目的 构建携带NT4-AcSDKP融合基因的重组腺相关病毒,为探讨AcSDKP(乙酰化-丝氨酰-天冬氨酰-赖氨酰-脯氨酸)的多样化功能提供基础.方法 以质粒载体pGEM-T Easy/NT4为模板,PCR技术获得NT4-AcSDKP基因片段,插入腺相关病毒载体穿梭质粒pSSHG-CMV,构建重组质粒pSSCMV-NT4-AcSDKP.用腺病毒全基因组质粒pFG140、辅助质粒pAAV/Ad和已构建的重组质粒,三质粒磷酸钙共沉淀法转染80％融合的293细胞,包装rAAV-NT4-AcSDKP,RT-PCR方法测定重组腺相关病毒滴度.结果 成功合成NT4-AcSDKP基因,构建重组质粒pSSCMV-NT4-AcSDKP,包装获得滴度为3.40×1010 copies/mL的重组腺相关病毒.结论 成功获得表达AcSDKP重组腺相关病毒,为抗炎、抗纤维化、器官修复等方面的进一步研究奠定了基础.%Objective To construct and produce the NT4-AcSDKP recombinant adeno-associated virus (AAV) and determinate the titer. Methods With plasmid vector pGEM-T Easy/NT4 as the template, NT4-AcSDK was obtained by polymerase chain reaction (PCR) and inserted into vector plasmid PSSHG-CMV to construct the recombinant plasmid pSSCMV-NT4-AcSDKP. The recombinant AAV-NT4-AcSDKP was packaged through co-transfecting 80% of confluent 293 cells with adenovirus full genome plasmid pFG140, helper plasmid pAAV/Ad and the recombinant plasmid. The recombinant AAV-NT4-AcSDKP was titrated by RT-PCR. Results The gene of NT4-AcSDKP was synthesized and the recombinant plasmid pSSCMV-NT4-AcSDKP was constructed successfully. RT-PCR results showed that we had obtained the rAAV of high titer (3. 40 × 1010 copies/mL). Conclusion We have successfully obtained the AcSDKP-expressing rAAV and paved the way for further studies on anti-inflammation, anti-fibrosis, and organ repair.
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