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目的:为研究人神经生长因子β(hNGFβ)基因对慢性神经病理性痛的治疗作用,构建人类神经生长因子β基因重组腺病毒载体。方法:将hNGFβ外源性核酸片段插入到pBluescriptⅡSK(+)的XmaⅠ与XbaⅠ位点,构建成pBluescriptⅡSK(+)hNGFβ,再将其与腺病毒穿梭质粒pShuttleCMV经KpnⅠ和NotⅠ双酶切后,构建成转移质粒pShuttleCMVhNGFβ,将该转移质粒经PmeⅠ酶切后,转化含腺病毒骨架质粒pAdEasy1的感受态菌BJ5183,重组为pAdEasy1hNGFβ,经PCR鉴定后,LipoVec介导pAdEasy1hNGFβ转染AD293细胞,将透析纯化后的腺病毒采用紫外分光光度计进行滴度测定。并进行自动测序鉴定。结果:线性化的pShuttleCMVhNGFβ转化含pAdEasy1的感受态菌BJ5183,24h后获得了30%阳性重组质粒,经酶切得到一个大于20kb的大片段和一个4.5kb的特征性条带,PCR扩增出731bp片段。pAdeasy1hNGFβ经PacI酶切线性化后,转染Ad293细胞包装成表达hNGFβ的重组腺病毒。260nm处吸光值测病毒滴度为2.24×1012OPU·L-1。重组AdhNGFβ腺病毒质粒经上海基康生物技术有限公司自动测序鉴定为目的基因。结论:应用细菌内同源重组能快速构建AdhNGFβ,为hNGFβ基因在神经病理性痛中的应用奠定了基础。
AIM: To study the therapeutic effect of hNGFβ on chronic neuropathic pain, a recombinant adenovirus vector containing human nerve growth factor β gene was constructed. Methods: The exogenous nucleic acid fragment of hNGFβ was inserted into the XmaⅠ and XbaⅠ sites of pBluescriptⅡSK (+) to construct pBluescriptⅡSK (+) hNGFβ. The recombinant plasmid pShuttleCMV was subcloned into adenovirus shuttle plasmid pShuttleCMV The plasmid pShuttleCMVhNGFβwas transfected, then the recombinant plasmid was digested with PmeⅠand transformed into competent cell strain BJ5183 containing adenovirus backbone plasmid pAdEasy1. The recombinant plasmid was transformed into pAdEasy1hNGFβ. After identification by PCR, LipoVec mediated pAdEasy1hNGFβ transfected AD293 cells, Adenovirus using ultraviolet spectrophotometer titer. And automatic sequencing identification. Results: The linearized pShuttleCMVhNGFβwas transformed into pAdEasy1-competent bacteria BJ5183, and 30% positive recombinant plasmids were obtained after 24 hours. A large fragment of more than 20kb and a 4.5kb characteristic band were obtained by PCR, and 731bp Fragment. pAdeasy1hNGFβ was linearized by PacI digestion and transfected into Ad293 cells and packaged into recombinant adenovirus expressing hNGFβ. The absorbance at 260 nm was 2.24 × 1012OPU · L-1. Recombinant AdhNGFβ adenovirus plasmid was identified by Shanghai Jikang Biotechnology Co., Ltd. as the target gene. Conclusion: The rapid construction of AdhNGFβ by homologous recombination in bacteria lays the foundation for the application of hNGFβ gene in neuropathic pain.