表达抗G250人源性抗体腺病毒对肾癌细胞的影响

The effects of recombinant adenovirus expressing anti - G250 fully human antibody on renal cancer cell

目的检测腺病毒Ad-G250表达的G250全长人源性抗体的生物学活性及其对肾癌细胞的影响。方法扩增纯化腺病毒Ad—G250、Ad—EGFP（对照病毒）。病毒感染LO-2细胞，ELISA法检测细胞培养液中抗体的表达量。收集病毒感染后的细胞上清液进行纯化，Westernblotting检测纯化抗体的相对分子质量及其与细胞表面G250抗原的结合能力。免疫组化法检测Ad—G250表达的抗体是否具有生物学活性。CCK-8法检测Ad—G250对肾癌Ketr-3及ACHN细胞增殖的影响。AnnexinV—PE／7-AAD法检测Ad—G250对‘肾癌Ketr-3及ACHN细胞凋亡的影响。结果病毒Ad—G250感染LO-2细胞后G250抗体的表达量随着感染天数的增加而增加。Westernblotting实验显示Ad—G250能够表达G250抗体的轻链和重链，电泳图上该抗体能使G250抗原阳性的Ketr-3和786-O细胞在相对分子质量58×10’附近出现反应条带，而G250抗原阴性的ACHN和HK-2细胞在相应位置无反应条带。细胞免疫组化实验显示Ketr-3细胞膜被染成棕黄色，而ACHN细胞未见着色。CCK-8实验结果显示，Ad—G250对Ketr-3细胞有抑制作用，而对ACHN细胞的抑制作用不明显；Ad—G250抑制Ketr-3细胞增殖存在浓度及时问依赖性。凋亡实验显示，Ad—G250诱导Ketr-3细胞凋亡作用和ACHN细胞相比差异具有统计学意义，Ad—G250诱导Kert-3凋亡作用和Ad—EGFP相比差异有统计学意义。结论腺病毒Ad—G250成功表达出具有生物学活性的人源性G250抗体，且Ad—G250可抑制表达G250抗原的肾癌细胞增殖，诱导其凋亡。

Objective To test the activity of Ad - G250 expressing anti - G250 antibody and its effects on the pro- liferation and apoptosis of renal cell carcinoma. Methods Ad - G250 and Ad - EGFP were propagated in HEK293 cells and then purified. LO- 2 cells were infected with Ad- G250, and the antibody＇s expression level was detected by ELISA. The cell culture liquid was purified. Western blotting was used to determine the molecular weight of the purified antibody and detect whether the purified antibody could bind to G250 protein specifically. Immunohistochemistry was used to detect the activity and specificity of the purified antibody. Cell proliferation was assayed by CCK - 8 method. The ap- optosis of tumor cells was measured by Annexin V - PE/7 - ADD. Results The expression level of the antibody in LO - 2 cells infected with Ad - G250 increased as days went by. Western blotting results showed that LO - 2 cells infected with Ad- G250 could express the light chain and heavy chain respectively. The purified antibodies could bind to G250 antigen of the Ketr-3 and 786 -O cells, but not in ACHN and HK -2 cells （G250 antigen -negative）. Immunohisto- chemistry showed that the cell membrane of the Ketr - 3 cells was stained, but not the ACHN cells. CCK - 8 assay showed Ad - G250 inhibited the proliferation in Kerr - 3 cells effectively, but not in ACHN ceils. And the effect of inhi- bition was dose and time - dependent. Ad - G250 could induce more apoptosis in Ketr - 3 and ACHN, but the efficacy in Ketr-3 was more obvious than that in ACHN. Both Ad -G250 and Ad -EGFP could induce the apoptosis in Kerr-3, but the effect produced by Ad - G250 was stronger than that induced by Ad - EGFP. Conclusion We had successfully constructed adenovims expressed the anti - G250 fully human antibody. Ad - G250 inhibits renal cancer cells prolifera- tion and induces apoptosis.