抗鼻咽癌单域抗体与硫酸博来霉素偶联物抗肿瘤特性的初步研究

发布时间：2018-02-02 13:02

本文关键词： 单域抗体鼻咽癌原核表达靶向抗肿瘤效应　出处：《中南大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的：对抗鼻咽癌噬菌体单域抗体HNSA039特异性进行鉴定,构建、表达抗鼻咽癌单域抗体HNSA039和大肠杆菌硫氧还蛋白(TrxA)融合蛋白,并对融合蛋白与硫酸博来霉素(Bleomycin, BLM)偶联物的靶向抗肿瘤特性进行初步研究。方法：利用鼻咽癌细胞系和非鼻咽癌细胞系对单域抗体HNSA039的特异性进行免疫细胞化学鉴定。构建单域抗体HNSA039与大肠杆菌硫氧还蛋白(TrxA)融合基因的原核表达载体pET-21 a(+)/TrxA-HNS A039,转化大肠杆菌BL21 (DE3),进行IPTG诱导表达。在变性条件下纯化融合蛋白TrxA-HNSA039,进行SDS-PAGE电泳鉴定和酶联免疫吸附法(ELISA)检测融合蛋白的免疫活性。以葡聚糖T40为中介体偶联融合蛋白TrxA-HNSA039与BLM,利用流式细胞周期分析、MTT、克隆形成实验检测其对鼻咽癌细胞系(CNE2)和人肺癌细胞系(A549)的抗肿瘤效应。结果：通过对噬菌体重链单域抗体HNSA039的免疫细胞化学鉴定,发现单域抗体HNSA039与CNE2呈较强阳性反应,而与其它人肿瘤细胞系和正常细胞系反应呈弱阳性或不反应。成功构建的重组表达载体pET-21 a(+)/TrxA-HNS A039,经IPTG诱导后表达形式分析显示,融合蛋白主要以包涵体的形式存在,SDS-PAGE电泳显示分子量大约为23KD；ELISA结果显示融合蛋白与CNE2具有较好的结合活性。通过流式细胞周期分析、MTT、克隆形成实验检测TrxA-HNSA039与硫酸博来霉素偶联物(TrxA-HNSA039-BLM)的抗肿瘤活性,结果显示偶联物的抗肿瘤活性比抗体药物混合物抗肿瘤活性显著下降,差别有统计学意义(P0.05),而对靶细胞系(CNE2)和人肺癌细胞系(A549)抗肿瘤活性差别没有统计学意义(P0.05)。结论：免疫细胞化学结果显示单域抗体HNSA039具有较好的鼻咽癌细胞特异性。成功构建TrxA-HNSA039的原核表达载体,然后表达并纯化TrxA-HNSA039融合蛋白。ELISA实验结果显示,融合蛋白TrxA-HNSA039具有较好的识别鼻咽癌细胞的免疫活性,而体外细胞实验结果显示TrxA-HNSA039-BLM没有明显的靶向抗鼻咽癌活性。为了更好地研究单域抗体HNSA039,我们准备融合单域抗体HNSA039基因或将单域抗体融合其它抗原识别结构,构建双价抗体增加其抗原识别特性,与“弹头”药物进行直接偶联进行靶向抗鼻咽癌研究。
[Abstract]:Objective: to identify and construct a HNSA039 specific antibody against nasopharyngeal carcinoma phage phage. The fusion protein of HNSA039 and TrxA was expressed, and the fusion protein was expressed with Bleomycin sulfate (Bleomycin). The target antitumor characteristics of BLM) conjugate were studied. Methods:. The specificity of single domain antibody (HNSA039) was identified by immunocytochemistry in nasopharyngeal carcinoma (NPC) cell line and non nasopharyngeal carcinoma cell line. Construction of single domain antibody HNSA039 and Escherichia coli thioredoxin (HNSA039). TrxA, the prokaryotic expression vector of TrxA fusion gene, was used as a prokaryotic expression vector pET-21 _ a (TrxA-HNS A039). The fusion protein TrxA-HNSA039 was purified under denaturation conditions by transformation of E. coli BL21 DE3 and IPTG induced expression. SDS-PAGE electrophoresis and enzyme-linked immunosorbent assay (Elisa). The immunological activity of the fusion protein was determined. The fusion protein TrxA-HNSA039 and BLM were mediated by dextran T40. The anti-tumor effects of MTT on nasopharyngeal carcinoma (NPC) cell line CNE2 and human lung cancer cell line A549) were detected by flow cytometry. Results: the immunocytochemical identification of phage heavy chain single domain antibody (HNSA039) showed that HNSA039 and CNE2 were strongly positive. The recombinant expression vector pET-21 _ a (TrxA-HNS A039) was successfully constructed and reacted weakly or not with other human tumor cell lines and normal cell lines. IPTG induced expression analysis showed that the fusion protein mainly existed in the form of inclusion body. SDS-PAGE electrophoresis showed that the molecular weight of the fusion protein was about 23kD. The results of ELISA showed that the fusion protein had good binding activity with CNE2. The antitumor activity of TrxA-HNSA039 combined with bleomycin sulfate (TrxA-HNSA039-BLM) was detected by clone formation assay. The results showed that the antitumor activity of the conjugate was significantly lower than that of the mixture of antibodies (P 0.05). However, there was no significant difference in antitumor activity between target cell line CNE2 and human lung cancer cell line A549. Conclusion: the results of immunocytochemistry show that the single-domain antibody HNSA039 has a good specificity of nasopharyngeal carcinoma cells. The prokaryotic expression vector of TrxA-HNSA039 was successfully constructed. Then the expression and purification of TrxA-HNSA039 fusion protein. Elisa results showed that the fusion protein TrxA-HNSA039 has a good recognition of NPC cells immune activity. The results of in vitro cell experiments showed that TrxA-HNSA039-BLM had no significant targeting activity against nasopharyngeal carcinoma. In order to better study single-domain antibody HNSA039. We intend to fuse the single-domain antibody HNSA039 gene or the single-domain antibody into other antigen-recognition structures to construct bivalent antibodies to enhance their antigen-recognition properties. Direct coupling with warhead drugs was carried out to study the targeting of nasopharyngeal carcinoma (NPC).
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.6

【参考文献】