本文關(guān)鍵詞： 單域抗體 鼻咽癌 原核表達(dá) 靶向抗腫瘤效應(yīng)　出處：《中南大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：對(duì)抗鼻咽癌噬菌體單域抗體HNSA039特異性進(jìn)行鑒定,構(gòu)建、表達(dá)抗鼻咽癌單域抗體HNSA039和大腸桿菌硫氧還蛋白(TrxA)融合蛋白,并對(duì)融合蛋白與硫酸博來霉素(Bleomycin, BLM)偶聯(lián)物的靶向抗腫瘤特性進(jìn)行初步研究。 方法：利用鼻咽癌細(xì)胞系和非鼻咽癌細(xì)胞系對(duì)單域抗體HNSA039的特異性進(jìn)行免疫細(xì)胞化學(xué)鑒定。構(gòu)建單域抗體HNSA039與大腸桿菌硫氧還蛋白(TrxA)融合基因的原核表達(dá)載體pET-21 a(+)/TrxA-HNS A039,轉(zhuǎn)化大腸桿菌BL21 (DE3),進(jìn)行IPTG誘導(dǎo)表達(dá)。在變性條件下純化融合蛋白TrxA-HNSA039,進(jìn)行SDS-PAGE電泳鑒定和酶聯(lián)免疫吸附法(ELISA)檢測(cè)融合蛋白的免疫活性。以葡聚糖T40為中介體偶聯(lián)融合蛋白TrxA-HNSA039與BLM,利用流式細(xì)胞周期分析、MTT、克隆形成實(shí)驗(yàn)檢測(cè)其對(duì)鼻咽癌細(xì)胞系(CNE2)和人肺癌細(xì)胞系(A549)的抗腫瘤效應(yīng)。 結(jié)果：通過對(duì)噬菌體重鏈單域抗體HNSA039的免疫細(xì)胞化學(xué)鑒定,發(fā)現(xiàn)單域抗體HNSA039與CNE2呈較強(qiáng)陽性反應(yīng),而與其它人腫瘤細(xì)胞系和正常細(xì)胞系反應(yīng)呈弱陽性或不反應(yīng)。成功構(gòu)建的重組表達(dá)載體pET-21 a(+)/TrxA-HNS A039,經(jīng)IPTG誘導(dǎo)后表達(dá)形式分析顯示,融合蛋白主要以包涵體的形式存在,SDS-PAGE電泳顯示分子量大約為23KD；ELISA結(jié)果顯示融合蛋白與CNE2具有較好的結(jié)合活性。通過流式細(xì)胞周期分析、MTT、克隆形成實(shí)驗(yàn)檢測(cè)TrxA-HNSA039與硫酸博來霉素偶聯(lián)物(TrxA-HNSA039-BLM)的抗腫瘤活性,結(jié)果顯示偶聯(lián)物的抗腫瘤活性比抗體藥物混合物抗腫瘤活性顯著下降,差別有統(tǒng)計(jì)學(xué)意義(P0.05),而對(duì)靶細(xì)胞系(CNE2)和人肺癌細(xì)胞系(A549)抗腫瘤活性差別沒有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論：免疫細(xì)胞化學(xué)結(jié)果顯示單域抗體HNSA039具有較好的鼻咽癌細(xì)胞特異性。成功構(gòu)建TrxA-HNSA039的原核表達(dá)載體,然后表達(dá)并純化TrxA-HNSA039融合蛋白。ELISA實(shí)驗(yàn)結(jié)果顯示,融合蛋白TrxA-HNSA039具有較好的識(shí)別鼻咽癌細(xì)胞的免疫活性,而體外細(xì)胞實(shí)驗(yàn)結(jié)果顯示TrxA-HNSA039-BLM沒有明顯的靶向抗鼻咽癌活性。為了更好地研究單域抗體HNSA039,我們準(zhǔn)備融合單域抗體HNSA039基因或?qū)斡蚩贵w融合其它抗原識(shí)別結(jié)構(gòu),構(gòu)建雙價(jià)抗體增加其抗原識(shí)別特性,與“彈頭”藥物進(jìn)行直接偶聯(lián)進(jìn)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).
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R739.6

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