小分子半抗原多价抗体的制备及其活性研究

发布时间：2018-09-08 07:43

【摘要】： 小分子半抗原单克隆抗体制备通常较复杂,且很多情况下,得到的抗体亲和力较差,效价很低,无法满足作为检测性抗体的要求,在很大程度上限制了单克隆抗体的应用。本研究以对硫磷为对象制备了单链抗体,旨在探索已知基因的半抗原基因工程抗体的构建,同时通过基因工程的方法构建了未知抗体基因的抗微囊藻毒素-LR(microcystin-LR, MC-LR)的单链抗体,并在此基础上构建了四价抗体,为基因工程抗体在小分子半抗原类物质检测方面的应用打下基础。参考GenBank中发表的对硫磷抗体序列,对其进行分析后在重链可变区基因(heavy chain V-region, VH)和轻链可变区基因(light chain V-region, VL)之间加上了45个核苷酸作为连接肽序列,由公司合成。构建单链抗体表达载体,经过原核表达得到了可溶性表达的目的蛋白。SDS-PAGE和Western Blot检测表明表达的单链抗体分子量为28 kD。用Ni-NTA金属亲和层析法对可溶性表达产物进行纯化,得到的目的蛋白纯度为74.8%,ELISA检测表明该scFv能够与对硫磷特异性结合。本实验为进一步制备高特异性、高亲和力抗体奠定了基础,并且为基因工程抗体在农药检测方面的应用提供依据。以分泌抗微囊藻毒素MC-LR单克隆抗体的杂交瘤细胞为材料,通过RT-PCR扩增出抗体的重链和轻链全编码区cDNA序列。序列分析表明,PCR得到的抗体重链序列共1473bp,包括部分VH,完整的CH1、CH2、CH3三个恒定区和3’端非编码区,其中VH大小为363bp,编码121个氨基酸,属小鼠IgH-V7183 VH5家族;得到的轻链序列共880bp,包括部分轻链可变区VL,完整的恒定区和3’非编码区,其中VL大小为336bp,编码112个氨基酸,属小鼠IGKV21亚组。VH和VL符合小鼠免疫球蛋白可变区基因特征,均含有4个框架区、3个抗原互补决定区及抗体特征性的2个半胱氨酸残基,且基因内无终止密码子,表明得到的序列为小鼠抗体重链和轻链基因序列。分别扩增VH和VL,通过重叠延伸PCR将二者拼接,构建原核表达载体pET-mc。序列分析表明,该scFv基因全长744bp,编码248个氨基酸。将表达载体转化大肠杆菌Origami 2(DE3)进行目的蛋白的诱导表达。经SDS-PAGE和Western Blot分析,单链抗体主要以包涵体的形式在大肠杆菌中表达。在IPTG为0.1 mM,培养温度为15℃时可溶性表达产物含量最高。利用Ni-NTA对可溶性表达产物进行纯化,回收到的目的蛋白浓度为0.115 mg/ml。ELISA检测表明该单链抗体能与MC-LR发生特异性结合。为提高单链抗体的亲和力,利用核心链霉亲和素的四聚化特性对scFv进行多聚化。将scFv基因与核心链霉亲和素基因拼接,并构建四价抗体表达载体pET-teab。经SDS-PAGE和Western Blot分析,四价抗体主要以不溶性的包涵体的形式表达。在IPTG为0.1 mM,培养温度为15℃时可溶性表达产物含量最高。利用Ni-NTA对可溶性表达产物进行纯化,回收到的目的蛋白浓度为0.179 mg/ml。非还原性SDS-PAGE及Western Blot检测表明,回收的目的蛋白以四聚体和单体的形式存在,没有检测到比四聚体分子量大的分子。ELISA检测结果表明,四价抗体与单链抗体和单克隆抗体相比,具有更高的亲和力。
[Abstract]:The preparation of monoclonal antibodies against small molecule hapten is usually complicated, and in many cases, the antibodies obtained have poor affinity and low titer, which can not meet the requirements of being used as detective antibodies. To a great extent, the application of monoclonal antibodies is limited. In this study, single chain antibodies against parathion were prepared to explore the semi-antibodies against known genes. At the same time, the single chain antibody against microcystin-LR (MC-LR) of unknown antibody gene was constructed by genetic engineering method. On this basis, the tetravalent antibody was constructed, which laid a foundation for the application of genetic engineering antibody in the detection of small molecule hapten substances.
Referring to the sequence of parathion antibody published in GenBank, 45 nucleotides were added between heavy chain V-region (VH) and light chain V-region (VL) genes as ligation peptides. The expression vector of single-chain antibody was constructed and soluble after prokaryotic expression. SDS-PAGE and Western Blot assays showed that the molecular weight of the expressed scFv was 28 kD. The purity of the soluble protein was 74.8% by Ni-NTA affinity chromatography. ELISA assay showed that the scFv could specifically bind to parathion. The high affinity antibody laid the foundation for the application of genetic engineering antibody in pesticide detection.
Hybridoma cells secreting monoclonal antibodies against microcystin MC-LR were used to amplify the full-coding region of the heavy chain and light chain of the antibody by RT-PCR. Sequence analysis showed that the heavy chain sequence of the antibody obtained by PCR was 1473 bp, including some VH, complete CH1, CH2 and CH3 constant regions and 3'-terminal non-coding regions, of which VH was 363 BP in size. It encodes 121 amino acids and belongs to the murine IgH-V7183 VH5 family. The obtained light-chain sequences consist of 880 bp, including partial light-chain variable region VL, complete constant region and 3'non-coding region, of which VL is 336 BP in size and encodes 112 amino acids, belonging to the murine IGKV21 subgroup. The two cysteine residues in the complementary determinant region of antigen and the characteristic antibody, and no termination codon in the gene, indicated that the obtained sequence was the heavy chain and light chain gene sequence of mouse antibody.
Prokaryotic expression vector pET-mc was constructed by splicing VH and VL, respectively. Sequence analysis showed that the scFv gene was 744 BP in length and encoded 248 amino acids. The expression vector was transformed into E. coli Origami 2 (DE3) to induce the expression of the target protein. SDS-PAGE and Western Blot analysis showed that the single chain antibody was mainly an inclusion body. When IPTG was 0.1 mM and culture temperature was 15 C, the soluble expression product was the highest. The soluble expression product was purified by Ni-NTA and the concentration of the recovered protein was 0.115 mg/ml. ELISA detection showed that the SCFv could bind specifically to MC-LR.
In order to improve the affinity of scFv, scFv was polymerized by tetramerization of core streptavidin. The scFv gene was spliced with core streptavidin gene and a tetravalent antibody expression vector pET-teab was constructed. The soluble protein was purified by Ni-NTA and the concentration of the recovered protein was 0.179 mg/ml. The results of non-reducing SDS-PAGE and Western Blot showed that the recovered protein existed in the form of tetramers and monomers, and the molecular weight of the recovered protein was not larger than that of tetramers. Molecular. ELISA results showed that tetravalent antibodies had higher affinity than single chain antibodies and monoclonal antibodies.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392.1

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