表达SARS-CoV-2 Omicron突变株RBD的重组4型腺病毒的构建与鉴定

Construction and evaluation of recombinant adenovirus serotype 4 expressing RBD of SARS-CoV-2 Omicron variant

目的构建和鉴定表达新型冠状病毒(severe acute respiratory symptom coronavirus 2,SARS-CoV-2)Omicron株刺突蛋白受体结合域(receptor binding domain,RBD)的重组4型腺病毒。方法使用重叠延伸PCR和无缝克隆构建增强型绿色荧光蛋白和RBD双顺反子表达盒,将PCR得到的ccdB片段和pBR322-Ad4质粒进行同源重组获得中间质粒pBR322-Ad4-ccdB,使用PacI和SpeI酶切质粒pBR322-Ad4-ccdB后回收载体片段,回收片段与双顺反子表达盒在E.coli GB08Red中同源重组获得重组质粒pBR322-Ad4-RBD,用AsiSI线性化后转染HEK293T细胞获得重组腺病毒Ad4-RBD,RT-PCR验证RBD蛋白的转录,Western blot验证重组腺病毒中RBD蛋白的表达。结果重组腺病毒Ad4-RBD的滴度达到5×108 PFU/mL,并能在细胞中稳定表达外源基因。结论成功构建了表达SARS-CoV-2 Omicron株RBD蛋白的重组4型腺病毒,为SARS-CoV-2 Omicron株疫苗的研发提供了科学有效的依据。

Objective To construct and identify a recombinant adenovirus type 4 expressing spike protein receptor binding domain(RBD)of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)omicron variant.Methods Enhanced green fluorescent protein and RBD dual cis-trans expression cassette was constructed by overlap extension PCR and seamless cloning.The intermediate plasmid pBR322-Ad4-ccdB was generated from ccdB fragment obtained by PCR and pBR322-Ad4 using homologous recombineering technique.After digestion with PacI and SpeI restriction enzymes,the vector fragment was recovered and homologously recombined with the dual cis-trans expression cassette in E.coli GB08Red to acquire the recombinant plasmid pBR322-Ad4-RBD.After linearisation with AsiSI,the recombinant pBR322-Ad4-RBD was transfected into HEK293T cells to generate recombinant Ad4-RBD.Transcription of the RBD gene was detected by RT-PCR and the expression of the RBD protein in transfected cells was assessed by western blot.Results The titer of the recombinant adenovirus Ad4-RBD was 5×108 PFU/mL and the expression of exogenous gene in infected cells was stable.Conclusions The recombinant adenovirus expressing the RBD protein of the SARS CoV-2 Omicron variant was constructed successfully.This may provide a scientific and effective basis for the vaccine development for SARS-CoV-2 Omicron variant.