PDGF-B免疫原的制备及其腹水抗体对HepG2细胞增殖的影响

PDGF-B immunogen preparation and the suppressive effect of anti-PDGF-B ascite antibody on the proliferation of hepG2 cells

摘要:

观察利用融合表达载体pET28-Trx合成的人血小板源性生长因子B (Human platelet-derived growth factor B，hPDGF-B) 免疫原的免疫原性，及其诱导小鼠产生针对hPDGF-B的腹水抗体对人HepG2细胞增殖的抑制作用。本研究首先选择hPDGF-B的第103–118及152–167氨基酸序列作为抗原表位，构建pET28-Trx-重组原核表达载体，表达并纯化获得6×his Trx-hPDGF-BΔ103-118及6×his Trx -hPDGF-BΔ152-167重组蛋白；然后使用纯化后的重组蛋白免疫小鼠，并给予腹腔注射H22肿瘤细胞制备腹水抗体，ELISA法检测抗体滴度，Western blotting法检测纯化腹水抗体与膜结合的PDGF-B结合能力；最后通过CCK8实验检测外源性PDGF-BB和两种纯化腹水抗体对肝癌HepG2细胞增殖的作用。研究发现上述两种重组蛋白作为免疫原均可诱导小鼠产生高滴度的PDGF-B中和性抗体；两种hPDGF-B纯化腹水抗体能明显抑制PDGF-BB对肝癌HepG2细胞增殖的促进作用。结果提示，Trx-PDGF-B重组蛋白作为免疫原有望为PDGF-B疫苗的制备提供新的方法，也为临床上肝癌的治疗提供了一种新的思路。

Abstract:

To observe the immunogenicity of hPDGF-B immunogens that were synthesized with the?fusional?expression vector pET28-Trx and to test the suppressive effect of these specific antibodies induced by both of immunogens on proliferation of human HepG2 hepatoma cells. First, we chose 2 antigenic epitopes hPDGF-BΔ103-118aa and hPDGF-BΔ152-167aa from human PDGF-B and inserted these 2 coding regions into the empty vector plasmid pET28-Trx, separately. Second, mice were immunized with purified recombinant proteins to generate polyclonal antibody. Then we intraperitoneally injected mice bearing hepatoma 22 (H22) tumor cells to prepare antibody ascites. ELISA and Western blot were used to detect the titer and the utility of the antibody, respectively. Finally, HepG2 cells were exposed to PDGF-BB protein or anti-PDGF-B ascite antibody in different dilution concentrations groups and the proliferation of HepG2 cells was quantified by CCK8 assay. As the results, we identified mice that could produce high drop of neutralizing antibodies against hPDGF-B induced by both two recombinant proteins. Two anti-PDGF-B ascite antibodies could markedly inhibit the proliferation of HepG2 cells by blocking the stimulating effect of PDGF-BB protein. Our results suggest that Trx-PDGF-B recombinant protein as immunogen provides a new method for the preparation of PDGF-B vaccine, and also a new idea for the treatment of hepatocellular carcinoma in clinical practice.