目的：利用鼠杂交瘤技术筛选和制备治疗性CD19单克隆抗体，探讨以CD19 scFv序列构建CD19-CAR（pCD19-CAR）修饰的NK细胞对CD19+B细胞淋巴瘤细胞的杀伤作用。 方法：用偶联多肽免疫小鼠制备CD19单克隆抗体，然后用基因测序法获得抗体的序列。分析抗体序列，并通过基因合成和分子克隆技术构建pCD19-CAR片段，然后将其克隆到慢病毒载体上，病毒包装制备后，转染NK-92MI细胞。最后，用流式细胞术检测不同的pCD9-CAR-NK-92MI细胞对CD19+细胞的杀伤率。 结果：（1）成功筛出特异性强的CD19单克隆抗体-pCD19；（2）抗体检测结果显示CD19+的Ramos细胞的阳性率为843%，Raji为85.6%，与商业化抗体结果相似；（3）被pCD19-CAR修饰的CD19-CAR阳性率为28.72%的NK-92MI细胞对CD19+的Ramos和Raji细胞的杀伤效率明显高于未被修饰的NK-92MI细胞株对Ramos和Raji细胞的杀伤率\[（47.1±17)% vs (24.7±6.2)%和(51.8±7.9)% vs (27.6±9.6)%，均P<0.05\]；对CD19-细胞Jurkat，不论是未被pCD19-CAR修饰的NK-92MI或是被修饰的NK-92MI细胞，几乎都不存在特异性杀伤作用\[(16.1±0.7)% vs (17.7±2.9)%, P>0.05\]。结论：成功构建pCD19-CAR，被pCD19-CAR修饰的NK-92MI细胞能特异性的识别CD19抗原并杀伤CD19+B细胞淋巴瘤细胞。

Objective:To screen and prepare theraputic monoclonal antibody CD19 using hybridoma technique in mice, and to explore killing effect of NK cells modified with pCD19-CAR which made by CD19 scFv sequence on B-cell lymphoma cells. Methods:CD19 monoclonal antibody was prepared in mice immunized with conjugated polypeptide, then sequence of the obtained antibody was obtained by gene sequencing method. The antibody sequence was analyzed and pCD19-CAR fragments were constructed via gene synthesis and molecular cloning PCR-based gene synthesis techniques. Then the pCD19-CAR fragments were cloned into lentiviral vectors, and transfected into NK-92MI cells after packaging preparation. and cloned into lenti-virus using molecular cloning method; and then the positive hybridoma cells were analyzed for scFv sequences. One of the scFv sequence was used to construct CAR (pCD19-CAR).Finally, the killing rates of various pCD19-CAR-NK-92MI cells on B-cell lymphoma cells were examined using Flow cytometry assay. Results: (1) CD19 monoclonal antibody pCD19 with high specificity was successfully screened out. (2) The measurement of the antibody showed that the positive rates of Ramos and Raji cells were 84.3% and 85.6% respectively, which were similar to commercial CD19 antibody. (3) The killing efficacy of pCD19-CAR-NK-92MI cells, of which modification rate was 28.72%, on CD+Ramos cells and CD+Raji cells was apparently higher than those of non-modified NK-92MI cells (\[47.0±1.7\]% vs \[24.7±6.2\]% and \[51.8±7.9\]% vs \[27.6±9.3\]%, all P<0.05). Specific killing effects of unmodified and modified NK-92 cells on CD19- Jurkat cells were not almost found (\[16.1±0.7\]% vs \[17.7±2.9\]%, P>0.05). Conclusion：pCD19-CAR was successfully constructed ; pCD19-CAR-NK-92MI cells could specifically recognize CD19 antigen and kill CD19+ B-cell lymphoma cells.

国家自然科学基金资助项目（No.31471283）