【摘要】：腫瘤壞死因子-α（TNF-α）是在類風(fēng)濕性關(guān)節(jié)炎（RA）等疾病的病理變化中起關(guān)鍵作用的細(xì)胞因子，臨床試驗(yàn)結(jié)果證實(shí)，TNF-α是治療該類疾病的合適靶點(diǎn)。針對TNF-α的抑制劑用于治療RA取得了良好的效果，其代表藥物有Enbrel，Remicade，Humira等。隨著TNF抗體類藥物臨床試驗(yàn)的開展，不斷有新的適應(yīng)癥出現(xiàn)，其市場空間不斷擴(kuò)大。這些藥物具有特異性高，靶向性好，副作用低的優(yōu)點(diǎn)，已逐漸成為與氨甲碟呤一起治療RA的一線用藥。但是它們也存在著不同的缺陷，主要有如下兩點(diǎn)：第一，Remicade是人鼠嵌合抗體，含有25%的鼠源片段，長期注射可能會(huì)引起人抗鼠抗體（HAMA）反應(yīng)；第二，這些抗體所攜帶的Fc段具有激活補(bǔ)體和ADCC等活性，會(huì)導(dǎo)致表達(dá)TNF的細(xì)胞凋亡，，產(chǎn)生一系列副作用。因此研發(fā)全人源小分子TNF-α抗體具有特別重要的意義。 本課題中全人源小分子TNF-α抗體的研發(fā)包括三個(gè)部分：TNF-α抗原的表達(dá)純化，抗體庫篩選和抗TNF-α Fab’抗體的表達(dá)純化。 TNF-α抗原的表達(dá)純化：根據(jù)GenBank中的人源TNF-α序列，全化學(xué)合成TNF-α基因，提取基因構(gòu)建TNF-α表達(dá)載體pET-23b/TNF-α并將其轉(zhuǎn)化大腸桿菌BL21(DE3)，獲得陽性克隆。將陽性克隆于37℃誘導(dǎo)表達(dá)16h得到發(fā)酵液上清中可溶性分泌表達(dá)的TNF-α，將其濃縮后再通過層析純化，最終得到了TNF-α純品，純度大于95%，比活性為4.4×10~6U/mg，產(chǎn)量為4mg/L。 抗體庫篩選：我們利用TNF-α作為抗原對噬菌體抗體庫進(jìn)行篩選，以期獲得對TNF-α有特異性親和力的抗體克隆。經(jīng)過六輪固相淘洗，與TNF-α特異性結(jié)合的噬菌體抗體克隆從1.1×10~3pfu上升至2×10~5pfu，產(chǎn)出投入比從3.2×10~(-8)上升至1.2×10~(-6)，盡管隨著篩選輪數(shù)的增加洗脫條件不斷嚴(yán)格，最終還是得到了200倍的富集。從第六輪淘洗獲得的克隆中隨機(jī)挑選200個(gè)克隆進(jìn)行ELISA篩選，得到5株陽性克隆。進(jìn)一步對這5株克隆進(jìn)行抗原結(jié)合特異性比較，最終確定了一株與TNF-α特異性最好的克隆G11。 抗TNF-α Fab’抗體的表達(dá)純化：將篩選得到的G11克隆進(jìn)行測序，獲得抗體基因序列，構(gòu)建Fab’抗體表達(dá)載體。將載體轉(zhuǎn)化大腸桿菌BL21(DE3)，獲得陽性克隆后，我們對其表達(dá)條件進(jìn)行優(yōu)化，比較了培養(yǎng)基和誘導(dǎo)時(shí)間對表達(dá)量的影響，結(jié)果顯示，用LB培養(yǎng)基在30℃，IPTG誘導(dǎo)濃度0.1mM下，誘導(dǎo)24h，F(xiàn)ab’抗體表達(dá)量最高。我們還對細(xì)菌破壁方法進(jìn)行了比較，在超聲，滲透壓休克、溶菌酶和反復(fù)凍融中我們選擇反復(fù)凍融法破碎細(xì)胞。純化后的Fab’抗體用SDS-PAGE電泳檢測，結(jié)果顯示抗體蛋白條帶單一，非競爭性ELISA測定抗體與TNF-α的親和常數(shù)為Ka=4×10-~(13)M~(-1)。另外體外中和試驗(yàn)顯示，F(xiàn)ab’抗體可以在一定程度上中和TNF-α對L929細(xì)胞的殺傷作用，在濃度為3.2ng/ml時(shí)對TNF-α細(xì)胞毒的中和率達(dá)到85%，在800pg/ml時(shí)對TNF-α細(xì)胞毒的中和率達(dá)到48%。 本研究以原核表達(dá)的TNF-α為抗原，從噬菌體抗體庫中篩選TNF-α抗體并對其進(jìn)行Fab’形式改造，最終獲得具有一定生物學(xué)活性的TNF-α人源性中和Fab’抗體，為相關(guān)疾病的治療奠定良好的基礎(chǔ)。
[Abstract]:Tumor necrosis factor - alpha (TNF- alpha) is a key cytokine in the pathological changes of rheumatoid arthritis (RA) and other diseases. The results of clinical trials have confirmed that TNF- alpha is a suitable target for the treatment of this disease. The inhibitors of TNF- alpha have achieved good results in the treatment of RA, and their representative drugs are Enbrel, Remicade, Humira and so on. The clinical trials of TNF antibody drugs have been developed with new indications and the market space is expanding. These drugs have the advantages of high specificity, good targeting and low side effects. They have gradually become the first-line drugs for the treatment of RA with methotrexate. But they also exist in different defects, including the following two points: first, Rem ICADE is human mouse chimeric antibody, which contains 25% mouse source fragments. Long-term injection may cause human anti mouse antibody (HAMA) reaction. Second, the Fc segments carried by these antibodies can activate complement and ADCC activity, which will lead to the expression of TNF cell apoptosis and produce a series of side effects. Therefore, it is particularly important to develop a whole human small molecule TNF- alpha antibody. Meaning.
The research and development of the TNF- alpha antibody of all human small molecules in this project includes three parts: expression and purification of TNF- alpha antigen, screening of antibody library and expression and purification of anti TNF- alpha Fab 'antibody.
The expression and purification of TNF- alpha antigen: according to the human TNF- alpha sequence in GenBank, the TNF- alpha gene was synthesized by the whole chemical synthesis, and the gene was extracted and transformed into the TNF- alpha expression vector pET-23b/TNF- A and transformed into the Escherichia coli BL21 (DE3) to obtain the positive clones. The positive clones were cloned at 37 C to express 16h to the soluble secretory expression of TNF- a in the supernatant of the fermentation liquid. After being concentrated and purified by chromatography, the purity of TNF- alpha was obtained. The purity was more than 95%, the specific activity was 4.4 * 10~6U/mg, and the yield was 4mg/L.
Antibody library screening: we use TNF- alpha as an antigen to screen the phage antibody library to obtain antibody clones that have specific affinity for TNF- alpha. After six rounds of solid phase scrub, the phage antibody clones with TNF- alpha specificity increased from 1.1 x 10~3pfu to 2 x 10~5pfu, and the output input ratio rises from 3.2 x 10~ (-8) to 1.2 x 10~ (-6). Although the elution conditions were more stringent with the increase of the number of screening wheels, 200 times of enrichment was finally obtained. 200 clones were selected randomly from sixth rounds of clones for ELISA screening and 5 positive clones were obtained. Further, the specificity of antigen binding was further compared to the 5 clones, and one of the most specific TNF- alpha specificity was determined. Good cloned G11.
The expression and purification of anti TNF- alpha Fab 'antibody: the screened G11 clones were sequenced, the antibody gene sequence was obtained, and the expression vector of Fab' antibody was constructed. After the vector was transformed into the Escherichia coli BL21 (DE3), the positive clones were obtained. The expression conditions were optimized and the effects of the medium and induction time on the expression were compared. The results showed that the expression of the antibody was compared. With the LB medium at 30 and IPTG induced concentration 0.1mM, the expression of 24h, Fab 'antibody was highest. We also compared the method of bacterial wall breaking. We selected the broken cells in ultrasonic, osmotic shock, lysozyme and repeated freezing and thawing. The purified Fab' antibody was detected by SDS-PAGE electrophoresis, and the result showed antibody protein. The affinity constant of the non competitive ELISA antibody and TNF- alpha was Ka=4 * 10-~ (13) M~ (-1). In addition, in vitro neutralization test showed that Fab 'antibody could neutralize the killing effect of TNF- a to L929 cells to a certain extent, and the neutralization rate of TNF- alpha cytotoxic at the concentration of 3.2ng/ml was 85%. And the rate is up to 48%.
In this study, the TNF- alpha expressed by prokaryotic expression was used as antigen to screen TNF- alpha antibody from phage antibody library and to transform it in Fab 'form. Finally, a certain biological activity of TNF- alpha human neutralization and Fab' antibody was obtained, which laid a good foundation for the treatment of related diseases.
【學(xué)位授予單位】：廣東藥學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 林麗珠，陳春妹，喻紅，呂群，陳曉龍，姚錫惠，陳海明，趙壽元;柱層析法純化重組人腫瘤壞死因子α及其抑癌作用[J];復(fù)旦學(xué)報(bào)(自然科學(xué)版);1994年05期

2 張振龍,張琰平,寧楊,竇金福,陳梅娣,崔春青,趙秀芬,樊曉翔,倪道明,李昌本;重組人腫瘤壞死因子α的分離純化及鑒定[J];中國生物制品學(xué)雜志;1998年02期

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