DNA-PKcs单链抗体DPK3-scFv的原核表达纯化及生物学作用

发布时间：2018-04-15 12:03

本文选题：单链抗体 + 原核表达　；参考：《安徽医科大学》2012年硕士论文

【摘要】：恶性肿瘤是当今威胁人类生命健康的头号杀手，而放射治疗是治疗恶性肿瘤的主要手段之一。但放射治疗过程中除了具有损伤正常组织包括骨髓毒性副作用外，另外一个主要问题就是肿瘤细胞产生放射耐受性。因此，增强肿瘤放疗的辐射敏感性就成为肿瘤研究领域的热点之一。放射治疗对肿瘤细胞的杀伤效应主要是通过电离辐射诱发肿瘤细胞DNA双链断裂(DNA double strand breaks, DSBs)，从而导致肿瘤细胞程序性死亡或坏死。但是细胞会对断裂的DNA进行修复，这是肿瘤细胞耐受辐射的主要机制之一，DNA修复能力越强，对放射的抵抗越强。哺乳动物细胞对DSBs损伤修复主要有两条途径，即：同源重组（homologous recombination，HR）和非同源末端连接（nonhomologous end-jioning，NHEJ）。DNA-PK （DNA-dependent protein kinase，DNA依赖的蛋白激酶）是NHEJ途径中重要的关键蛋白。 DNA-PK属于磷脂酰肌醇3-激酶相关蛋白激酶（Phosphatidylinositol-3-kinase-like kinase, PIKK）超家族成员，由调节亚单位Ku70/Ku80异源二聚体和催化亚基DNA-PKcs（DNA-dependent protein kinasecatalytic subunit）组成。DNA-PKcs在DSB修复中发挥重要作用主要是通过自磷酸化及磷酸化其他效应蛋白，DNA-PKcs还参与细胞周期G2期调控，细胞凋亡和自吞噬死亡，并且参与V（D）J重组过程和维护端粒长度。另外，研究表明DNA-PKcs在多种癌组织中异常高表达，且具有高转移特性和放化疗耐受的癌细胞中DNA-PKcs活性显著增高；特异性抑制DNA-PKcs可增强细胞对辐射的敏感性；表明DNA-PKcs具有作为放射增敏抗癌分子靶标的重要价值。目前已有多种抑制剂用以抑制DNA-PKcs功能，但是这些抑制剂或特异性差、或可导致肝毒性、或引起免疫反应，使其实际应用受限。如今，人源抗体制备技术尤其是通过噬菌体抗体库筛选单链抗体技术的发展，为癌症治疗的生物制药提供了新的技术方法。单链抗体是由免疫球蛋白的重链可变区（VH）和轻链可变区(VL)通过一段包含约10-25个氨基酸的连接肽连接而成的重组蛋白，是具有完全抗原结合位点的最小抗体片段。单链抗体具有很多优点：分子小，免疫原性低；容易进入实体瘤周围的微环境；半衰期短，不易蓄积；无Fc段，不易与具有Fc受体的非靶细胞结合；易于基因操作和基因工程大量生产。本实验室前期已成功地从人源化噬菌体抗体库中筛选到针对DNA-PKcs蛋白DPK3片段(AA1960-2227)的特异性人源化单链抗体DPK3-scFv,并将该抗体基因转染HeLa细胞，得出DPK3-scFv能显著提高其形成的肿瘤对放射治疗敏感性的结果。本研究拟通过原核表达单链抗体DPK3-scFv并纯化后，研究其透膜进入细胞和对DNA-PKcs自身磷酸化活性的影响，，为发展具有放射增敏作用的单链抗体提供依据。获得如下主要结果： 1、运用PCR扩增目的基因HA-DPK3-scFv,并将其克隆到原核表达载体pET28a，转化E.coli BL21(DE3)，经优化表达条件以包涵体形式成功表达出单链抗体HA-DPK3-scFv，经过包涵体蛋白变性复性得到纯化的HA-DPK3-scFv。 2、通过DNA依赖蛋白激酶分析系统检测单链抗体HA-DPK3-scFv的蛋白活性，大容量表达纯化单链抗体HA-DPK3-scFv，并观察HA-DPK3-scFv的跨膜转运；结果显示单链抗体HA-DPK3-scFv未跨细胞膜进入细胞内。 3、运用PCR扩增目的基因DPK3-scFv,并将其克隆到原核表达载体pET28a，转化E.coli BL21(DE3)，经优化表达条件以可溶性表达形式成功表达出单链抗体DPK3-scFv，经过亲和层析法在体外纯化出高纯度的单链抗体DPK3-scFv，并通过DNA依赖蛋白激酶分析系统检测单链抗体DPK3-scFv的蛋白活性。 4、将表达纯化出的单链抗体DPK3-scFv以终浓度70μg/ml加入到培养基中，与肿瘤细胞孵育，通过细胞免疫荧光染色和Western blotting检测不同时间点单链抗体DPK3-scFv透膜进入细胞的状况；结果显示，在无血清培养基的培育下单链抗体DPK3-scFv能有效透膜进入细胞并主要定位在细胞核中，单链抗体DPK3-scFv在体内的半衰期大约是12小时。 5、基于单链抗体DPK3-scFv在孵育1小时后就进入细胞的研究结果，肿瘤细胞与单链抗体DPK3-scFv孵育后2小时进行4Gy γ射线处理，并通过细胞免疫荧光染色和Western blotting检测不同时间点DNA-PKcs/pS2056磷酸化水平；结果显示，与对照组（未与单链抗体DPK3-scFv孵育的肿瘤细胞）相比，单链抗体DPK3-scFv会使照射后的肿瘤细胞DNA-PKcs/pS2056磷酸化水平下降。综上所述，本课题成功表达了并纯化出高浓度的针对DNA损伤修复关键蛋白DNA-PK的单链抗体DPK3-scFv，且检测了其蛋白活性；单链抗体DPK3-scFv可有效透膜进入细胞并稳定存在；单链抗体DPK3-scFv抑制照射后肿瘤细胞的DNA-PKcs/pS2056磷酸化水平。
[Abstract]:Cancer is one of the leading killers threatening the health of human life , and radiotherapy is one of the primary means of treating malignant tumors . However , in addition to the side effects of bone marrow toxicity in the course of radiotherapy , one of the main problems is the radiation tolerance of tumor cells . Therefore , it is one of the hot spots in the field of tumor research to enhance the radiation sensitivity of radiotherapy .

The killing effect of radiotherapy on tumor cells is mainly caused by DNA double strand breaks ( Downs ) induced by ionizing radiation , which leads to apoptosis or necrosis of tumor cells . However , the stronger the DNA repair ability , the stronger the resistance to radiation , the stronger the ability of DNA repair , the stronger the resistance to radiation . DNA - dependent protein kinase ( DNA - dependent protein kinase ) is a key protein in the NHEJ pathway .

DNA - PKcs ( DNA - PKcs ) plays an important role in regulation of cell cycle G2 , apoptosis and autophagy of DNA - PKcs . DNA - PKcs plays an important role in regulation of cell cycle G2 , apoptosis and autophagy death , and is involved in V ( D ) J recombination process and maintenance of telomeric length .
Specific inhibition of DNA - PKcs enhances cell sensitivity to radiation ;
It is shown that DNA - PKcs has the important value as the target of radiosensitization anti - cancer molecular target . There are many inhibitors to inhibit DNA - PKcs function , but these inhibitors or specific differences may lead to liver toxicity , or cause immune response , so that its practical application is limited . Nowadays , the preparation technology of human antibody , especially the development of screening single - chain antibody technology by phage antibody library , provides a new technical method for the biological pharmacy for cancer treatment .

The single - chain antibody is a recombinant protein formed by connecting a heavy chain variable region ( VH ) and a light chain variable region ( VL ) of an immunoglobulin through a connection peptide comprising about 10 to 25 amino acids , and is a minimum antibody fragment having a complete antigen binding site .
easy access to the microenvironment around the solid tumor ;
the half - life is short and is not easy to accumulate ;
no Fc fragment is difficult to bind to non - target cells with Fc receptors ;
The specific humanized single chain antibody DPK3 - scFv against DNA - PKcs protein DPK3 fragment ( AA1960 - 2227 ) was successfully screened from humanized phage antibody library , and the antibody gene was transfected into HeLa cells .

In order to develop a single - chain antibody with radiosensitivity , it is proposed to study the influence of its permeable membrane on the phosphorylation of DNA - PKcs by prokaryotic expression of single - chain antibody DPK3 - scFv . The results are as follows :

1 . The HA - DPK3 - scFv was amplified by PCR and cloned into the prokaryotic expression vector pET28a . The recombinant plasmid pET28a was transformed into E . coli BL21 ( DE3 ) . The HA - DPK3 - scFv was successfully expressed in inclusion body by the optimized expression condition , and the purified HA - DPK3 - scFv was purified by inclusion body protein denaturation .

2 , detecting the protein activity of the single - chain antibody HA - DPK3 - scFv by a DNA - dependent protein kinase assay system , expressing and purifying the single - chain antibody HA - DPK3 - scFv in large capacity , and observing the transmembrane transport of the HA - DPK3 - scFv ;
The results showed that the single chain antibody HA - DPK3 - scFv did not enter the cell membrane across the cell membrane .

3 . The target gene DPK3 - scFv was amplified by PCR and cloned into E . coli BL21 ( DE3 ) . The single - chain antibody , DPK3 - scFv , was successfully expressed in soluble form by optimized expression conditions , and the high - purity single - chain antibody DPK3 - scFv was purified by affinity chromatography in vitro , and the protein activity of single - chain antibody DPK3 - scFv was detected by DNA - dependent protein kinase assay .

4 . The purified single - chain antibody DPK3 - scFv was added to the culture medium at a final concentration of 70.mu . g / ml , incubated with tumor cells , and the conditions of entry of the single - chain antibody DPK3 - scFv permeable membrane at different time points were detected by cell immunofluorescence staining and Western blotting ;
The results showed that the single - chain antibody DPK3 - scFv was able to effectively penetrate into the cell and mainly locate in the nucleus without serum - free medium , and the half - life of single - chain antibody DPK3 - scFv in vivo was about 12 hours .

5 . After 1 hour incubation of single - chain antibody DPK3 - scFv , the tumor cells were treated with 4Gy 纬 - ray at 2 hours after incubation with single chain antibody DPK3 - scFv , and the phosphorylation level of DNA - PKcs / pS2056 was detected by immunofluorescence staining and Western blotting .
The results showed that the single - chain antibody DPK3 - scFv decreased the phosphorylation level of DNA - PKcs / pS2056 after irradiation compared to the control group ( tumor cells not incubated with single - chain antibody DPK3 - scFv ) .

In conclusion , the single - chain antibody DPK3 - scFv with high concentration of DNA - PK for DNA damage repair was successfully expressed and purified , and its protein activity was detected .
Single - chain antibody DPK3 - scFv can effectively penetrate into cells and stably exist ;
Single - chain antibody DPK3 - scFv inhibited the phosphorylation of DNA - PKcs / pS2056 of tumor cells after irradiation .

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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