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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。
Objective: To construct and express anti-human seminal plasma protein / anti-CD3 bispecific single chain antibody (BsscFv) and to detect its biological activity. Methods: The anti-human seminal plasma protein scFv gene and anti-CD3 scFv gene were spliced by overlap extension splicing PCR. A flexible short peptide (GlySerGly) 2 was introduced in the middle to construct BsscFv gene of anti-human seminal plasma protein / anti-CD3. After sequencing, the fusion gene was subcloned into the eukaryotic expression vector and expressed in HeLa cells. The flow cytometry (FCM) and 51Cr release assay were used to evaluate the antigen-binding activity and in vitro-mediated specificity of BsscFv Killing target cells, as well as the use of nude mouse model of prostate cancer observed in vivo antitumor effect. Results: Sequencing analysis confirmed that the size of Bss-cFv gene fragment was 1.5kb and encoded 500 amino acids. The sequence was completely consistent with the designed one. SDS-PAGE and Western blot analysis demonstrated that the expressed product was present in the culture supernatant of HeLa cells with a relative molecular mass (Mr) of 61,000. The results of FCM showed that BsscFv specifically binds to LNCaP, a prostate cancer cell line, and Jurkat, a CD3 + lymphoma cell line with binding rates of 54.1% and 53.7%, respectively. In vitro experiments show that BsscFv can mediate CTL killing of LNCaP cells. Compared with the control group, the growth of tumor in nude mice inoculated with LNCaP was significantly inhibited (P <0.05) after CTL and BsscFv were activated in vivo. CONCLUSION: BsscFv anti-human seminal plasma protein / anti-CD3 has certain biological activity, and can mediate CTL killing target cell LNCaP in vitro and in vivo.