表达传染性支气管炎病毒S1重组腺病毒的构建及免疫原性分析

Construction of a recombinant adenovirus expression vector for the S1 gene of infectious bronchitis

目的 构建表达传染性支气管炎病毒S1蛋白的重组腺病毒,并进行初步免疫原性分析。方法 本研究利用腺病毒表达载体系统,利用同义点突变技术,消除IBV-S1基因中的PacⅠ和PmeⅠ限制性酶切位点;将已突变的S1基因克隆至pShuttle-CMV-N-DsRed穿梭质粒中;使用Pme I线性化处理携带目的基因的穿梭质粒,并与pAdEasy-1质粒同时转化进行同源重组;后将重组质粒转染HEK 293细胞,包装成重组Adv,构建S1-CMV-pAd(293)重组病毒;并进行免疫实验验证抗体。结果 PCR扩增出S1基因,大小约为1 700 bp。S1和pShuttle-CMV-N-DsRed穿梭质粒经酶切及测序结果表明,成功构建S1-CMV表达载体;在BJ5183感受态中进行同源重组,测序结果表明成功构建S1-CMV-Pad重组质粒。利用HEK-293细胞包装成重组腺病毒,能稳定产生红色荧光;经Western blot和IFA验证重组病毒成功表达S1 (RBD)蛋白;以106TCID50/mL滴度肌肉免疫雏鸡,7~21 d后均可检测到S1抗体,且抗体浓度仍呈增长趋势。结论 本研究所构建的S1-CMV-pAd重组Adv为之后的免疫保护试验研究及IBV-Adv疫苗构建提供了基础实验支撑。

Objective The objective of this study was to construct a recombinant adenovirus that expresses the S1 protein of infectious bronchitis virus(IBV)and conduct preliminary analysis of its immunogenicity.Methods The adenovirus expression vector system was utilized to remove the PacⅠand PmeⅠrestriction enzyme sites in the IBV-S1 gene through synonymous mutations.The mutated S1 gene was then cloned into the pShuttle-CMV-N-DsRed shuttle plasmid.The shuttle plasmid carrying the target gene was linearized using PmeⅠand co-transformed with the pAdEasy-1 plasmid for homologous recombination.The resulting recombinant plasmid was transfected into HEK 293 cells to package the recombinant adenovirus,leading to the construction of the S1-CMV-pAd(293)recombinant virus.Immunological experiments were conducted to verify the antibody response.Results The S1 gene was successfully amplified through PCR,yielding a fragment of approximately 1700 bp.Enzyme digestion and sequencing confirmed the successful construction of the S1-CMV expression vector.Homologous recombination was accomplished in BJ5183 competent cells,as evidenced by sequencing results of the S1-CMV-pAd recombinant plasmid.The recombinant adenovirus,packaged in HEK-293 cells,consistently expressed red fluorescence.Utilizing the polyclonal anti-S1 rabbit antibodies prepared in our laboratory as a primary antibody in this experiment.Western blot and immunofluorescence assays confirmed the successful expression of the S1 protein by the recombinant virus.Immunization of chicken muscles with a titer of 106 TCID50/mL resulted in detectable S1 antibodies from 7 to 21 days,with antibody concentrations displaying an upward trend.Conclusion The constructed S1-CMV-pAd recombinant adenovirus serves as a foundation for future immunoprotection experiments and the development of IBV-Adv vaccines.