核糖体展示天然鼠源scFv文库筛选己烯雌酚抗体

发布时间：2018-05-18 04:35

本文选题：核糖体展示 + 天然抗体库　；参考：《华中农业大学》2011年硕士论文

【摘要】：己烯雌酚(Diethylstilbestrol, DES)是第一个有活性可口服的人工合成非甾体雌激素物质。但是,随着研究的深入,发现DES有对人致癌,致畸以及对生殖系统的危害。针对上述情况,发展检测DES残留的简便、快速、灵敏的方法是有效遏制滥用DES的重要技术手段,也是维护法规制度的必要保障。与其它检测方法相比,具有快速、灵敏、简便特性的检测方法是免疫学分析方法。但是此法需要以特异性的抗体作为检测基础。利用核糖体展示技术筛选单链抗体(singe chain variable fragment, scFv),成本低,方便进行大规模生产等。从未经免疫的动物或人的免疫组织或B细胞中,构建的天然抗体文库,从理论上讲,只要使用合适的筛选方法,任何抗原都可以从中筛选到相应的抗体,具有较强的通用性。利用核糖体展示技术筛选针对小分子半抗原单链抗体的研究较少,尤其是从天然抗体文库中筛选更是少之又少。目前,还未见到利用此技术从天然抗体文库中筛选DES单链抗体的报道。本研究利用重叠引物延伸法(splicing by overlap extension, SOE) PCR技术构建了天然鼠源单链抗体文库,应用核糖体展示技术筛选己烯雌酚特异的单链抗体。主要研究结果如下：主要研究结果如下： 1.从未免疫的6-8周龄Balb/c小鼠的脾脏细胞中提取总RNA,利用Oligo(dT)15引物反转出单链cDNA,再扩增VH、VL基因片段,大小分别约为380 bp,350 bp;合成(G4S)3linker,经序列测定,与理论序列完全一致。利用SOE-PCR构建单链抗体文库,大小约为750 bp,经序列比对,单链抗体的文库中约80%的单链抗体可以正确翻译,无移码突变和致死突变,无终止密码子；测序正确的单链抗体互补决定区氨基酸序列均不相同,证明构建的单链抗体文库具有良好的多样性,可以进行下一步的单链抗体筛选。 2.核糖体展示元件T(包含T7启动子,5’茎环,核糖体识别位点)以及间隔序列P(包含3’茎环,间隔序列)是从已构建好的载体pT7PD上扩增,其大小分别为102bp和298bp,两片段分别经序列测定,与理论序列完全一致。以重叠引物延伸法(splicing by overlap extension, SOE)将T片段与scFv文库连接,再将P片段与其拼接,构建成核糖体展示单链抗体文库,大小为1200 bp左右。 3.鼠源天然scFv文库质量的鉴定。 (1)将构建好的核糖体展示单链抗体文库连接pMD18-T克隆载体后,随机挑取13株送Invitrogen公司测序鉴定文库的完整性和多样性。由测序结果可知,共有9株序列与文库理论大小相符,且能完全正确的翻译,内部无终止密码子,无移码突变和点突变。由序列分析可知文库仍保持着多样性,经Bioedit软件对文库的氨基酸序列分析,CDR区仍然保持着高度的可变性。以上结果进一步验证了利用SOE-PCR构建核糖体展示单链抗体文库的方法是可行的,为下一步文库的筛选打下了坚实的基础。 (2)经鉴定,文库的可扩增性,转录活性,反转录活性良好。 4.使用固相和液相交替筛选的方法对文库进行七轮筛选,第一轮筛选后RT-PCR得到的条带比较弱,说明原始抗体文库中能与DES结合的抗体较少。经过七轮核糖体展示筛选后,RT-PCR扩增得到的DNA条带亮度明显增加,说明筛选后的抗体文库中抗原阳性的抗体得到了富集。 5.将七轮筛选后单链抗体文库进行序列鉴定,其中有43株无致死突变,可以进行DES结合活性的测定。 6.随机挑选26个克隆子,将单链抗体基因与pTIG-TRX连接成表达载体,在大肠杆菌BL21(DE3)中表达,经SDS-PAGE和Western Blot鉴定,在约30KDa处有目的蛋白条带,与理论蛋白大小一致,确定表达产物存在于破碎菌体后的上清液中。 7.将26株克隆子的表达上清液用间接ELISA和间接竞争ELISA分别鉴定,结果筛选出5株克隆子对DES有特异性的结合。 8.对5株克隆子进行序列比对,结果表明CDR区有相似性,推断抗DES单链抗体的抗体识别位点相似。用SPR分析筛出的一株30-3的单链抗体的亲和力为3.79×10-6M。利用Anti-His tag亲和层析柱对表达的单链抗体进行了纯化,使目的条带得到了明显的浓缩。
[Abstract]:Diethylstilbestrol (Diethylstilbestrol, DES) is the first synthetic non steroidal estrogenic substance with active and oral administration. However, with the development of research, it is found that DES has a carcinogenic, teratogenic and harmful effects on the reproductive system. In this case, the development of a simple, rapid and sensitive method for detecting DES residues is an effective way to prevent the abuse of DES. An important technical means is also a necessary safeguard for the maintenance of the regulatory system. Compared with other testing methods, the rapid, sensitive and simple detection method is immunological analysis. However, this method needs specific antibodies as the basis of detection. Singe chain variable fragment (scFv) is selected by the ribosome display technique. Low cost, convenient for large-scale production, and so on. The natural antibody library which has never been immune to animals or human immune tissues or B cells. In theory, as long as the appropriate screening method is used, any antigen can be screened from the corresponding antibody, which is more versatile. There are few studies on the single chain antibody of molecular hapten, especially in the screening of natural antibody library. At present, there has not been a report on the screening of DES single chain antibody from natural antibody library by this technique.
In this study, a single chain antibody library of natural rat source was constructed by using the splicing by overlap extension (SOE) PCR technique, and the specific single chain antibody of diethylstilbestrol was screened by ribosome display technique. The main results were as follows:
The main results are as follows:
1. the total RNA was extracted from the spleen cells of the Balb/c mice of 6-8 weeks old, which was never immune. Using Oligo (dT) 15 primers to reverse the single chain cDNA, then amplify the VH and VL gene fragments, the size was about 380 BP, 350 BP, and the synthesis (G4S) 3linker was identical with the theoretical sequence. The size of the single chain antibody library was about 750, and the sequence was about 750. About 80% of single chain antibody in the library of single chain antibody can be translated correctly, without transmutation and fatal mutation, without terminating codon, and the sequence of amino acids in the correct single chain antibody complementary determination region of sequencing is different, which proves that the constructed single chain antibody library has good versatility and can be screened for the next single chain antibody.
2. ribosome display element T (including T7 promoter, 5 'stem ring, ribosome identification site) and spacer sequence P (including 3' stem ring, interval sequence) are amplified from the constructed carrier pT7PD, the size of which is 102bp and 298bp respectively, and the two fragments are sequenced respectively. The overlap primer extension method (splicing by ove) is used. Rlap extension (SOE) connects T fragments to scFv libraries, and then splits P fragments into a library of ribosome scFv, which is about 1200 BP.
3. identification of the quality of the natural scFv Library of the rat.
(1) after the constructed ribosome display single chain antibody library was connected to the pMD18-T cloning vector, the integrity and diversity of the 13 Invitrogen sequencing identification library were randomly selected. From the sequencing results, a total of 9 sequences were conformed to the size of the library theory and could be completely translated, internal no terminating codons, no shift code mutation and points were found. From the sequence analysis, we know that the library remains diverse. After the analysis of the amino acid sequence of the library by the Bioedit software, the CDR region remains highly variable. The above results further verify that the method of using SOE-PCR to construct the ribosome to display the single chain antibody library is feasible, and has laid a solid foundation for the screening of the next library. Foundation.
(2) the amplification, transcriptional activity and reverse transcription activity of the library were good.
4. using the solid-phase and liquid phase alternate screening method to select the library for seven rounds. After the first round of screening, the band of the RT-PCR is weaker. It shows that the antibody that can be combined with DES in the original antibody library is less. After seven rounds of ribosome display, the DNA strip brightness of the RT-PCR amplification is obviously increased, indicating the antibody library after screening. The antigen positive antibody was enriched.
5. sequence identification of single strand FV library after seven rounds of screening. 43 of them had no lethal mutation, and DES binding activity could be detected.
6. randomly selected 26 clones, connected the single chain antibody gene with pTIG-TRX as an expression vector, expressed in Escherichia coli BL21 (DE3), identified by SDS-PAGE and Western Blot, and had the target protein band at about 30KDa, consistent with the size of the theoretical protein, and determined the expression product in the supernatant after the broken body.
7. the expression supernatants of 26 clones were identified by indirect ELISA and indirect competitive ELISA respectively. The results showed that 5 clones had specific binding to DES.
8. the sequence alignment of 5 clones showed that the CDR region was similar. It was concluded that the antibody recognition site of the anti DES single chain antibody was similar. The affinity of a 30-3 single chain antibody screened by SPR was 3.79 x 10-6M. using Anti-His tag affinity chromatography column to purify the expressed single chain antibody. Concentrate.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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