本文選題：表位 + 質(zhì)譜��； 參考：《第四軍醫(yī)大學(xué)》2010年碩士論文

【摘要】： 眾所周知,腫瘤是最致命的疾病之一。自20世紀(jì)70年代以來,我國癌癥患者的人數(shù)逐年升高,約有百分之八十的癌癥患者死于肺癌、肝癌、胃癌等常見腫瘤。通常的治療方法包括外科手術(shù)、放射治療、化療、生物治療等,這些治療方法有時(shí)單獨(dú)使用,有時(shí)聯(lián)合使用。 抗體治療是生物治療的一種,它的重要性越發(fā)明顯�？贵w治療的前景是光明的,目前已批準(zhǔn)20余種抗體藥物用于治療各種疾病,在臨床上已取得預(yù)期效果。抗體藥物是利用以細(xì)胞工程技術(shù)和基因工程技術(shù)為主體的抗體工程技術(shù)制備的藥物,但由于其伴隨的人抗鼠抗體反應(yīng)(HAMA),抗體藥物的應(yīng)用往往受到了一定程度的限制。為了降低抗體藥物的HAMA反應(yīng),基于蛋白質(zhì)結(jié)構(gòu)信息發(fā)展了一系列的改造抗體的方法。隨著分子生物學(xué)的發(fā)展,嵌合抗體和人源化抗體的出現(xiàn),成為了解決抗體應(yīng)用中降低HAMA反應(yīng)的有效途徑,但改造的抗體往往伴隨著親和力的大幅下降。 本研究建立了一種新型的抗體人源化方法,首次聯(lián)合基于質(zhì)譜的表位確定技術(shù)和分子對接對抗體進(jìn)行人源化改造。通過鑒定出HAb18G/CD147的抗原表位進(jìn)行HAb18抗體的人源化設(shè)計(jì),這樣就在保證了抗體HAb18親和力的基礎(chǔ)上進(jìn)行人源化改造,之后通過基因重組技術(shù)合成新的抗體片段并檢測該抗體片段的生物學(xué)活性,下面將從以下三部分分別說明。 第一部分:MALDI-TOF MS確定HAb18G/CD147的抗原表位 目的:確定HAb18G/CD147的表位序列 方法:利用配套的磁架洗滌新的磁珠,洗去磁珠上的防腐劑。加入適量的HAb18抗體,在室溫的條件下與磁珠結(jié)合1~2小時(shí)并輕微搖晃,使抗體與磁珠充分結(jié)合。之后用PBS(0.1% BSA,pH=7.4)洗滌磁珠4~5次,以封閉未結(jié)合抗體磁珠的活性位點(diǎn)。將適量的HAb18G/CD147加入到制備好的抗體磁珠混懸液中,室溫下結(jié)合1~2小時(shí),使抗原與抗體充分結(jié)合。加入胰酶溶液酶解HAb18G/CD147,用PBS洗滌掉未結(jié)合的抗原肽段,并用0.1㳠TFA(pH=2.5)洗脫HAb18G/CD147抗原表位肽并迅速凍干,用MALDI-TOF MS檢測制備的樣品。 結(jié)果:制備了HAb18G/CD147抗原表位肽,說明了利用免疫磁珠酶解抗原的辦法是可行的,并且制備出的樣品符合MALDI-TOF MS檢測的標(biāo)準(zhǔn)。對制備的HAb18G/CD147抗原表位肽進(jìn)行MALDI-TOF MS分析,并與PeptideCutter軟件預(yù)測的胰酶酶切HAb18G/CD147各肽段分子量進(jìn)行對比,得到了HAb18G/CD147抗原表位肽的序列信息。 第二部分:應(yīng)用確定的表位與分子對接對抗體HAb18人源化設(shè)計(jì) 目的:確定突變的氨基酸位點(diǎn) 方法:根據(jù)已有的HAb18抗體模型和得到的HAb18G/CD147抗原表位序列,應(yīng)用DOCKING軟件建立HAb18G/CD147與HAb18的分子對接模型,并預(yù)測表位的關(guān)鍵氨基酸殘基。在模擬出HAb18G/CD147同其抗體HAb18的對接模型后,用表面重塑的方法對HAb18可變區(qū)進(jìn)行人源化設(shè)計(jì)。利用序列分析和結(jié)構(gòu)分析確定鼠源抗體外露的差異殘基,選定將要突變的氨基酸殘基位點(diǎn),以便能夠合成低免疫原性又具有抗原結(jié)合活性的人源化抗體。 結(jié)果:建立了一套新的人源化抗體的方法,首次將基于MALDI-TOF MS確定抗原表位技術(shù)引入到分子對接中,建立了HAb18G/CD147和HAb18的分子對接模型,完成了HAb18可變區(qū)的人源化設(shè)計(jì),確定突變的氨基酸位點(diǎn)為H42E,H90A和L43S。 第三部分:人源化抗體片段HAb18-huscFv的表達(dá)與鑒定 目的:驗(yàn)證該人源化方法 方法:根據(jù)設(shè)計(jì)的人源化方案,用overlapping PCR的方法合成人源化形式HAb18-huscFv的基因,克隆至表達(dá)載體pCANTAB-6His-HAb18-huscFv中,轉(zhuǎn)化至E. coli中誘導(dǎo)表達(dá)。經(jīng)Ni2+柱純化表達(dá)的蛋白后,采用SDS-PAGE、Western blot、細(xì)胞免疫熒光等方法對表達(dá)產(chǎn)物進(jìn)行檢測、鑒定,同步構(gòu)建表達(dá)其親代鼠源形式抗體為對照。采用間接競爭ELISA法分析抗體HAb18-huscFv與HAb18G/CD147的結(jié)合活性, SPR法測定抗體HAb18-huscFv與HAb18G/CD147的親和力,并用同步表達(dá)的鼠源形式抗體作為對照,分析兩種抗體有無差異。最后采用HAMA陽性血清,對比了HAb18-huscFv和鼠源HAb18-scFv的免疫原性差異。 結(jié)果:根據(jù)已選擇的突變位點(diǎn),用overlapping PCR的方法合成了新的基因并克隆至表達(dá)載體,測序驗(yàn)證正確后轉(zhuǎn)化到E. coli中誘導(dǎo)表達(dá),經(jīng)Ni2+柱純化后獲得了較純的HAb18-huscFv。SDS-PAGE和Western blot檢測目標(biāo)蛋白分子量約為27kDa。經(jīng)SPR法測定,HAb18-huscFv與HAb18-scFv相比親和力無明顯差異。用細(xì)胞免疫熒光法檢測HAb18-huscFv與肝癌細(xì)胞SMMC-7721具有一定結(jié)合能力。間接競爭ELISA法結(jié)果顯示:抗體HAb18-huscFv與HAb18-scFv相比具有較低的免疫原性。 結(jié)論:基于利用新方法建立的抗體人源化方案,改造后的抗體HAb18-huscFv與HAb18-scFv相比,在降低了免疫原性的同時(shí),又保留了抗體的抗原結(jié)合能力,證明了該方法是有效可行的,為抗體人源化設(shè)計(jì)提供了新的思路。
[Abstract]:As we all know, cancer is one of the most deadly diseases. Since 1970s, the number of cancer patients in our country has increased year by year, and about eighty percent of cancer patients die from common tumors such as lung, liver and stomach cancer. The common treatment methods include surgery, radiation therapy, chemotherapy, biological treatment and so on. These treatments are sometimes alone. Use, sometimes combined.
Antibody therapy is a kind of biological therapy, its importance is more and more obvious. The prospect of antibody treatment is bright. At present, more than 20 kinds of antibody drugs have been approved for the treatment of various diseases, and the expected effect has been achieved. Antibody drugs are made by using antibody engineering technology based on cell engineering and genetic engineering technology. But because of its anti mouse antibody response (HAMA), the application of antibody drugs is often limited to a certain extent. In order to reduce the HAMA reaction of antibody drugs, a series of methods for transforming antibodies have been developed based on protein structure information. With the development of molecular biology, the emergence of chimeric and humanized antibodies has become the result of the development of molecular biology. The effective way to reduce the HAMA reaction in the application of antibodies is to solve the problem, but the modified antibody is often accompanied by a sharp decrease in affinity.
In this study, a new method of humanization of antibody was established. It was first combined with mass spectrometry based epitope determination technique and molecular docking for humanization of antibody. By identifying the epitopes of HAb18G/CD147, the humanized design of HAb18 antibody was carried out, so that the humanized transformation was made on the basis of guaranteeing the affinity of antibody HAb18. After that, the new antibody fragment was synthesized by gene recombination technology and the biological activity of the antibody fragment was detected. The following three parts will be explained below.
Part I: MALDI-TOF MS determines the epitope of HAb18G/CD147.
Objective: to determine the epitope sequence of HAb18G/CD147
Methods: wash the new magnetic beads and remove the antiseptic on the magnetic beads. Add appropriate HAb18 antibody, combine the magnetic beads with the magnetic beads for 1~2 hours at room temperature and gently shake them, so that the antibody and magnetic beads are fully combined. Then PBS (0.1% BSA, pH=7.4) is used to wash the magnetic beads 4~5 times to close the active site of unconjugated magnetic beads. HAb18G/CD147 was added to the prepared antibody magnetic bead suspension. The antigen was combined with the antibody at room temperature for 1~2 hours at room temperature. The trypsin solution HAb18G/CD147 was added to the enzyme solution and the unbound antigen peptide was washed out with PBS, and the HAb18G/CD147 antigen epitopes were eluted with 0.1 TFA (pH=2.5) and freeze-dried quickly, and the samples were prepared by MALDI-TOF MS Goods.
Results: the HAb18G/CD147 epitope peptide was prepared, indicating that the method of using immunomagnetic beads to solve the antigen was feasible, and the prepared samples were in conformity with the standard of MALDI-TOF MS detection. The MALDI-TOF MS analysis of the prepared HAb18G/CD147 epitopes was carried out and the peptide segments of the pancreatin were predicted by the PeptideCutter software. The sequence information of HAb18G/CD147 epitope peptide was obtained by comparing the subunits.
The second part: the application of designated epitope and molecular docking to the humanization design of antibody HAb18.
Objective: to determine the amino acid site of the mutant
Methods: Based on the existing HAb18 antibody model and the HAb18G/CD147 epitope sequence obtained, the molecular docking model of HAb18G/CD147 and HAb18 was established by DOCKING software, and the key amino acid residues in the epitopes were predicted. After simulating the docking model of HAb18G/CD147 with its antibody HAb18, the HAb18 variable region was carried out by surface remodeling. Source design. Using sequence analysis and structural analysis to determine the differential residues of mouse source antibody exposure, select the amino acid residues that will be mutated, in order to be able to synthesize human derived antibodies with low immunogenicity and antigen binding activity.
Results: a new method of humanized antibody was established. The MALDI-TOF MS based epitope technique was first introduced into the molecular docking, and the molecular docking model of HAb18G/CD147 and HAb18 was established. The humanized design of the HAb18 variable region was completed, and the mutant amino acid sites were H42E, H90A and L43S..
The third part: expression and identification of humanized antibody fragment HAb18-huscFv.
Objective: to verify the humanization method
Methods: according to the designed humanization scheme, the gene of human derived form HAb18-huscFv was synthesized by overlapping PCR, and was cloned into the expression vector pCANTAB-6His-HAb18-huscFv and transformed into E. coli to induce expression. After purification of the expressed protein by Ni2+ column, the expression products were introduced by SDS-PAGE, Western blot, cell immunofluorescence and so on. An indirect competitive ELISA method was used to analyze the binding activity of antibody HAb18-huscFv and HAb18G/CD147, and the affinity between the antibody HAb18-huscFv and HAb18G/CD147 was measured by SPR method, and the antibody of the mouse source was used as the control, and the two kinds of antibodies were analyzed. HAMA positive serum was used to compare the immunogenicity of HAb18-huscFv and mouse HAb18-scFv.
Results: according to the selected mutation sites, a new gene was synthesized and cloned to the expression vector by overlapping PCR. After the sequencing verification was correct, the expression was transformed into E. coli. After the purification of the Ni2+ column, the molecular weight of the pure HAb18-huscFv.SDS-PAGE and Western blot detection target protein was about 27kDa. by SPR method and HAb18-h. There was no significant difference in affinity between uscFv and HAb18-scFv. The binding ability of HAb18-huscFv to SMMC-7721 cells was detected by cell immunofluorescence. The result of indirect competitive ELISA showed that the antibody HAb18-huscFv had a lower immunogenicity compared with HAb18-scFv.
Conclusion: Based on the antibody humanization scheme established by the new method, the modified antibody HAb18-huscFv is compared with HAb18-scFv, while reducing the immunogenicity and retaining the antigen binding ability of the antibody, it is proved that this method is effective and feasible, and provides a new idea for the antibody humanization design.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

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本文編號：2031021