五种检测嵌合抗原受体表达方法的比较

Comparison of 5 Methods of Evaluating the Expressions of Chimeric Antigen Receptors

比较检测新型嵌合抗原受体(CAR)的5种方法。通过慢病毒感染得到稳定表达嵌合抗原受体的细胞。使用免疫球蛋白G抗体染色、蛋白质L染色、绿色荧光蛋白共表达法、实时荧光定量PCR的绝对定量和相对定量法5种方法检测不同嵌合抗原受体在Jurkat细胞中的表达。成功构建6种CAR慢病毒表达载体(Meso-CAR、Met-CAR、R132H-CAR1-2-GFP、R132HCAR2-4-GFP、Dupixent-HL-CAR-GFP、Dupixent-LH-CAR-GFP),并得到其对应的CAR稳定表达Jurkat细胞。发现Meso-CAR和Met-CAR可同时用免疫球蛋白G抗体和蛋白质L染色鉴定其表达(免疫球蛋白G抗体染色效果较优),而其余4种CAR均不可以。对荧光法染色不可行的CAR可使用绿色荧光蛋白共表达法、实时荧光定量PCR的绝对定量和相对定量法间接检测CAR的表达,3种方法的结果之间存在一定的对应性。不同的CAR分子,可用不同方法确定细胞中CAR分子的表达情况。

This work is to compare 5 different methods for detection of novel chimeric antigen receptor(CAR).Stably expressing CAR cells were generated through lentiviral infection.Five methods,immunoglobulin G antibody staining,protein L staining,green fluorescent protein(GFP)co-expression,real-time fluorescent quantitative PCR and relative quantitative qPCR,were used to detect different novel CAR in Jurkat cells.Six lentiviral vectors expressing different CARs of Meso-CAR,Met-CAR,R132H-CAR1-2-GFP,R132H-CAR2-4-GFP,Dupixent-HL-CAR-GFP,and Dupixent-LH-CAR-GFP were successfully constructed,and their corresponding CAR stably-expressed Jurkat cells were obtained.The expressions of Meso CAR and Met CAR were detected by both immunoglobulin G antibody and protein L staining(better result with immunoglobulin G antibody staining),but the detection of the rest 4 CARs failed.GFP co-expression,absolute quantitative and relative quantitative methods of real-time fluorescent quantitative PCR can be utilized to indirectly detect the expression of CAR that was impossible with fluorescent staining,and there was certain correspondence among 3 methods.In conclusion,different approaches should be used to determine the expressions of CAR molecules for different novel CAR molecules,.