【摘要】： 研究背景急性髓系白血病(AML)是一種常見的惡性血液系統(tǒng)疾病,雖然化放療和造血干細(xì)胞移植技術(shù)的應(yīng)用提高了患者的生存率,但仍有60%-70%遭受該疾病痛苦的病人死于AML。放化療后的造血干細(xì)胞移植只是為許多耐藥白血病類型病人提供了一種治愈的機(jī)會,盡管有許多研究團(tuán)隊已證明了這種治療方法的有效、安全和可行性。研究證明針對白血病前體細(xì)胞和骨髓造血干細(xì)胞進(jìn)行靶向放療最安全的方法就是用放射物標(biāo)記的抗體直接靶向白血病細(xì)胞或正常骨髓造血干細(xì)胞。用單克隆抗體治療急性髓性白血病早已有所報道,常用的抗體靶向標(biāo)志有CD33、CD66和CD45,而現(xiàn)行研究認(rèn)為抗CD45的抗體是最具研發(fā)前景的。 CD45抗原是一種穩(wěn)定、高表達(dá)于所有白細(xì)胞和他們的前體細(xì)胞以及超過70%的骨髓有核細(xì)胞表面上的單鏈細(xì)胞膜糖蛋白,CD45又被稱為GP180、T200或白細(xì)胞共同抗原(LCA),是一種分子量約為200 kDa的酪氨酸磷酸酶,它穩(wěn)定表達(dá)于除成熟紅細(xì)胞和血小板外的所有造血細(xì)胞,且不存在于非造血組織中,使之成為治療白血病的一個誘人的靶點(diǎn)。 CD45能廣泛的表達(dá)于幾乎所有的白細(xì)胞,包括骨髓的髓系前體細(xì)胞和淋巴結(jié)中的成熟淋巴細(xì)胞以及90%的AML細(xì)胞,這些細(xì)胞和組織為病人治療的緩解或復(fù)發(fā)提供了大量抗體結(jié)合位點(diǎn)。由于CD45作為白細(xì)胞的標(biāo)記物能出現(xiàn)在正常和惡性細(xì)胞,所以在病人體內(nèi)應(yīng)用抗CD45抗體可以通過代謝分布到骨髓、脾臟和淋巴結(jié)中與靶抗原結(jié)合,用于治療急性白血病。 在放射免疫治療中,通常的做法是有選擇地針對一個特定的抗原將放射性物質(zhì)運(yùn)輸?shù)侥[瘤細(xì)胞。由于造血組織來源的細(xì)胞對放射線敏感和其上的靶抗原的高度可接近性,對血液系統(tǒng)腫瘤的放免治療一直被認(rèn)為是最成功的。在AML和MDS的放免治療中,CD45抗原由于具有高表達(dá)和特異結(jié)合性而成為極具吸引力的靶向目標(biāo)。放射性標(biāo)記的抗CD45抗體可將輻射物質(zhì)運(yùn)輸?shù)娇乖栃约?xì)胞和周邊環(huán)繞的抗原陰性細(xì)胞上。因此,放射免疫偶聯(lián)物不需要以高濃度綁定到每一個白血病細(xì)胞上或滲入骨髓或腫瘤細(xì)胞中引發(fā)致死性的DNA損傷。在復(fù)發(fā)的白血病病人的放免治療中,即使腫瘤細(xì)胞不表達(dá)CD45抗原,也會由于圍繞其周圍的高表達(dá)CD45抗原的非惡性造血細(xì)胞所介導(dǎo)的旁觀者效應(yīng)而被殺滅。在造血干細(xì)胞移植領(lǐng)域,CD45抗原的這些特性,能促進(jìn)研究者針對它的放射性標(biāo)記的系列抗體進(jìn)行更加深入的研究,以便用于臨床的治療。 隨著CD45分子異構(gòu)體的研究、流式細(xì)胞儀免疫分型的應(yīng)用及抗CD45單抗的靶向治療研究等深入發(fā)展,CD45分子在免疫學(xué)和血液學(xué)方面的應(yīng)用研究受到進(jìn)一步的關(guān)注�？梢灶A(yù)期,CD45必將會在臨床相關(guān)疾病的診斷、治療和預(yù)后等方面發(fā)揮更大的作用。然而抗CD45單克隆抗體來源于小鼠,用于人體可誘導(dǎo)抗鼠抗體的產(chǎn)生。因此進(jìn)行基因工程改造抗體具有重要實(shí)踐意義。我們實(shí)驗(yàn)組已報道過了抗CD45單抗在AML治療中良好前景,由于治療中藥物仍存在毒性使我們一直致力于不斷的改進(jìn)。 在這篇文章中,我們首次報告關(guān)于抗CD45人-鼠嵌合抗體的構(gòu)建,并描述了該基因工程抗體對血液系統(tǒng)疾病治療的獨(dú)特優(yōu)勢。為后續(xù)將該嵌合抗體和放射性物質(zhì)如碘-131偶聯(lián),用于預(yù)定位放射免疫治療自體和異體造血干細(xì)胞移植的進(jìn)一步研究奠定了堅實(shí)的基礎(chǔ)。 人源化或嵌合單克隆抗體(MoAbs)能與造血細(xì)胞表達(dá)的抗原產(chǎn)生反應(yīng),他們可以直接(“親本”或“裸”抗體)或通過與毒素、放射性同位素或抗腫瘤藥物偶聯(lián)而用于臨床研發(fā)中。抗體治療由于抗體小分子的特異性和親和力在循環(huán)系統(tǒng)中容易與腫瘤細(xì)胞結(jié)合是急性的骨髓性白血病治療的理想方案。根據(jù)其是否攜帶其他連接物分為未結(jié)合型單抗、抗癌藥物結(jié)合型單抗及同位素結(jié)合型單抗。目前已有五個藥物獲美國FDA的批準(zhǔn)用于臨床,其中未結(jié)合型均為人-鼠嵌合抗體或人源化抗體。但單獨(dú)使用未結(jié)合型CD45單抗的在人體內(nèi)應(yīng)用效果甚微和受到鼠源性的限制,目前國外關(guān)于CD45單抗的研究主要是與放射性核素131I及213Bi連接作為造血干細(xì)胞移植預(yù)處理方案的組成部分,以期減少TBI的劑量,從而減少TBI所導(dǎo)致的全身毒副作用。動物試驗(yàn)及Ⅰ/Ⅱ期臨床試驗(yàn)均證實(shí),CD45單抗能選擇性的將放射性核素131I及213Bi導(dǎo)入骨髓、肝脾和淋巴結(jié),可部分代替TBI參與預(yù)處理方案。Vallera等用90Y標(biāo)記CD45單抗進(jìn)行了淋巴瘤動物模型的治療試驗(yàn),結(jié)果顯示,給予適當(dāng)90Y-Anti-CD45劑量的所有小鼠,第6天腫瘤消失,觀察到第135天無1例復(fù)發(fā),肝、腎、小腸病理切片檢查均未發(fā)現(xiàn)明顯損害。我們已采用用90Y標(biāo)記的CD45單抗進(jìn)行急性白血病的治療研究,前期實(shí)驗(yàn)中選擇CDTPA螯合90Y與CD45單抗。CDTPA是一種經(jīng)典的金屬離子鰲合劑,早在1985年Hnatowich就用CDTPA螯合IgG與90Y,所得的產(chǎn)物比活度為1.7μCi/μg,放化純度達(dá)90%以上,24小時的解離率為13%。我們的標(biāo)記產(chǎn)物90Y-CDTPA-CD45單抗在CDTPA/IgG為20：1條件下,標(biāo)記率為95%,放化純度達(dá)99%以上；穩(wěn)定性良好,24小時解離率為8.32%,與國內(nèi)張錦明等報道的每天11.9%的解離率也相近。通過間接免疫熒光試驗(yàn)也證明,該螯合物的免疫活性沒有受到核素的明顯影響,與AML細(xì)胞的結(jié)合率較高,是一個較理想的靶向治療制劑,為進(jìn)行下一步的研究奠定了基礎(chǔ)。 鼠標(biāo)源性全分子抗體由于具有高度免疫原性和巨大分子量在臨床治療應(yīng)用局限很多,而且長期使用鼠單抗能誘發(fā)人抗鼠抗體(HAMA)的反應(yīng)導(dǎo)致過敏性反應(yīng)和損傷對人體健康造成危害。為了減少親本鼠單抗的免疫原性,提高抗體在體內(nèi)增強(qiáng)免疫機(jī)制的能力,我們設(shè)想改造其成為人-鼠嵌合的基因工程抗體。我們用人源抗體的恒定區(qū)去取代親本鼠單抗的C區(qū),由于嵌合抗體中的C區(qū)為人源性的,所以該人-鼠嵌合抗體在保留了鼠單抗特異性的同時,又降低了鼠抗體對人體的免疫原性；而且還具有比小鼠抗體更強(qiáng)的介導(dǎo)補(bǔ)體和細(xì)胞對靶抗原的殺傷和吞噬作用。此外,在構(gòu)建嵌合抗體時,我們有目的地選擇了抗體的類型,使之能更有效的發(fā)揮抗體的效用。我們將該單抗的可變區(qū)基因插入到含人恒定區(qū)的嵌合抗體專用表達(dá)載體pFUSE-CHIg和pFUSE2-CLIg,轉(zhuǎn)染CHO細(xì)胞表達(dá)、純化嵌合抗體。 現(xiàn)在我們首次研究并報導(dǎo)這種新型抗CD45的鼠／人嵌合抗體(命名Chi-CD45),目前國內(nèi)外尚無將CD45單抗進(jìn)行人源化改造的相關(guān)報道。體外試驗(yàn)研究證明,抗體的競爭抑制試驗(yàn)結(jié)果隨著單克隆抗體濃度的改變發(fā)生梯度變化。而補(bǔ)體介導(dǎo)的細(xì)胞殺傷作用發(fā)揮了嵌合抗體恒定區(qū)的功能,它能通過增加嵌合抗體的濃度而抑制.Jurkat/人PBMC增殖。 本課題利用PCR技術(shù)擴(kuò)增抗CD45單抗的輕鏈與重鏈基因,并插入pGEM-T載體中,將陽性克隆進(jìn)行核苷酸序列分析,目的是獲得正確地基因,為進(jìn)一步研究奠定基礎(chǔ)。 目的構(gòu)建抗CD45嵌合抗體的真核表達(dá)載體并實(shí)現(xiàn)在真核細(xì)胞中的表達(dá)。 方法通過PCR技術(shù)分別擴(kuò)增抗CD45單克隆抗體的輕鏈與重鏈可變區(qū)基因片段；利用DNAtools,IMGT/QUEST及EBI TOOLS:ClustalW2分析軟件對輕鏈和重鏈基因分別進(jìn)行同源性比較。將經(jīng)過測序確認(rèn)的基因序列分別構(gòu)建入嵌合抗體的表達(dá)載體中轉(zhuǎn)染CHO細(xì)胞,通過Western blotting檢測嵌合抗體的表達(dá)。對該嵌合抗體進(jìn)行分子建模,模擬其蛋白質(zhì)二級、三級結(jié)構(gòu),并通過相關(guān)效應(yīng)檢測進(jìn)一步驗(yàn)證該基因工程抗體四級結(jié)構(gòu)的完整和功能的有效性。收集細(xì)胞培養(yǎng)上清后,通過Protein A親和層析對表達(dá)產(chǎn)物進(jìn)行純化；通過FACS檢測純化后的嵌合抗體與靶細(xì)胞特異性結(jié)合的能力；與親本單抗的競爭抑制活性；在補(bǔ)體存在條件下的嵌合抗體Fc段介導(dǎo)的CDC效應(yīng)能力,以及嵌合抗體抑制靶細(xì)胞增殖的活力。 結(jié)果PCR擴(kuò)增出的VL、VH基因片段經(jīng)1%瓊脂糖凝膠電泳鑒定,其片段大小與理論值相符,DNA測序鑒定顯示其序列正確。經(jīng)分析軟件鑒定功能性輕鏈結(jié)構(gòu)域：從第16位堿基(ATG)開始,其后為57個bp的信號肽序列,編碼19個氨基酸；可變區(qū)全長為333個堿基,抗CD45單抗有功能的輕鏈均屬于IGκV1-117'01家族,V區(qū)匹配率為99.32%,J區(qū)為100%。 重鏈結(jié)構(gòu)域分析：從第16位堿基(ATG)開始,其后為57個bp的信號肽序列,編碼19個氨基酸；可變區(qū)全長為348個堿基,功能性重鏈屬于IGHV2-9-1'01家族。V區(qū)匹配率為96.84%,J區(qū)為87.23%。 重組嵌合抗體的表達(dá)載體分別通過特異性引物經(jīng)PCR擴(kuò)增鑒定顯示其產(chǎn)物片段大小與理論值相符,DNA測序鑒定顯示其序列和閱讀框正確。共轉(zhuǎn)染CHO細(xì)胞經(jīng)篩選后顯示嵌合抗體表達(dá)量低,后續(xù)實(shí)驗(yàn)結(jié)果不理想。重組抗體產(chǎn)量低是制備基因工程抗體最大的障礙,高表達(dá)細(xì)胞株相對于整個轉(zhuǎn)染細(xì)胞群是非常稀少的,在穩(wěn)定培養(yǎng)時產(chǎn)量可形成梯度下降,過度生長的非表達(dá)細(xì)胞亦可使整個穩(wěn)轉(zhuǎn)細(xì)胞產(chǎn)量下降更明顯,甚至形成非表達(dá)細(xì)胞群。 大多數(shù)細(xì)胞系產(chǎn)生的IgG可合成更多的輕鏈基因,在對人鼠嵌合抗體雜交細(xì)胞系研究中,發(fā)現(xiàn)輕鏈分泌的比率和細(xì)胞內(nèi)的輕鏈含量存在相關(guān)性,它合成的速度比重鏈要快幾倍,而且需要的量更多。有文獻(xiàn)報道分步轉(zhuǎn)染的方法,可以使表達(dá)有功能的細(xì)胞增加5-30%,先用高濃度抗生素穩(wěn)轉(zhuǎn)輕鏈基因待該細(xì)胞穩(wěn)定后再進(jìn)一步轉(zhuǎn)染重鏈基因。在后續(xù)培養(yǎng)過程適量減少培養(yǎng)基用量,延長轉(zhuǎn)染細(xì)胞培養(yǎng)時間達(dá)7-10天以上均能相對提高抗體濃度,或選用DMSO,丁酸鈉等在能抑制細(xì)胞增長同時,促進(jìn)抗體分泌尤其對CHO細(xì)胞重組蛋白質(zhì)有促進(jìn)效果(產(chǎn)量提高約2倍)。 參考相關(guān)文獻(xiàn)后我們采用了分步轉(zhuǎn)染的方法并用丁酸鈉間隔刺激轉(zhuǎn)染細(xì)胞,ELISA結(jié)果顯示嵌合抗體產(chǎn)量有極大提高；分子模擬的各項檢測指標(biāo)顯示了構(gòu)建的抗CD45人-鼠嵌合抗體符合完整有功能的基因工程抗體,Insight II軟件模擬了抗CD45抗體的Fab段結(jié)構(gòu)域,該建模模型表明該抗體具有完整的抗原結(jié)合凹槽能穩(wěn)定的發(fā)揮抗體的相關(guān)功能。 轉(zhuǎn)染瘤細(xì)胞經(jīng)檢測證實(shí)表達(dá)成功以后,部分轉(zhuǎn)染瘤細(xì)胞放入液氮中凍存,5個月后取出凍存的細(xì)胞復(fù)蘇培養(yǎng),先后經(jīng)過一次常規(guī)有限稀釋克隆和多次ELISA方法鑒定,證明該轉(zhuǎn)染瘤細(xì)胞經(jīng)過液氮凍存及多次傳代,仍能夠保持良好的體外生長狀態(tài)。 Western blotting結(jié)果顯示培養(yǎng)3天的轉(zhuǎn)染瘤細(xì)胞分泌的上清中有嵌合抗體表達(dá)。收集培養(yǎng)的上清經(jīng)Protein A親和層析純化后,SDS-PAGE鑒定顯示在純化產(chǎn)物中僅見嵌合抗體重鏈和輕鏈條帶,其分子量大小與理論值相符。FCM檢測顯示純化后的嵌合抗體分子的V區(qū)能夠與多株腫瘤細(xì)胞及人PBMC細(xì)胞表面CD45抗原特異性的結(jié)合,且其結(jié)合能力與相同濃度的鼠源性親本抗體相似。與親本單抗的競爭抑制作用隨著嵌合抗體的濃度增加而梯度增加；嵌合抗體所介導(dǎo)的CDC效應(yīng)及抑制腫瘤細(xì)胞增殖的能力均隨該抗體濃度的增加而增強(qiáng)。 結(jié)論成功地構(gòu)建了表達(dá)抗CD45人-鼠嵌合抗體真核表達(dá)載體,并獲得了具有生物學(xué)活性的嵌合抗體分子。為后續(xù)研究該嵌合抗體對血液系統(tǒng)疾病的治療提供了堅實(shí)的基礎(chǔ)。
[Abstract]:Background acute myeloid leukemia (AML) is a common malignant blood system disease. Although the application of chemoradiotherapy and hematopoietic stem cell transplantation improves the survival rate of patients, there is still a hematopoietic stem cell transplant after AML. radiotherapy and chemotherapy for patients with 60%-70% suffering from the disease only for a number of patients with drug-resistant leukemia. It provides a chance to cure, although many research teams have demonstrated the effectiveness, safety and feasibility of this treatment. The safest way to target radiotherapy for leukemic precursor cells and bone marrow hematopoietic stem cells is to target leukemia cells or normal bone marrow hematopoiesis with a radiated antibody. Stem cells. The treatment of acute myeloid leukemia with monoclonal antibodies has long been reported. The commonly used antibody targeting markers are CD33, CD66 and CD45, and the current research suggests that anti CD45 antibodies are the most promising.
CD45 antigen is a stable, highly expressed single strand cell membrane glycoprotein on the surface of all leukocytes and their precursors and more than 70% of the bone marrow nucleated cells. CD45 is also known as GP180, T200, or leukocyte common antigen (LCA), a tyrosine phosphatase with a molecular weight of about 200 kDa, which is stably expressed in the removal of mature red cells. All hematopoietic cells outside the platelets do not exist in non hematopoietic tissues, making them an attractive target for the treatment of leukemia.
CD45 can be widely expressed in almost all leukocytes, including myeloid precursor cells in bone marrow and mature lymphocytes in lymph nodes, and 90% AML cells. These cells and tissues provide a large number of antibody binding sites for the relief or recurrence of treatment. As CD45 as a marker for leukocyte, it can appear in normal and malignant cells. Therefore, the use of anti CD45 antibodies in patients can be combined with target antigens through metabolic distribution to bone marrow, spleen and lymph nodes for the treatment of acute leukemia.
In radioimmunotherapy, the usual practice is to selectively transport radioactive substances to a tumor cell for a specific antigen. Because the cells of the hematopoietic tissue are sensitive to the radiating line and the target antigen is highly approachable, the treatment of hematological cancer is always considered the most successful. In AML and MDS In radioimmunoassay, CD45 antigen is a highly attractive target target because of its high expression and specificity. Radioactivity labeled anti CD45 antibodies can transport radiant substances to antigen positive cells and surrounding surrounding antigen negative cells. Therefore, radioimmunoconjugate does not need to bind to every leukemia at high concentration. Lethal DNA damage in or infiltrating into bone marrow or tumor cells. In the radioimmunoassay of recurrent leukemia patients, even if the tumor cells do not express CD45 antigen, they will be killed by the bystander response mediated by non malignant hematopoietic cells surrounding the high expression of CD45 antigen. In the field of hematopoietic stem cell transplantation, C These characteristics of D45 antigens can facilitate researchers to further study their radiolabeled antibodies in order to be used for clinical treatment.
With the research of the CD45 isomer, the application of the immunophenotyping of flow cytometer and the research on the targeting therapy of anti CD45 monoclonal antibody, the application of CD45 in immunology and hematology has been paid more attention. It is expected that CD45 will play a greater role in the diagnosis, treatment and prognosis of clinical related diseases. However, anti CD45 monoclonal antibodies are derived from mice and are used to induce the production of anti mouse antibodies in the human body. Therefore, it is of great practical significance to carry out genetically engineered antibodies. Our experimental group has reported that the anti CD45 monoclonal antibody has a good prospect in the treatment of AML, and we have been committed to the constant toxicity of drugs in the treatment. Improvement.
In this article, we first report on the construction of anti CD45 human mouse chimeric antibody and describe the unique advantage of this gene engineering antibody in the treatment of blood system diseases. The research laid a solid foundation.
Human or chimeric monoclonal antibodies (MoAbs) can react with antigens expressed in hematopoietic cells. They can be used directly ("parent" or "naked" antibody) or in clinical research and development by coupling with toxins, radioisotopes or antitumor drugs. Antibody therapy is in the circulatory system due to the specificity and affinity of anti body small molecules. Combining with tumor cells is an ideal solution for the treatment of acute myeloid leukemia. According to whether or not it carries other connections, it is divided into uncombined monoclonal antibody, anticancer drug binding monoclonal antibody and isotope binding monoclonal antibody. Currently, five drugs have been approved by the FDA in the United States for clinical use, of which unconjugated type is human mouse chimeric antibody or Human derived antibody. But the use of unbound CD45 monoclonal antibody in human body is very small and is limited by mouse origin. The current research on CD45 mAb is mainly composed of the connection of radionuclide 131I and 213Bi as a preconditioning scheme for hematopoietic stem cell transplantation, in order to reduce the dose of TBI and thus reduce TBI Both animal tests and phase I / II clinical trials have confirmed that CD45 mAb can selectively transfer radionuclide 131I and 213Bi into the bone marrow, liver and spleen and lymph nodes, and can partially replace TBI in the pre treatment.Vallera with 90Y labeled CD45 McAbs for the treatment of lymphoid tumor animal models, and the results show that it is given. All mice with appropriate 90Y-Anti-CD45 dose disappeared in sixth days. No 1 cases of recurrence were observed on the 135th day. No obvious damage was found in the liver, kidney, and small intestine pathological sections. We have used the 90Y labeled CD45 monoclonal antibody for the treatment of acute leukemia. In the earlier experiment, the selection of CDTPA chelating 90Y and CD45 McAb.CDTPA is a classic. Metal chelating agent, as early as in 1985, Hnatowich was chelated with CDTPA and IgG and 90Y, the specific activity of the product was 1.7 mu Ci/ mu g, the radiochemical purity was above 90%, and the 24 hour dissociation rate was 13%.. The labeling product 90Y-CDTPA-CD45 McAb was 95% and the radiochemical purity was more than 99% under the CDTPA/IgG 20:1 condition, and the stability was good and 24 was small. The dissociation rate was 8.32%, which was similar to the 11.9% dissociation rate per day reported by Zhang Jinming in China. Indirect immunofluorescence test also proved that the immune activity of the chelate was not obviously affected by the nuclide, and the binding rate with AML cells was higher. It was an ideal target treatment preparation, which laid the foundation for the next step of research.
The mouse derived total molecular antibody is limited in clinical treatment because of its high immunogenicity and large molecular weight, and the response to human anti mouse antibody (HAMA) induced by rat mAb induces hypersensitivity and damage to human health. In order to reduce the immunogenicity of the monoclonal antibody to the parent, improve the antibody in the body. In order to enhance the ability of the immune mechanism, we conceive to transform it into a human mouse chimeric gene engineering antibody. We use the constant region of the human antibody to replace the C region of the parent mouse mAb. Because the C region of the chimeric antibody is humanized, the human mouse chimeric antibody retains the specificity of the mouse monoclonal antibody while reducing the mouse antibody against the human body. The immunogenicity and the killing and phagocytosis of the mediated complement and cell against the target antigen are also stronger than that of the mice. In addition, in the construction of chimeric antibodies, we have selected the type of antibody to make it more effective to use the antibody. We insert the variable region gene of the monopulse into the constant area. The antibody specific expression vector pFUSE-CHIg and pFUSE2-CLIg were transfected into CHO cells to express and purify chimeric antibody.
For the first time, we have studied and reported this new anti CD45 mouse / human chimeric antibody (named Chi-CD45). At present, there is no related report on the human transformation of CD45 McAbs at home and abroad. In vitro studies have shown that the results of the competitive inhibition test of antibodies change with the change of the concentration of monoclonal antibodies. Cytotoxicity plays a role in the constant region of chimeric antibody. It can inhibit the proliferation of.Jurkat/ human PBMC by increasing the concentration of chimeric antibody.
In this study, the PCR technique was used to amplify the light chain and heavy chain genes of anti CD45 monoclonal antibodies and inserted into the pGEM-T vector to analyze the positive clones for nucleotide sequences. The aim is to obtain the correct genes and lay the foundation for further research.
Objective to construct a eukaryotic expression vector against CD45 chimeric antibody and to express it in eukaryotic cells.
Methods the light chain and heavy chain variable region gene fragment of anti CD45 monoclonal antibody was amplified by PCR technique, and the homology of light chain and heavy chain gene was compared with DNAtools, IMGT/QUEST and EBI TOOLS:ClustalW2 analysis software. The gene sequence confirmed by sequencing was constructed into the expression vector of chimeric antibody and transfected to CHO respectively. Cell, the expression of chimeric antibody was detected by Western blotting. Molecular modeling of the chimeric antibody was used to simulate the protein two and three structure, and the integrity and function of the four stage structure of the gene engineering antibody were verified by correlation effect detection. After collecting cell culture supernatant, Protein A affinity chromatography was used to table the table. The product was purified; the ability to detect the specific binding of the purified chimeric antibody to the target cells by FACS, the competitive inhibition activity with the parent monoclonal antibody, the CDC effect mediated by the chimeric antibody Fc segment under the presence of complement, and the activity of the chimeric antibody to inhibit the proliferation of the target cells.
Results the VL, VH gene fragment amplified by PCR was identified by 1% agarose gel electrophoresis, and the size of the fragment was in accordance with the theoretical value. The sequence of DNA sequencing showed that the sequence was correct. The functional light chain domain was identified by the analysis software: from the sixteenth base base (ATG), the sequence of the 57 BP peptide and the 19 amino acids were encoded, and the variable region length was 333. The light chain of anti CD45 monoclonal antibody belongs to the IG kappa V1-117'01 family. The matching rate of V region is 99.32%, and the J area is 100%..
Heavy chain domain analysis: starting from sixteenth base (ATG), followed by 57 BP signal peptide sequences, encoding 19 amino acids, variable region full length of 348 bases, functional heavy chains belonging to the IGHV2-9-1'01 family.V region matching rate of 96.84%, J region 87.23%.
The expression vector of recombinant chimeric antibody was identified by PCR amplification by specific primers. The fragment size of the product was consistent with the theoretical value. DNA sequencing showed that the sequence and reading frame were correct. After screening, the co transfected CHO cells showed that the expression of chimeric antibody was low and the subsequent experimental results were not ideal. The low yield of recombinant antibody was a preparation gene. The highest expression of engineering antibody is that the high expression cell line is very rare compared with the whole transfected cell group. The yield can form a gradient in stable culture, and the overgrowth of non expressed cells can also decrease the output of the whole cell, even form a non expressed cell group.
The IgG produced by most cell lines can synthesize more light chain genes. In the study of human chimeric antibody hybrid cell lines, it is found that the ratio of light chain secretion is correlated with the light chain content in cells. It synthesizes a few times faster and needs more. The functional cells increase 5-30%, and then use high concentration antibiotics to stabilize the light chain gene and then further transfect the heavy chain gene after the cell is stable. In the subsequent culture, a proper amount of the medium is reduced and the cell culture time is more than 7-10 days, or DMSO, or sodium butyrate can be used to inhibit the cell growth. At the same time, it promotes the secretion of antibodies, especially for CHO cell recombinant protein (the yield is increased by about 2 times).
Reference correlation
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 扶云碧;李貴平;孟凡義;;~(90)Y標(biāo)記單克隆抗體CD45的研究[J];南方醫(yī)科大學(xué)學(xué)報;2006年08期

2 李貴平;扶云碧;黃凱;劉峰;孟凡義;;~(90)Y間接法標(biāo)記單克隆抗體的實(shí)驗(yàn)研究[J];放射免疫學(xué)雜志;2008年06期

3 朱志剛,沈關(guān)心,朱惠芬,王曉林,張悅,龔非力,毛平;抗CD_4單克隆抗體可變區(qū)基因克隆及測序[J];廣東醫(yī)學(xué);2000年08期

4 肖溶,張日;CD45分子及在急性白血病中應(yīng)用的研究進(jìn)展[J];國外醫(yī)學(xué)(內(nèi)科學(xué)分冊);2004年03期

5 朱志剛,沈關(guān)心,王曉林;抗CD4單克隆抗體可變區(qū)基因克隆及序列分析[J];免疫學(xué)雜志;1999年02期

6 王秀紅;周素芳;;基因工程抗體研究進(jìn)展[J];生物技術(shù)通訊;2007年02期

7 王玉剛,馮健男,沈倍奮;用RLM-RACE法克隆抗CD20單克隆抗體可變區(qū)基因及其信號肽基因[J];細(xì)胞與分子免疫學(xué)雜志;2005年04期

8 瞿秋霞;陳永井;葛彥;王勤;陳成;楊明峰;張學(xué)光;;抗人CD40人-鼠嵌合抗體的構(gòu)建及其表達(dá)[J];細(xì)胞與分子免疫學(xué)雜志;2006年02期

9 王玉剛;黃英;谷欣;于鳴;馮健男;孫瑛勛;黎燕;沈倍奮;;抗CD20嵌合抗體的構(gòu)建與表達(dá)[J];細(xì)胞與分子免疫學(xué)雜志;2006年03期

10 葛彥;陳永井;瞿秋霞;張學(xué)光;;抗人CD154人-鼠嵌合抗體Fab段的構(gòu)建和表達(dá)[J];細(xì)胞與分子免疫學(xué)雜志;2007年06期

，

本文編號：2143008