抗人精浆蛋白/抗CD3双特异性单链抗体的活性研究

Biologic activity of bispecific single chain antibody against human γ-seminoprotein and CD3 molecule

目的:构建并表达抗人精浆蛋白/抗CD3的双特异性单链抗体(BsscFv),并检测其生物学活性。方法:利用重叠延伸拼接PCR,拼接抗人精浆蛋白scFv基因和抗CD3scFv基因,并在中间引入柔性短肽(GlySerGly)2,构建抗人精浆蛋白/抗CD3的BsscFv基因。测序正确后,将融合基因亚克隆入真核表达载体中,并在HeLa细胞中进行表达,采用流式细胞术(FCM)和51Cr释放试验,评价BsscFv的抗原结合活性和体外介导的特异性杀伤靶细胞的效应,以及利用裸鼠前列腺癌模型观察其在体内的抑瘤作用。结果:测序分析证实,Bss-cFv基因片段的大小为1.5kb,编码500个氨基酸,该序列与设计的完全一致。SDS-PAGE和Westernblot分析证明:表达产物存在于HeLa细胞的培养上清中,其相对分子质量(Mr)为61000。FCM结果显示:BsscFv可特异性的结合前列腺癌细胞LNCaP和CD3+淋巴瘤细胞Jurkat,结合率分别为54.1%和53.7%。体外实验表明,BsscFv可介导CTL对LNCaP细胞的杀伤。与对照组相比较,接种LNCaP的裸鼠在体内注射激活的CTL和BsscFv治疗后,肿瘤的生长明显受到抑制(P<0.05)。结论:抗人精浆蛋白/抗CD3的BsscFv具有一定的生物学活性,在体内、体外均可介导CTL杀伤靶细胞LNCaP。

AIM： To construct a recombinant vector which expresses bispecific single chain antibody （BsscFv） against human γ-seminoprotein and CD3 molecule and evaluate its biologic activity. METHODS： The BsscFv gene was constructed by the splicing overlap extensive （SOE） PCR and then a flexible peptide linker was inserted between antihuman γ-seminoprotein single chain Fv gene and anti-CD3 single chain Fv gene. The fusion gene was subcloned into the pSectag2-B plasmid and was expressed in HeLa cell lines. After being analyzed by SDS-PAGE and Western blot, the expressed product was purified through a Ni^2＋ -NTAsuperflow affinity chromatography column. Flow cytometry （FCM） was used to detect the binding activity of BsscFv to CD3^＋ cell line Jurkat cells and prostate carcinoma cells LNCaP. In vitro killing effect on target cells （LNCaP） mediated by BsscFv was determined by chromium^51-release test. The effect of CTLs mediated by BsscFv on inhibiting tumor growth was observed by utilizing nude mice bearing prostate cancer cells. RESULTS： DNA sequencing indicated that BsscFv gene consisted of 1 500 bp, encoding 500 amino acids. SDS-PAGE and Western blot analysis showed that the expressed product with relative molecular mass of 61 000 existed in culture supernatant of Hela cells. Flow cytometry analysis demonstrated that the binding rate of BsscFv to LNCaP cells and Jurkat cells was 54. 1% and 53.7%, respectively. In vitro, BsscFv mediated cytotoxicity of CTLs to LNCaP cells as confirmed by chromium^51-release assay. In prostate cancer nude mouse model, BsscFv inhibited tumor＇s growth as compared with control group. CONCLUSION： The BsscFv against human γ-seminoprotein and CD3 molecule possesses certain biologic activity, and in vitro and in vivo it can mediate cytotoxicity of CTLs to prostate cancer cells.