VP1 expressing BNeV VP1was successfully constructed.It has a good immunogenicity and can stimulate mice to rapidly produce specific antibodies against BNeV VP1,which provides a basis for the vaccine development for bovine nebovirus infection.