本文選題：核糖體展示 + 天然抗體庫�。� 參考：《華中農(nóng)業(yè)大學(xué)》2011年碩士論文

【摘要】：己烯雌酚(Diethylstilbestrol, DES)是第一個(gè)有活性可口服的人工合成非甾體雌激素物質(zhì)。但是,隨著研究的深入,發(fā)現(xiàn)DES有對(duì)人致癌,致畸以及對(duì)生殖系統(tǒng)的危害。針對(duì)上述情況,發(fā)展檢測(cè)DES殘留的簡(jiǎn)便、快速、靈敏的方法是有效遏制濫用DES的重要技術(shù)手段,也是維護(hù)法規(guī)制度的必要保障。與其它檢測(cè)方法相比,具有快速、靈敏、簡(jiǎn)便特性的檢測(cè)方法是免疫學(xué)分析方法。但是此法需要以特異性的抗體作為檢測(cè)基礎(chǔ)。利用核糖體展示技術(shù)篩選單鏈抗體(singe chain variable fragment, scFv),成本低,方便進(jìn)行大規(guī)模生產(chǎn)等。從未經(jīng)免疫的動(dòng)物或人的免疫組織或B細(xì)胞中,構(gòu)建的天然抗體文庫,從理論上講,只要使用合適的篩選方法,任何抗原都可以從中篩選到相應(yīng)的抗體,具有較強(qiáng)的通用性。利用核糖體展示技術(shù)篩選針對(duì)小分子半抗原單鏈抗體的研究較少,尤其是從天然抗體文庫中篩選更是少之又少。目前,還未見到利用此技術(shù)從天然抗體文庫中篩選DES單鏈抗體的報(bào)道。 本研究利用重疊引物延伸法(splicing by overlap extension, SOE) PCR技術(shù)構(gòu)建了天然鼠源單鏈抗體文庫,應(yīng)用核糖體展示技術(shù)篩選己烯雌酚特異的單鏈抗體。主要研究結(jié)果如下： 主要研究結(jié)果如下： 1.從未免疫的6-8周齡Balb/c小鼠的脾臟細(xì)胞中提取總RNA,利用Oligo(dT)15引物反轉(zhuǎn)出單鏈cDNA,再擴(kuò)增VH、VL基因片段,大小分別約為380 bp,350 bp;合成(G4S)3linker,經(jīng)序列測(cè)定,與理論序列完全一致。利用SOE-PCR構(gòu)建單鏈抗體文庫,大小約為750 bp,經(jīng)序列比對(duì),單鏈抗體的文庫中約80%的單鏈抗體可以正確翻譯,無移碼突變和致死突變,無終止密碼子；測(cè)序正確的單鏈抗體互補(bǔ)決定區(qū)氨基酸序列均不相同,證明構(gòu)建的單鏈抗體文庫具有良好的多樣性,可以進(jìn)行下一步的單鏈抗體篩選。 2.核糖體展示元件T(包含T7啟動(dòng)子,5’莖環(huán),核糖體識(shí)別位點(diǎn))以及間隔序列P(包含3’莖環(huán),間隔序列)是從已構(gòu)建好的載體pT7PD上擴(kuò)增,其大小分別為102bp和298bp,兩片段分別經(jīng)序列測(cè)定,與理論序列完全一致。以重疊引物延伸法(splicing by overlap extension, SOE)將T片段與scFv文庫連接,再將P片段與其拼接,構(gòu)建成核糖體展示單鏈抗體文庫,大小為1200 bp左右。 3.鼠源天然scFv文庫質(zhì)量的鑒定。 (1)將構(gòu)建好的核糖體展示單鏈抗體文庫連接pMD18-T克隆載體后,隨機(jī)挑取13株送Invitrogen公司測(cè)序鑒定文庫的完整性和多樣性。由測(cè)序結(jié)果可知,共有9株序列與文庫理論大小相符,且能完全正確的翻譯,內(nèi)部無終止密碼子,無移碼突變和點(diǎn)突變。由序列分析可知文庫仍保持著多樣性,經(jīng)Bioedit軟件對(duì)文庫的氨基酸序列分析,CDR區(qū)仍然保持著高度的可變性。以上結(jié)果進(jìn)一步驗(yàn)證了利用SOE-PCR構(gòu)建核糖體展示單鏈抗體文庫的方法是可行的,為下一步文庫的篩選打下了堅(jiān)實(shí)的基礎(chǔ)。 (2)經(jīng)鑒定,文庫的可擴(kuò)增性,轉(zhuǎn)錄活性,反轉(zhuǎn)錄活性良好。 4.使用固相和液相交替篩選的方法對(duì)文庫進(jìn)行七輪篩選,第一輪篩選后RT-PCR得到的條帶比較弱,說明原始抗體文庫中能與DES結(jié)合的抗體較少。經(jīng)過七輪核糖體展示篩選后,RT-PCR擴(kuò)增得到的DNA條帶亮度明顯增加,說明篩選后的抗體文庫中抗原陽性的抗體得到了富集。 5.將七輪篩選后單鏈抗體文庫進(jìn)行序列鑒定,其中有43株無致死突變,可以進(jìn)行DES結(jié)合活性的測(cè)定。 6.隨機(jī)挑選26個(gè)克隆子,將單鏈抗體基因與pTIG-TRX連接成表達(dá)載體,在大腸桿菌BL21(DE3)中表達(dá),經(jīng)SDS-PAGE和Western Blot鑒定,在約30KDa處有目的蛋白條帶,與理論蛋白大小一致,確定表達(dá)產(chǎn)物存在于破碎菌體后的上清液中。 7.將26株克隆子的表達(dá)上清液用間接ELISA和間接競(jìng)爭(zhēng)ELISA分別鑒定,結(jié)果篩選出5株克隆子對(duì)DES有特異性的結(jié)合。 8.對(duì)5株克隆子進(jìn)行序列比對(duì),結(jié)果表明CDR區(qū)有相似性,推斷抗DES單鏈抗體的抗體識(shí)別位點(diǎn)相似。用SPR分析篩出的一株30-3的單鏈抗體的親和力為3.79×10-6M。利用Anti-His tag親和層析柱對(duì)表達(dá)的單鏈抗體進(jìn)行了純化,使目的條帶得到了明顯的濃縮。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張林,劉杞;核糖體展示技術(shù)[J];重慶醫(yī)學(xué);2004年06期

2 楊勝林,蔣誠(chéng)績(jī);己烯雌酚對(duì)山羊育肥增重效果的研究[J];當(dāng)代畜牧;2001年02期

3 邵奎仲;;高效液相色譜測(cè)定畜禽肉中己烯雌酚殘留量方法研究[J];黑龍江科技信息;2009年14期

4 徐英宏,孫亞欣;薄層色譜法同時(shí)鑒別復(fù)方己烯雌酚栓的四種成分[J];遼寧藥物與臨床;2002年S1期

5 林紅英;王偉強(qiáng);沈子龍;蔣天梅;;鱔魚中己烯雌酚殘留量HPLC測(cè)定的研究[J];南京曉莊學(xué)院學(xué)報(bào);2006年04期

6 胡鯤,楊先樂,張菊;酶聯(lián)免疫法檢測(cè)中華鱉肌肉中己烯雌酚[J];上海水產(chǎn)大學(xué)學(xué)報(bào);2002年03期

7 馬紀(jì)偉;;雞肉中己烯雌酚殘留量的HPLC法測(cè)定研究[J];現(xiàn)代預(yù)防醫(yī)學(xué);2009年17期

8 李德元;己烯雌酚、維生素E綜合治療母豬乏情不孕[J];畜牧與獸醫(yī);1995年05期

9 趙國(guó)成;己烯雌酚誘導(dǎo)母兔發(fā)情試驗(yàn)[J];畜牧與獸醫(yī);1996年03期

10 劉清,劉軍,李志強(qiáng),馮菊英;甘肅省豬肉、豬肝中激素類生長(zhǎng)促進(jìn)劑殘留量檢測(cè)[J];預(yù)防醫(yī)學(xué)文獻(xiàn)信息;2002年06期

相關(guān)博士學(xué)位論文 前1條

1 袁青;柑橘潰瘍病菌重組單鏈抗體研究[D];重慶大學(xué);2007年

相關(guān)碩士學(xué)位論文 前1條

1 王恒;己烯雌酚免疫分析化學(xué)研究[D];揚(yáng)州大學(xué);2006年

，

本文編號(hào)：1904413