针对前列腺癌的PSMA-CAR慢病毒表达载体的构建及病毒包装

Construction and virus packaging of PSMA-CAR lentivirus expression vector for prostate cancer

目的:构建针对前列腺特异性膜抗原(PSMA)的CAR慢病毒表达载体,并包装病毒颗粒。方法:设计并合成针对PSMA的CAR基因序列,将设计的CAR片段连入pCDH-CMV-MCS-EF1-CopGFP-T2A-puro载体中,经酶切、PCR及测序鉴定构建是否成功。应用慢病毒三质粒包装系统将PSMA-CAR载体进行包装,通过荧光显微镜观察及流式细胞仪检测GFP鉴定包装结果及包装时的转染效率。通过超速离心方法浓缩病毒颗粒。结果:成功构建了PSMA-CAR慢病毒表达载体,成功包装了PSMA-CAR慢病毒颗粒且包装时的转染效率为70.43%,经测定获得的PSMA-CAR慢病毒滴度为5×105TU/mL。结论:PSMA-CAR慢病毒表达载体的成功构建及病毒颗粒的包装为应用CAR技术治疗前列腺癌奠定了基础。

Objective:To construct a CAR lentivirus expression vector for prostate specific membrane antigen(PSMA)and package the virus particles.Methods:We designed the CAR gene sequence for PSMA and synthesized.The designed CAR fragment was connected into the pCDH-CMV-MCS-EF1-CopGFP-T2A-puro vector,and the construction was confirmed by enzyme digestion,PCR and sequencing.We packed the PSMA-CAR vector by lentivirus triplasmid packaging system,and GFP was examined by fluorescence microscope and flow cytometry to identify the packaging results and transfection efficiency.The virus particles were concentrated by means of overspeed centrifugation.Results:We successfully constructed the PSMA-CAR lentivirus expression vector.The lentivirus praticles of PSMA-CAR were successfully packaged and the transfection efficiency was 70.43%.The titer of PSMA-CAR lentivirus was 5×105TU/mL.Conclusion:The successful construction of PSMA-CAR lentivirus expression vector and the packaging of virus particles lay a foundation for the application of CAR technology in the treatment of prostate cancer.