分泌人源性單克隆抗體永生化淋巴細(xì)胞系的構(gòu)建

1、    分泌人源性單克隆抗體永生化淋巴細(xì)胞系的構(gòu)建*        摘要目的：構(gòu)建分泌人源性單克隆抗體(McAb)的永生化B淋巴細(xì)胞系。方法：用正丁醇法提取的人卵巢癌細(xì)胞系ao10/17抗原在含免疫活化劑的無血清培基中免疫人淋巴細(xì)胞；將制備ao 10/17細(xì)胞DNA轉(zhuǎn)染經(jīng)體外免疫的淋巴細(xì)胞。采用ELISA篩選和有限稀釋法克隆化。結(jié)果：建成4株分泌抗卵巢癌McAb的轉(zhuǎn)染體細(xì)胞AT1、AT2、AT3和AT4；McAb相加試驗(yàn)表明，它們識(shí)別卵巢癌抗原分子的同一表位。AT4已在體外培

2、養(yǎng)460多天，凍存復(fù)蘇后仍保持抗體產(chǎn)生能力。夾心ELISA發(fā)現(xiàn)，人源性McAb AT4能與卵巢癌細(xì)胞表面可溶性抗原發(fā)生免疫反應(yīng)。結(jié)論：體外免疫和DNA轉(zhuǎn)染的結(jié)合，可建成分泌人源性McAb的永生化B淋巴細(xì)胞系，為人源性McAb的產(chǎn)生提供了一條技術(shù)途徑。主題詞淋巴細(xì)胞；抗體，單克??；轉(zhuǎn)染；免疫法；卵巢腫瘤 The construction of immortalized lymphoid cell lines secreting human monoclonal antibodiesWU Su-Yun, WEN Gan-Sheng, XU Fang-Yun, LIU Qiu-Ru, GONG We

3、n-Hua, Yu Ying, YI Wei-Min, LIU Si-SunDepartment of Pathophysiology and Tumor Immunology Research, Jiangxi Medical College, Nanchang(330006)Department of Obstetrics and Gynecology First Affiliated Hospital, Jiangxi Medical College Abstract AIM:To construct immortalized lymphoid cell lines secreting

4、human monoclonal antibodies. METHODS：Human lymphocytes were immunized in serum-free media containing immunoactive reagents using ovarian cancer cell line ao10/17 antigen extracted by 1-butanol. In vitro immunized lymphocytes were transfected with DNA preparations from ao 10/17. RESULTS：Four transfec

5、tant cell lines designated AT1, AT2, AT3 and AT4 secreting monoclonal antibodies against ovarian cancer were established by ELISA-based screening and limiting dilution. All of monoclonal antibodies were reactive with the identical epitopes of ovarian cancer-associated antigen molecule in the additio

6、n test. The transfectant cell line AT4 has grown continuously in vitro culture for more than 460 days and maintained the ability of secreting antibody after frozen and recovered. Human monoclonal antibody AT4 had immunoreactions with a soluble antigen from the cell surface of ovarian cancer by using

7、 sandwich ELISA.CONCLUSION：A combination of in vitro immunization and transfection with DNA induced the construction of B-cell-derived cell lines which secrete human monoclonal antibodies, and provide a technological approach for producing human monoclonal antibody.MeSHLymphocytes; Antibodies, monoc

8、lonal;Transfection; Immunization; Ovarian neoplasms人源性單克隆抗體(human monoclonal antibody, HMcAb)優(yōu)于鼠源性單克隆抗體(monoclonal antibody, McAb)，但HMcAb制備的困難頗多，特別是人B淋巴細(xì)胞的抗原致敏和永生化。近年來采用體外免疫以致敏人B細(xì)胞，但其所需要條件還不很了解。EBV轉(zhuǎn)化雖可使致敏B細(xì)胞永生化，但因其存在諸多缺陷而甚少單獨(dú)采用。新近興起的基因工程抗體技術(shù)，人們寄予期望1。我們曾用DNA轉(zhuǎn)染技術(shù)構(gòu)建了兩株永生化鼠淋巴細(xì)胞轉(zhuǎn)染體，它們能不斷產(chǎn)生抗宮頸癌McAb，并能在含血清

9、和無血清培基中長期生長2。本文報(bào)道，以低分子量卵巢癌細(xì)胞表面可溶性抗原體外免疫人脾細(xì)胞，用卵巢癌細(xì)胞DNA轉(zhuǎn)染經(jīng)體外免疫的人淋巴細(xì)胞；將所形成的轉(zhuǎn)化細(xì)胞進(jìn)行篩選和克隆，構(gòu)建能產(chǎn)生抗卵巢癌HMcAb的永生化人轉(zhuǎn)染體淋巴細(xì)胞系。材料與方法(一)培養(yǎng)液、細(xì)胞和細(xì)胞系：1.基礎(chǔ)培養(yǎng)液：RPMI 1640培養(yǎng)液(Gibco),每升含5 mg胰島素，5 mg轉(zhuǎn)鐵蛋白，10 g硒，2 L 乙醇胺，0.3 g谷氨酰胺，0.11 g丙酮酸鈉，10 mg慶大霉素。2.人卵巢癌細(xì)胞系ao10/17：購自中國科學(xué)院細(xì)胞生物學(xué)研究所細(xì)胞庫，生長于含10%(V/V)FCS的RPMI 1640培基中。3.人卵巢癌細(xì)胞OV：

10、來自江西省婦產(chǎn)醫(yī)院經(jīng)確診的卵巢癌病人的腹水，離心后用PBS洗3次，備用或-70凍存。4.人脾細(xì)胞：來自一附院外科脾破裂病人手術(shù)切除的標(biāo)本。切碎、通過200目不銹鋼網(wǎng)，Tris-NH4Cl處理5 min，制成懸浮細(xì)胞，備用或-70凍存。(二)人卵巢癌細(xì)胞表面可溶性抗原的制備：按本室報(bào)道的LeGrue正丁醇提取法的改良法3，將ao 10/17和OV細(xì)胞分別用2.5%正丁醇處理5 min，然后離心10 min，收集上清；將上清離心(15 000×g)30 min，透析，制得人卵巢癌細(xì)胞表面可溶性抗原，ao CBE和OVCBE，將前者進(jìn)行SDS-PAGE測定。(三)人淋巴細(xì)胞體外免疫：1.體

11、外免疫培基制備：將基礎(chǔ)培基加入IL-2(2.5×104 U/L)和IFN-(4×105U/L)。2.體外抗原刺激：將制得的人脾細(xì)胞(3×109/L)懸浮于體外免疫培基中；加入ao CBE(6 mg/L)，24 h后加入FCS(終濃度為15%)；置于37，7% CO2孵育箱內(nèi)培養(yǎng)15 d，其間每隔4 d加入20%體外免疫培基。獲得的致敏人淋巴細(xì)胞供DNA轉(zhuǎn)染用。(四)卵巢癌細(xì)胞ao 10/17 DNA轉(zhuǎn)染致敏淋巴細(xì)胞：按我們發(fā)表的Jonak等的方法的改良法2并參照Sambrook等的方法4制備DNA和DNA共沉淀物并將后者轉(zhuǎn)染致敏淋巴細(xì)胞。(五)淋巴細(xì)胞轉(zhuǎn)染體產(chǎn)生的

12、特異性抗體的檢測：按間接ELISA3：用包被緩沖液稀釋ao CBE至5 mg/L，酶標(biāo)板每孔加入50 L，4孵育過夜；加入50 L轉(zhuǎn)染體細(xì)胞培養(yǎng)上清液(以正常人脾細(xì)胞培養(yǎng)上清液為陰性對(duì)照)，37孵育2 h；最后加入底物(OPD)溶液50 L，37孵育30 min。終止后，以酶標(biāo)儀測定A490 值。(六)轉(zhuǎn)染體細(xì)胞克隆化：96孔培養(yǎng)板加入BALB/C小鼠脾細(xì)胞，將針對(duì)ao CBE的特異性IgG抗體陽性反應(yīng)轉(zhuǎn)染體細(xì)胞，按有限稀釋法做單克隆細(xì)胞培養(yǎng)。(七)轉(zhuǎn)染體McAb的制備：將克隆3次的轉(zhuǎn)染體細(xì)胞在含10%小牛血清培基中擴(kuò)大培養(yǎng)，其培養(yǎng)上清液用硫酸銨沉淀。(八)HRP標(biāo)記McAb AT4：按Win

13、ston等5的方法，制成HRP-AT4。質(zhì)量鑒定后，分裝、凍存。(九)McAb夾心ELISA的建立：按本室以前報(bào)道的方法3。(十)McAb的表位特異性分析：按Friguet等6報(bào)道的ELISA McAb相加實(shí)驗(yàn)測定McAb的抗原識(shí)別表位。(十一)McAb的效價(jià)測定：用間接ELISA測定轉(zhuǎn)染體細(xì)胞培養(yǎng)上清液的效價(jià)。(十二)McAb的特異性鑒定：用間接和夾心ELISA測定McAb與ao CBE和OVCBE的免疫反應(yīng)。結(jié)果(一)癌細(xì)胞DNA轉(zhuǎn)染誘導(dǎo)體外免疫人淋巴細(xì)胞永生化：從人卵巢癌細(xì)胞系ao 10/17提取的DNA，其A260/A280比值為1.8-1.9；用其制得的DNA共沉淀物轉(zhuǎn)染經(jīng)ao CB

14、E(SDS-PAGE測定為低分子量抗原，24.552.3 kD)體外致敏的人淋巴細(xì)胞。1. DNA轉(zhuǎn)染淋巴細(xì)胞的形態(tài)學(xué)：轉(zhuǎn)染后第8 d，在倒置相關(guān)顯微鏡下可見轉(zhuǎn)化細(xì)胞灶，轉(zhuǎn)化細(xì)胞體積較淋巴細(xì)胞大，略顯大小不一，密度較未轉(zhuǎn)化細(xì)胞高，但仍保持典型的淋巴樣細(xì)胞形態(tài)。第2周可見簇狀的轉(zhuǎn)化細(xì)胞集落，其細(xì)胞數(shù)量迅速增加。第8周可見多種形態(tài)的淋巴樣細(xì)胞集落(1)。8個(gè)月后，細(xì)胞緊密貼壁，其間有明顯基質(zhì)(2)。Fig 1 Inverted phase-contrast photomicrograph of immortalized human lymphoid cell line established by

15、 in vitro immunization and DNA transfection. Eight weeks after transfection (magnification ×33) 1體外免疫和DNA轉(zhuǎn)染建立的永生化人淋巴細(xì)胞系(轉(zhuǎn)染后8周)Fig 2 Inverted phase-contrast photomicrograph of immortalized human lymphoid cell line established by in vitro immunization and DNA transfection. Eight months after tran

16、sfection (magnification ×132) 2體外免疫和DNA轉(zhuǎn)染過后的永生化人淋巴細(xì)胞系(轉(zhuǎn)染后8個(gè)月)2.DNA轉(zhuǎn)染體淋巴細(xì)胞的篩選、克隆化和永生化：在轉(zhuǎn)染后第2周出現(xiàn)轉(zhuǎn)化細(xì)胞時(shí)，用間接ELISA檢測DNA轉(zhuǎn)染體淋巴細(xì)胞培養(yǎng)上清液與卵巢癌抗原ao CBE的免疫反應(yīng)。將IgG抗體陽性反應(yīng)轉(zhuǎn)染體細(xì)胞按有限稀釋法進(jìn)行3次克隆化，獲得4株DNA轉(zhuǎn)染體淋巴細(xì)胞系，命名為AT1、AT2、AT3和AT4。經(jīng)ELISA McAb相加試驗(yàn)證明，它們均針對(duì)卵巢癌細(xì)胞表面可溶性抗原分子同一表位。選擇其中1株AT4轉(zhuǎn)染體細(xì)胞系進(jìn)行研究；迄今，AT4已經(jīng)過460多天連續(xù)培養(yǎng)，McAb的分泌

17、持續(xù)穩(wěn)定，凍存復(fù)蘇后依然如此。上述結(jié)果說明，體外免疫和DNA轉(zhuǎn)染人淋巴細(xì)胞，可使其永生化，并產(chǎn)生McAb。二、卵巢癌轉(zhuǎn)染體McAb的特性：1.McAb的效價(jià)：將轉(zhuǎn)染體細(xì)胞系A(chǔ)T4的培養(yǎng)上清液用PBS進(jìn)行倍比系列稀釋后，用間接ELISA測定，測得其效價(jià)為164。2.McAb的特性：在間接和夾心ELISA中，McAb AT4能與人卵巢癌細(xì)胞表面可溶性抗原ao CBE和卵巢癌病人腹水中癌細(xì)胞表面可溶性抗原OVCBE發(fā)生免疫反應(yīng)(表1)。說明人源性 McAb AT4能識(shí)別卵巢癌相關(guān)抗原。表1ELISA測定McAb AT4與卵巢癌細(xì)胞CBE和正常人血清的反應(yīng)Tab 1 Reactivity of McA

18、b AT4 with CBE of ovarian cancer cellsand normal serum in ELISASample obsorbanceRatio of sample absorbanceat 490to control absorbanceao CBE0.532.7OVCBE0.9254.87NS0.19ao=ovarian cancer cell line ao10/17; OV=ovarian cancer cells of ascites from a patient with ovarian cancer;CBE=crude butanol extract;

19、NS=serum of normal females討論(一)DNA轉(zhuǎn)染誘導(dǎo)B細(xì)胞永生化：人源性McAb雖難以采用雜交瘤技術(shù)制得，但可用DNA轉(zhuǎn)染、EBV感染、人淋巴細(xì)胞在SCID小鼠中構(gòu)建以及基因工程技術(shù)特別近年發(fā)展的抗體組合文庫技術(shù)以制備適于臨床應(yīng)用的HMcAb;這些技術(shù)各有特點(diǎn)，可相互取長補(bǔ)短。本研究采用DNA轉(zhuǎn)染經(jīng)ao CBE體外免疫的人淋巴細(xì)胞，構(gòu)建4株永生化轉(zhuǎn)染體細(xì)胞系，它們能不斷分泌針對(duì)人卵巢癌細(xì)胞表面可溶性抗原的McAb，說明DNA轉(zhuǎn)染技術(shù)可構(gòu)建永生化抗體產(chǎn)生淋巴細(xì)胞系。(二)體外免疫構(gòu)建McAb產(chǎn)生細(xì)胞：Kawahara等7研究了人淋巴細(xì)胞體外致敏所需的免疫活化劑，認(rèn)為胞壁肽

20、，白介素類是必需的；用此法獲得的McAb, IgG比IgM多1倍以上，抗體產(chǎn)生效率比體內(nèi)自發(fā)免疫大10倍。Niedbata等8應(yīng)用EBV感染技術(shù)制備人源性McAb時(shí)，以含IL-2(2.5×104U/L)和低劑量IFN-的培基進(jìn)行體外致敏。本研究采用體外免疫人淋巴細(xì)胞時(shí)，在無血清培基中只加了很低劑量的IL-2和IFN-，建成的4株淋巴細(xì)胞轉(zhuǎn)染體均分泌IgG McAb。(三)體外致敏抗原：目前制得的HMcAb通常是識(shí)別胞質(zhì)抗原而不是細(xì)胞表面抗原。本研究用正丁醇法3制得的低分子量ao CBE經(jīng)體外免疫人淋巴細(xì)胞，建成了轉(zhuǎn)染體淋巴細(xì)胞系，其所分泌的McAb能與卵巢癌細(xì)胞表面抗原和可溶性抗原以

21、及卵巢癌組織，發(fā)生免疫反應(yīng)9，10，并能通過CDC和ADCC殺仿靶腫瘤細(xì)胞(另文發(fā)表)。本研究結(jié)果表明，卵巢癌細(xì)胞表面可溶性抗原體外免疫和DNA轉(zhuǎn)染人淋巴細(xì)胞，可建成分泌HMcAb的永生化B淋巴細(xì)胞系。因此，體外免疫技術(shù)和分子生物學(xué)永生化的結(jié)合有希望成為產(chǎn)生HMcAb的一條技術(shù)途徑。* 國家自然科學(xué)基金(39460031)和江西省自然科學(xué)基金(94516)資助作者單位：鄔淑云溫淦升徐方云劉秋如龔文華余鷹江西醫(yī)學(xué)院病理生理教研室、腫瘤免疫研究室(南昌330006)婦產(chǎn)科易為民劉絲蓀江西醫(yī)學(xué)院第一附屬醫(yī)院參考文獻(xiàn)1Glassy MC. Production methodes for generat

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23、1997, 13:437.4Sambrook J, Fritsch EF, Maniatis T.eds. Molecular clonong, a laboratory manual. 2nd ed. New York:Cold Spring Harbor Laboratory Prees, 1989. 1638.5Winston S E, Fuller S A, Evelegh M J, et al. Conjugation of enzymes to antibodies. In:Ausuble FM, Brent R, Kingston RE, et al.eds. Short protocols in molecular biology.3rd ed. Vo