流式细胞术快速测定逆转录病毒滴度的研究

Rapid Retroviral Titration Based on the Analysis of Antigen Expression Using Flow Cytometry

目的 建立一种快速测定逆转录病毒滴度的方法。方法 用通过乒乓转导获得的逆转录病毒 (G1FP ,LGSN ,CD87)分别感染NIH 3T3受体细胞 ,以流式细胞术 (FCM )定量分析受体细胞中绿色荧光蛋白 (EGFP)或CD87的表达 ,并与常规的耐药集落形成法进行比较。结果 PT6 7/G1FP细胞高度表达EGFP ,荧光强度较对照细胞提高 10 5 1倍 ;G1FP - 8,Am12 /G1FP ,PT6 7/G1FP ,Am12 /LGSN和Am12 /CD87细胞上清中病毒滴度分别为 2 .9× 10 6,1.1× 10 5,2 .0× 10 6,1.5× 10 5和 4.3× 10 5感染性颗粒 /ml,比集落形成法测得的滴度低 3～ 6倍。结论 FCM分析抗原表达是测定逆转录病毒滴度的有效方法 ,特别适宜于缺乏选择性基因的载体。

Objective To develop a procedure for rapid retroviral titration. Methods Retrovirus vectors, viz. G1FP, LGSN, and CD87, were collected from superinfected producer cells and infected target cell line NIH 3T3. The enhanced green fluorescent protein (EGFP) or CD87 antigen expression in NIH 3T3 cells was quantitatively analyzed using flow cytometry (FCM). Retroviral vectors containing a drug resistance gene were also titrated by colony-forming assay. Results PT67/G1FP cells were overexpressed high level of EGFP, in which mean cellular fluorescence was improved to 1051-fold as compared with that in PT67 cells. Based on antigen expression, the titer in supernatants of G1FP-8, Am12/G1FP, PT67/G1FP, Am12/LGSN and Am12/CD87 cells was 2.9×10 6, 1.1×10 5, 2.0×10 6, 1.5×10 5 and 4.3×10 5 infectious particles/ml, respectively. The titer measured by antigen expression was 3～6 times lower than the respective titer obtained after drug selection of target cells. Conclusion Retroviral titer assessment by FCM analysis of antigen expression is effective, especially for vectors lacking a selectable marker.