CRISPR/Cas9介导的T4噬菌体Soc基因敲除及突变体构建

CRISPR/Cas9-mediated knockout of the Soc gene in T4 bacteriophage and mutant construction

摘要:

为提升病毒样颗粒疫苗的抗原展示密度与稳定性，开发T4噬菌体作为新型的高容量展示与递送平台。T4噬菌体具有结构稳定、载量高及易于生产等优势，但其衣壳表面高拷贝内源Soc蛋白的存在严重限制了外源蛋白的有效展示。本研究采用CRISPR/Cas9系统对T4噬菌体的Soc基因进行定向敲除，以克服上述空间限制。通过将pCas、pTargetF-sgRNA与pMD19-T-Soc-arm三质粒系统共转化至大肠杆菌TG1，经l-阿拉伯糖诱导Cas9表达后，利用野生型T4噬菌体(命名为T4 WT)侵染工程菌，成功实现Soc基因的敲除。实验结果表明，经PCR、SDS-PAGE及测序验证，成功构建Soc基因缺失型T4噬菌体突变株(命名为T4ΔSoc)。该突变株在生长、热稳定及pH稳定性方面与野生型相当，并保持完整感染活性；连续传代5代后，其基因组、蛋白质组成及噬菌斑表型均保持稳定，未发生回复突变。综上，T4ΔSoc遗传与表型稳定，有效突破了T4 WT展示空间受限的限制。本研究为构建高效、稳定的抗原展示与疫苗递送平台提供了可靠的载体工具与技术基础，在疫苗研发及靶向递送系统构建中具有较好的应用潜力。

Abstract:

To address the antigen display limitations of current vaccine carriers, we engineered the T4 bacteriophage into a high-capacity platform. The T4 bacteriophage has advantages such as structural stability, high loading capacity, and easy production. However, the presence of high-copy endogenous Soc protein on its capsid surface severely restricts the effective display of exogenous proteins. To overcome the aforementioned spatial limitations, we employed the CRISPR/Cas9 system to precisely knockout the Soc gene of T4 bacteriophage. We successfully achieved the knockout of the Soc gene by co-transferring the three plasmid systems—pCas, pTargetF-sgRNA, and pMD19-T-Soc-arm—into Escherichia coli TG1, inducing the expression of Cas9 with L-arabinose, and then infecting the engineered bacteria with the wild-type T4 bacteriophage (named T4 WT). The PCR, SDS-PAGE, and sequencing results confirmed a Soc gene-deficient T4 bacteriophage mutant strain (named T4ΔSoc) was successfully constructed. This mutant strain had comparable growth, thermal stability, and pH stability to the wild type, and maintained complete infectivity. After continuous passage for five generations, it remained stable in terms of genome, protein composition, and phage plaque phenotype, with no occurrence of revertant mutations. In conclusion, T4ΔSoc is stable both genetically and phenotypically, effectively breaking through the limitation of the restricted display space of T4 WT. This study provides a reliable vector tool and technical foundation for constructing an efficient and stable antigen display and vaccine delivery platform and has good application potential in vaccine research and the construction of targeted delivery systems.