【摘要】： 鼠源性單抗在人體使用后可產(chǎn)生人抗鼠抗體(Human Anti-Mouse Antibody, HAMA)反應(yīng),而且分子量大,不能有效進(jìn)入病灶部位,臨床使用受到限制。單鏈抗體(single chain Fv, ScFv)是由一條連接肽將IgG分子的重鏈和輕鏈的可變區(qū)連接而成的,減少了免疫原性,分子小,實(shí)體組織穿透力強(qiáng),血漿半衰期短,因而在臨床疾病的診斷和治療中具有重要的應(yīng)用價(jià)值。 將抗人CD28的鼠源單鏈抗體基因(mouse anti-human CD28 ScFv )亞克隆到桿狀病毒轉(zhuǎn)移載體pFastBacHTa中,得到重組轉(zhuǎn)移載體pFastBacHTa-ScFv轉(zhuǎn)化大腸桿菌DH10Bac,獲得重組桿粒rBacmid-ScFv。脂質(zhì)體介導(dǎo)轉(zhuǎn)染Sf9昆蟲細(xì)胞后獲得重組桿狀病毒rBV-ScFv。將該重組病毒感染Sf9細(xì)胞, SDS-PAGE和Weatern blot分析表明:抗人CD28 ScFv在昆蟲細(xì)胞中獲得高效表達(dá),分子量約36 kDa。細(xì)胞裂解及產(chǎn)物可溶性分析表明:該單鏈抗體基因在Sf9細(xì)胞中超高量表達(dá)并以不溶性集聚體形式存在,表達(dá)產(chǎn)物可達(dá)細(xì)胞總蛋白的25.7%。經(jīng)變性溶解集聚體、Ni-NTA親和層析,獲得純化抗人CD28 ScFv表達(dá)產(chǎn)物。流式細(xì)胞分析,當(dāng)CD28 SvFc結(jié)合了T-28細(xì)胞表面抗原結(jié)合表位后,單克隆抗體2F5與T-28細(xì)胞的結(jié)合率從86.9%下降至18.1%,表明抗人CD28 ScFv可有效地與CD28單克隆抗體競爭性結(jié)合CD28細(xì)胞表面抗原。 HyNPV是BmNPV和AcNPV通過基因重組后得到的一個(gè)具有BmNPV和AcNPV雙重優(yōu)點(diǎn)的新型雜交病毒,本研究還利用家蠶-HyNPV表達(dá)系統(tǒng),研究了其在家蠶中的表達(dá)。 本研究在成功拼接抗人CD28單鏈抗體基因的基礎(chǔ)上,應(yīng)用二種Bac-to-Bac BEVS系統(tǒng)構(gòu)建并獲得了抗人CD28-ScFv的轉(zhuǎn)移載體和重組病毒,并在Sf9細(xì)胞和家蠶中表達(dá)了CD28-ScFv,首次對(duì)Sf9細(xì)胞中表達(dá)的外源蛋白抗CD28 ScFv的可溶性問題進(jìn)行了研究,獲得了具有免疫學(xué)活性的高純度的抗人CD28單鏈抗體。為抗CD28 ScFv的抗腫瘤藥物的開發(fā)奠定了基礎(chǔ)。
[Abstract]:Human Anti-Mouse Antibody (Hama) reaction can be produced after human use of murine monoclonal antibody, and its molecular weight is too large to enter the lesion effectively, so its clinical use is restricted. The single chain antibody (single chain Fv (scFv) is made of a ligated peptide that connects the heavy chain and the variable region of the light chain of the molecule. It reduces immunogenicity, is small, has strong penetration of solid tissue, and has a short plasma half-life. Therefore, it has important application value in the diagnosis and treatment of clinical diseases. The mouse scFv gene (mouse anti-human CD28 scFv) was subcloned into baculovirus transfer vector pFastBacHTa. The recombinant transfer vector pFastBacHTa-ScFv was transformed into E. coli DH10Bac.Recombinant rBacmid-ScFv was obtained. Recombinant baculovirus rBV-ScFv was obtained after transfection of Sf9 insect cells by liposome. The recombinant virus was infected with Sf9 cells. SDS-PAGE and Weatern blot analysis showed that anti-human CD28 scFv was highly expressed in insect cells with a molecular weight of about 36 kDa. Cell cleavage and soluble product analysis showed that the single chain antibody gene was expressed in high quantity in Sf9 cells and existed in the form of insoluble aggregates, and the expressed product could reach 25.775% of the total cell protein. The anti-human CD28 scFv expression product was purified by denaturing solubilizing agglomeration Ni-NTA affinity chromatography. By flow cytometry, when CD28SvFc binds to T-28 cell surface antigen binding epitopes, The binding rate of monoclonal antibody 2F5 to T-28 cells decreased from 86.9% to 18.1%, which indicated that anti-human CD28 scFv could effectively compete with CD28 monoclonal antibody to bind to CD28 cell surface antigen. HyNPV was the result of gene recombination of BmNPV and AcNPV. A novel hybrid virus with the advantages of BmNPV and AcNPV was obtained. The expression of silkworm-HyNPV in silkworm was studied by using silkworm-HyNPV expression system. On the basis of successfully splicing the single chain antibody gene against human CD28, two kinds of Bac-to-Bac BEVs system were used to construct and obtain the transfer vector and recombinant virus against human CD28-ScFv. CD28-ScFv was expressed in Sf9 cells and silkworm. The soluble expression of foreign protein against CD28 scFv in Sf9 cells was studied for the first time. A high purity anti-CD28 scFv antibody with immunological activity was obtained. It lays a foundation for the development of anti-tumor drugs for anti-CD 28 scFv.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 胡靜平;抗人B7-2單鏈抗體基因在Tn5細(xì)胞、家蠶及哺乳動(dòng)物細(xì)胞中的表達(dá)研究[D];蘇州大學(xué);2010年

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本文編號(hào)：2133162