【摘要】： 免疫球蛋白(Ig)是重要的免疫分子,其兩種存在方式分別為:膜型和分泌型,前者作為B淋巴細(xì)胞表面的抗原受體在抗原識(shí)別、細(xì)胞的活化、分化甚至程序性細(xì)胞死亡中起重要作用;后者主要存在與血液及其它體液中的,能夠與相應(yīng)抗原相結(jié)合發(fā)揮抗體的效應(yīng)功能。經(jīng)典的免疫學(xué)理論認(rèn)為,Ig的來(lái)源為B淋巴細(xì)胞及漿細(xì)胞,Ig基因僅在B淋巴細(xì)胞發(fā)育過(guò)程中成功進(jìn)行選擇性重排,而在其它體細(xì)胞中該基因處于胚系狀態(tài),或者僅能發(fā)生不完全的重排,即不發(fā)生功能性基因重排,故不會(huì)有Ig分子的產(chǎn)生。 2003年,北京大學(xué)邱曉彥教授課題組在Cancer Research發(fā)表論文證實(shí)上皮來(lái)源腫瘤細(xì)胞可產(chǎn)生Ig分子,該工作被評(píng)為2004年中國(guó)醫(yī)藥研究領(lǐng)域十大新聞之一。其工作證明了除B淋巴細(xì)胞及漿細(xì)胞外,在一些非免疫細(xì)胞,如上皮來(lái)源的腫瘤細(xì)胞(如肺癌、卵巢癌、腸癌、鼻咽癌、乳腺癌、胃癌、胰腺癌等)及部分正常的上皮細(xì)胞內(nèi)Ig基因同樣存在功能性基因重排,并可以產(chǎn)生Ig分子,因此現(xiàn)將其稱(chēng)為非B細(xì)胞來(lái)源Ig(non B-Ig);此外,更重要的是該課題組通過(guò)對(duì)non B-Ig分子功能的研究,表現(xiàn)在基因及蛋白質(zhì)水平抑制癌細(xì)胞Ig分子的表達(dá)或封閉其活性,則癌細(xì)胞增殖能力明顯減弱。上述現(xiàn)象將有可能使人們重新定義Ig分子的來(lái)源及其潛在的、有別于經(jīng)典Ig的生物學(xué)活性,具有重要的學(xué)術(shù)價(jià)值。同時(shí),也將對(duì)腫瘤發(fā)生與發(fā)展的生物學(xué)研究以及以腫瘤細(xì)胞表達(dá)的Ig分子做為靶點(diǎn)的腫瘤生物治療研究產(chǎn)生重要的影響�？上驳氖�,非B細(xì)胞能夠產(chǎn)生Ig的現(xiàn)象已逐漸被國(guó)內(nèi)外學(xué)者所證實(shí)與認(rèn)同。 已知,B細(xì)胞來(lái)源的Ig具獨(dú)特的結(jié)構(gòu)和功能,主要表現(xiàn)在:其可變區(qū)(V區(qū))識(shí)別特定的抗原決定簇,不同的B淋巴細(xì)胞克隆產(chǎn)生不同的IgV區(qū)結(jié)構(gòu),從而構(gòu)成了Ig分子的多樣性,使得免疫系統(tǒng)能夠應(yīng)對(duì)自然界不同的抗原;其恒定區(qū)與一組特定的分子(如Fc受體、信號(hào)傳導(dǎo)分子、補(bǔ)體的級(jí)聯(lián)成分)相結(jié)合,行使Ig分子的效應(yīng)功能。根據(jù)恒定區(qū)的不同,B淋巴細(xì)胞產(chǎn)生的Ig分子根據(jù)重鏈不同被分為五類(lèi),分別是IgG、IgM、IgA、IgD和IgE;其輕鏈則分為κ與λ型。 B細(xì)胞來(lái)源的Ig分子的可變區(qū)序列呈高度多樣性,這是構(gòu)成抗體及BCR多樣性的結(jié)構(gòu)基礎(chǔ)。對(duì)非B細(xì)胞來(lái)源的Ig基因研究結(jié)果顯示,與經(jīng)典的Ig分子相似,這些non B-Ig分子的重鏈及輕鏈編碼基因均發(fā)生了典型的基因重排及轉(zhuǎn)錄。但在其V-D-J或V-J序列、體細(xì)胞突變特點(diǎn)和類(lèi)別轉(zhuǎn)換等方面均呈現(xiàn)出與經(jīng)典Ig分子不同的特征。邱曉彥教授課題組借助激光顯微切割(LCM)及RT-PCR技術(shù),證明所有腫瘤細(xì)胞來(lái)源的Ig分子V-D-J或V-J片段組合方式有明顯的傾向性。表現(xiàn)在同一個(gè)體的不同細(xì)胞克隆、甚至不同個(gè)體腫瘤來(lái)源的Ig序列之間高度同源,甚至完全相同,并且這些序列在B淋巴細(xì)胞表達(dá)的Ig數(shù)據(jù)庫(kù)中均無(wú)記錄,提示這些序列的確是腫瘤細(xì)胞產(chǎn)生的。更令人驚奇的是,所有腫瘤細(xì)胞均表達(dá)序列完全相同或極其相似的(個(gè)別堿基的替換)重排模式,即Vκ4—1/Jκ3型Igκ轉(zhuǎn)錄本(專(zhuān)利申請(qǐng)?zhí)枮?00510107833.9)。這一現(xiàn)象提示,這些序列特點(diǎn)與組織類(lèi)型無(wú)關(guān),它們可能在不同的組織中發(fā)揮著相似的作用。 根據(jù)上述發(fā)現(xiàn),作為與北京大學(xué)邱曉彥教授的合作項(xiàng)目,本研究的主要目的是制備這種保守的κ鏈可變區(qū)抗體。我們根據(jù)其特有Vκ4—1/Jκ3氨基酸序列,對(duì)比胚系基因可變區(qū)序列,綜合考慮氨基酸組成、疏水性、抗原性、表面暴露性等因素,利用生物信息學(xué)方法分析預(yù)測(cè)了Vκ4—1/Jκ3型Igκ兩個(gè)較為特異的B細(xì)胞識(shí)別表位,設(shè)計(jì)并合成三個(gè)短肽AL13(序列ASINCKSSQRVSL)、QF20(QAEDVAVYYCQQYYDTPVTF),AL13B(TQSPDSLVVSLGERASINCKSSQRVSLG,即AL13的延長(zhǎng)序列)。同時(shí)獲得由邱曉彥教授課題組提供的原核表達(dá)Vκ4—1/Jκ3型Igκ純化融合蛋白(GST-V_κ4-1-J_κ3)。本實(shí)驗(yàn)分別以合成肽AL13、QF20交聯(lián)載體免疫家兔獲得單表位特異性多克隆抗體;分別以合成肽AL13B-KLH、原核蛋白GST-V_κ4-1-J_κ3免疫小鼠獲得系列單克隆抗體抗。再通ELISA、WesternBlot、流式細(xì)胞術(shù)等方法鑒定了抗體的效價(jià)和特異性,為后續(xù)研究提供了有效工具。 第一章Vκ4-1-Jκ3型IgκV區(qū)特異性單表位多抗的制備及鑒定 將合成肽AL13、QF20分別與載體蛋白交聯(lián)制備免疫原,免疫新西蘭大白兔獲得相應(yīng)抗血清,經(jīng)ELISA檢測(cè)其抗體滴度分別為0.3×10~(-5)和0.6×10~(-5)。運(yùn)用親和層析方法對(duì)兔血清進(jìn)行純化,得到anti-AL13和anti-QF20單表位特異性多抗。ELISA結(jié)果顯示,所得抗體與合成肽特異性結(jié)合,但不與正常人IgG反應(yīng)。為了進(jìn)一步證明所得抗體的特異性,以原核表達(dá)蛋白GST-Vκ4-1-Jκ3(39kD)和V5-51-Vκ4-1-Jκ3-pRA(34kD)作為抗原進(jìn)行Western Blot檢測(cè),結(jié)果顯示anti-AL13和anti-QF20都能夠正確識(shí)別含Vκ4-1-Jκ3序列的原核表達(dá)蛋白,在預(yù)期位置上都有明確的條帶出現(xiàn),但基本不與商品化的人IgG的輕鏈結(jié)合;用anti-AL13檢測(cè)HT-29細(xì)胞中內(nèi)源性Igκ的表達(dá),結(jié)果顯示anti-AL13的陽(yáng)性信號(hào)主要在HT-29細(xì)胞不可溶成分中(僅少量的存在于可溶性組分中),這種與經(jīng)典Ig分子完全不同的細(xì)胞內(nèi)定位,提示其可能具有不同于經(jīng)典Ig分子的生物學(xué)活性。 第二章Vκ4-1-Jκ3型IgκV區(qū)特異性單克隆抗體的制備及鑒定 以合成肽AL13B-KLH作為免疫原免疫Balb/c小鼠,取其脾細(xì)胞與NS-1細(xì)胞融合制備雜交瘤,經(jīng)合成肽和原核表達(dá)蛋白(GST-Vκ4-1-Jκ3)篩選和三次克隆化,并從制備的腹水中純化單克隆抗體,篩選后共獲得3株(6G5、3H9.2、5D3)穩(wěn)定分泌針對(duì)Vκ4-1-Jκ3型Igκ的抗體的雜交瘤細(xì)胞;同時(shí)以原核重組蛋白(GST-Vκ4-1-Jκ3)作為免疫原免疫Balb/c小鼠,取其脾細(xì)胞與NS-1細(xì)胞進(jìn)行融合,經(jīng)篩選、克隆化后獲得1株(4E9)穩(wěn)定分泌針對(duì)Vκ4-1-Jκ3型Igκ的抗體的雜交瘤細(xì)胞。這4株單抗的亞類(lèi)分別為IgG1、IgG1、IgG1和IgG2b。將腹水單抗經(jīng)辛酸—硫酸銨沉淀法純化,并用改良過(guò)碘酸鈉法對(duì)4株抗體標(biāo)記了辣根過(guò)氧化物酶。ELISA鑒定結(jié)果顯示所得單抗均與合成肽及原核表達(dá)蛋白(GST-Vκ4-1-Jκ3)發(fā)生特異性結(jié)合。對(duì)合成肽AL13B-KLH來(lái)源的3株單抗的近一步ELISA鑒定結(jié)果顯示:這3株單抗與常用的載體蛋白KLH、BC、BSA、OVA均無(wú)交叉反應(yīng),同時(shí)與商品化的人IgG、羊IgG、兔IgG、鼠IgG均不反應(yīng);用流式細(xì)胞術(shù)鑒定上述單抗可與以人結(jié)腸癌細(xì)胞系HT-29和人肺癌細(xì)胞系A(chǔ)549的細(xì)胞內(nèi)蛋白發(fā)生明顯的特異性結(jié)合,驗(yàn)證了Vκ4-1-Jκ3型Igκ的胞內(nèi)表達(dá);與人末梢血淋巴細(xì)胞、單核細(xì)胞、粒細(xì)胞均有高比例的結(jié)合。鑒于人與小鼠Igκ的同源性高達(dá)70%,我們對(duì)靶抗原序列與小鼠IgκV區(qū)進(jìn)行了比對(duì),用流式細(xì)胞術(shù)證明上述4株單抗可與小鼠腫瘤細(xì)胞系的胞內(nèi)蛋白有明顯結(jié)合;與小鼠脾細(xì)胞的部分細(xì)胞膜結(jié)合。用上述4株單抗鑒定人和小鼠細(xì)胞的Vκ4-1-Jκ3型Igκ表達(dá)譜工作仍在進(jìn)行中。 第三章抗藍(lán)載體單克隆抗體的制備及特性鑒定 該部分是作為碩士研究生進(jìn)入實(shí)驗(yàn)室初期進(jìn)行免疫學(xué)技能訓(xùn)練時(shí)完成的一項(xiàng)工作,并得以發(fā)表。藍(lán)載體(Blue Carrier,BC)是從軟體動(dòng)物Concholepasconcholepas中分離出的血藍(lán)蛋白,是鑰孔戚血藍(lán)素(Keyhole limpethemocyanin,KLH)的一種類(lèi)似物。BC的溶解性與均一性明顯優(yōu)于KLH,作為合成肽以及半抗原免疫時(shí)的載體蛋白已被成功用于多克隆抗體以及單克隆抗體的制備。但此前國(guó)內(nèi)并無(wú)針對(duì)BC單抗的工作報(bào)道。本研究在制備抗短肽(以藍(lán)載體為交聯(lián)載體)單克隆抗體的同時(shí),有目的地篩選了抗藍(lán)載體單克隆抗體,所獲單抗585只與藍(lán)載體特異性結(jié)合,與鑰孔戚血藍(lán)素(KLH)、牛血清白蛋白(BSA)、雞卵清白蛋白(OVA)等載體蛋白不發(fā)生交叉反應(yīng)。該單抗可成為深入研究藍(lán)載體抗原、以及短肽或半抗原交聯(lián)抗原的相關(guān)研究的有效工具。
[Abstract]:Immunoglobulin (Ig) is an important immune molecule. It exists in two ways: membrane type and secretory type. The former plays an important role in antigen recognition, cell activation, differentiation and even programmed cell death as an antigen receptor on the surface of B lymphocyte; the latter mainly exists in blood and other body fluids and can be associated with the corresponding antigen phase. The classical immunological theory holds that Ig is derived from B lymphocytes and plasma cells, and the Ig gene is selectively rearranged only during the development of B lymphocytes, whereas in other somatic cells the gene is in embryonic state, or can only undergo incomplete rearrangement, i.e. no functional gene rearrangement. Therefore, there will be no generation of Ig molecules.
In 2003, Professor Qiu Xiaoyan of Peking University published a paper in Cancer Research confirming that epithelial-derived tumor cells can produce Ig molecules. The work was named one of the top ten news reports in the field of Chinese medicine research in 2004. The work demonstrated that in addition to B-lymphocytes and plasma cells, some non-immune cells, such as epithelial-derived tumor cells, such as Lung cancer, ovarian cancer, intestinal cancer, nasopharyngeal cancer, breast cancer, gastric cancer, pancreatic cancer, etc.) and some normal epithelial cells also have functional gene rearrangement and can produce Ig molecules, so it is now called non-B-cell-derived Ig (non-B-Ig); moreover, more importantly, through the study of the function of non-B-Ig molecule, the team showed in the base. These phenomena will make it possible to redefine the origin and potential of Ig molecules, which are different from the biological activity of classical Ig and have important academic value. Researches and tumor biotherapy targeting Ig molecules expressed by tumor cells have important implications. Fortunately, the phenomenon that non-B cells can produce Ig has been gradually confirmed and recognized by scholars at home and abroad.
It is known that B-cell-derived Ig has a unique structure and function, mainly manifested in: its variable region (V region) recognizes specific antigen determinants, different B-lymphocyte clones produce different IgV region structure, thus constituting the diversity of Ig molecules, so that the immune system can respond to different antigens in nature; its constant region and a group of specific components. According to the constant region, the Ig molecules produced by B lymphocytes are classified into five categories according to the different heavy chains: IgG, IgM, IgA, IgD and IgE, and the light chains are classified into kappa and lambda.
Variable region sequences of B-cell-derived Ig molecules are highly diverse, which is the structural basis of antibody and BCR diversity. Similar to classical Ig molecules, the results of non-B-cell-derived Ig gene studies show that the heavy chain and light chain coding genes of these non-B-Ig molecules have undergone typical gene rearrangement and transcription, but in their V-D-J or V-J genes, both of them have undergone typical gene rearrangement and transcription. Professor Qiu Xiaoyan's research team demonstrated that the combination of V-D-J or V-J fragments of Ig molecules derived from all tumor cells had obvious tendency by means of laser microdissection (LCM) and RT-PCR. Highly homologous, or even identical, Ig sequences from different individual tumor sources were not recorded in the Ig database of B lymphocyte expression, suggesting that these sequences were indeed produced by tumor cells. More surprisingly, all tumor cells expressed identical or very similar sequences (individual bases). The pattern of rearrangement, i.e. V-kappa 4-1/J-kappa 3 Ig-kappa transcript (patent number 2005 10107833.9), suggests that these sequence characteristics are not related to the type of tissue, and they may play a similar role in different tissues.
According to the above findings, as a cooperative project with Professor Qiu Xiaoyan of Peking University, the main purpose of this study is to prepare the conserved variable region antibody of the kappa chain. Two specific B-cell recognition epitopes of Vkappa 4-1/J kappa 3 Ig kappa were analyzed and predicted by bioinformatics. Three short peptides, AL13 (ASINCKSSQRVSL), QF20 (QAEDVAVYCQYDTPVTF), AL13B (TQSPDSLVSLGERASINCKSSQRVSLG, or AL13 extension sequence), were designed and synthesized. The prokaryotic table provided by Professor Qiu Xiaoyan's research group was also obtained. The purified fusion protein (GST-V_kappa_4-1-J_kappa_3) was obtained by immunizing rabbits with synthetic peptides AL13 and QF20 respectively, and mice immunized with synthetic peptides AL13B-KLH and prokaryotic protein GST-V_kappa_4-1-J_kappa_3 were immunized with a series of monoclonal antibodies. The titers and specificities of the antibodies were identified by the method and so on, which provided an effective tool for further research.
Chapter one preparation and identification of V kappa 4-1-J kappa 3 Ig kappa V region specific single epitope polyclonal antibody
The synthetic peptides AL13 and QF20 were cross-linked with the carrier protein to prepare the immunogen. The corresponding antisera were obtained from New Zealand white rabbits immunized with these peptides. The titers of antibodies were 0.3 (-5) and 0.6 (-5) respectively by ELISA. The rabbit sera were purified by affinity chromatography, and anti-AL13 and anti-QF20 single epitope specific polyantibodies were obtained. To further prove the specificity of the antibody, the prokaryotic expression proteins GST-Vkappa 4-1-J kappa 3 (39kD) and V5-51-Vkappa 4-1-J kappa 3-pRA (34kD) were used as antigens for Western Blot assay. The results showed that both anti-AL13 and anti-QF20 could correctly recognize the Vkappa 4-1-J kappa 3 sequence. Prokaryotic expression proteins showed clear bands at expected sites, but did not bind to commercialized light chains of human IgG. Anti-AL13 was used to detect the expression of endogenous Ig-kappa in HT-29 cells. The results showed that the positive signal of anti-AL13 was mainly found in the insoluble components of HT-29 cells (only a small amount in the soluble components). Different intracellular localization of classical Ig molecules suggests that they may have different biological activities from classical Ig molecules.
The second chapter is the preparation and identification of V kappa 4-1-J type 3 Ig kappa V specific monoclonal antibody.
Balb/c mice were immunized with synthetic peptide AL13B-KLH as immunogen. The spleen cells were fused with NS-1 cells to prepare hybridoma. The synthesized peptide and prokaryotic expression protein (GST-Vkappa 4-1-J kappa 3) were screened and cloned for three times. Monoclonal antibodies were purified from the ascites. Three strains (6G5,3H9.2,5D3) secreted stably Ig kappa against Vkappa 4-1-J kappa 3. Balb/c mice were immunized with prokaryotic recombinant protein (GST-Vkappa 4-1-J kappa 3) as immunogen, and their spleen cells were fused with NS-1 cells. After screening and cloning, a hybridoma cell line (4E9) secreting antibodies against Vkappa 4-1-J kappa 3 Ig kappa stably was obtained. 2b. Ascites monoclonal antibodies were purified by octanoic acid-ammonium sulfate precipitation method and labeled with horseradish peroxidase by modified sodium periodate method. The results of ELISA identification showed that the monoclonal antibodies were specifically bound to synthetic peptides and prokaryotic expression protein (GST-Vkappa 4-1-J kappa 3). The results showed that the three monoclonal antibodies did not cross-react with carrier proteins KLH, BC, BSA and OVA, and did not react with commercialized human IgG, sheep IgG, rabbit IgG and mouse IgG. In view of the 70% homology between human and mouse Ig-kappa, we compared the target antigen sequence with mouse Ig-kappa V region and confirmed that the four McAbs could bind to the intracellular protein of mouse tumor cell line by flow cytometry. The identification of Vkappa 4-1-J kappa 3 Ig kappa expression profiles in human and mouse cells by these four monoclonal antibodies is still in progress.
The third chapter is preparation and characterization of monoclonal antibodies against blue carriers.
This part is a job done as a postgraduate student in the early stage of the laboratory immunological skills training, and published. Blue Carrier (BC) is a hemocyanin isolated from the mollusk Concholepas concholepas, is a key hole limpethemocyanin (KLH) analogue. As a carrier protein for synthetic peptides and hapten immunization, it has been successfully used in the preparation of polyclonal antibodies and monoclonal antibodies. However, there have been no reports on the preparation of monoclonal antibodies against BC. In this study, monoclonal antibodies against short peptides (using blue carriers as cross-linking carriers) were prepared and screened purposefully. A total of 585 monoclonal antibodies against blue vectors were obtained. The monoclonal antibodies specifically bind to blue vectors and do not cross-react with carrier proteins such as key hole hemocyanin (KLH), bovine serum albumin (BSA), chicken ovalbumin (OVA). The monoclonal antibodies can be used as an effective tool for further study of blue vectors antigens, short peptides or hapten cross-linked antigens. It is.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R392

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相關(guān)期刊論文 前7條

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2 唐磊,朱平,羅琛,徐湘民,富寧;抗人ζ珠蛋白鏈不同表位單克隆抗體的制備和鑒定[J];第一軍醫(yī)大學(xué)學(xué)報(bào);2005年11期

3 余利紅,高艷,王利紅,韓洪彥,孫志賢,張成崗;β-Netrin抗原表位的分析及其抗體的制備與鑒定[J];細(xì)胞與分子免疫學(xué)雜志;2005年03期

4 汪泱,張叔人,何祖根,邱曉彥,張友會(huì);免疫球蛋白樣物質(zhì)在上皮來(lái)源的惡性腫瘤中的表達(dá)[J];中國(guó)腫瘤臨床;2001年03期

5 邱曉彥,侯春梅,沈繼銘,張叔人,唐佩弦,毛寧;HL-60細(xì)胞Ig樣蛋白的表達(dá)及IgH V-D-J序列分析[J];中華血液學(xué)雜志;1999年06期

6 邱曉彥,楊貴貞;上皮性惡性腫瘤細(xì)胞的Ig基因分析[J];中國(guó)腫瘤生物治療雜志;1997年02期

7 邱曉彥，楊貴貞;惡性腫瘤細(xì)胞內(nèi)呈現(xiàn)Ig樣物質(zhì)的特性及其基因結(jié)構(gòu)分析[J];中國(guó)免疫學(xué)雜志;1996年05期

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