非B细胞来源IgκV区保守序列特异性抗体的制备及鉴定
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摘要
免疫球蛋白(Ig)是重要的免疫分子,其两种存在方式分别为:膜型和分泌型,前者作为B淋巴细胞表面的抗原受体在抗原识别、细胞的活化、分化甚至程序性细胞死亡中起重要作用;后者主要存在与血液及其它体液中的,能够与相应抗原相结合发挥抗体的效应功能。经典的免疫学理论认为,Ig的来源为B淋巴细胞及浆细胞,Ig基因仅在B淋巴细胞发育过程中成功进行选择性重排,而在其它体细胞中该基因处于胚系状态,或者仅能发生不完全的重排,即不发生功能性基因重排,故不会有Ig分子的产生。
2003年,北京大学邱晓彦教授课题组在Cancer Research发表论文证实上皮来源肿瘤细胞可产生Ig分子,该工作被评为2004年中国医药研究领域十大新闻之一。其工作证明了除B淋巴细胞及浆细胞外,在一些非免疫细胞,如上皮来源的肿瘤细胞(如肺癌、卵巢癌、肠癌、鼻咽癌、乳腺癌、胃癌、胰腺癌等)及部分正常的上皮细胞内Ig基因同样存在功能性基因重排,并可以产生Ig分子,因此现将其称为非B细胞来源Ig(non B-Ig);此外,更重要的是该课题组通过对non B-Ig分子功能的研究,表现在基因及蛋白质水平抑制癌细胞Ig分子的表达或封闭其活性,则癌细胞增殖能力明显减弱。上述现象将有可能使人们重新定义Ig分子的来源及其潜在的、有别于经典Ig的生物学活性,具有重要的学术价值。同时,也将对肿瘤发生与发展的生物学研究以及以肿瘤细胞表达的Ig分子做为靶点的肿瘤生物治疗研究产生重要的影响。可喜的是,非B细胞能够产生Ig的现象已逐渐被国内外学者所证实与认同。
已知,B细胞来源的Ig具独特的结构和功能,主要表现在:其可变区(V区)识别特定的抗原决定簇,不同的B淋巴细胞克隆产生不同的IgV区结构,从而构成了Ig分子的多样性,使得免疫系统能够应对自然界不同的抗原;其恒定区与一组特定的分子(如Fc受体、信号传导分子、补体的级联成分)相结合,行使Ig分子的效应功能。根据恒定区的不同,B淋巴细胞产生的Ig分子根据重链不同被分为五类,分别是IgG、IgM、IgA、IgD和IgE;其轻链则分为κ与λ型。
B细胞来源的Ig分子的可变区序列呈高度多样性,这是构成抗体及BCR多样性的结构基础。对非B细胞来源的Ig基因研究结果显示,与经典的Ig分子相似,这些non B-Ig分子的重链及轻链编码基因均发生了典型的基因重排及转录。但在其V-D-J或V-J序列、体细胞突变特点和类别转换等方面均呈现出与经典Ig分子不同的特征。邱晓彦教授课题组借助激光显微切割(LCM)及RT-PCR技术,证明所有肿瘤细胞来源的Ig分子V-D-J或V-J片段组合方式有明显的倾向性。表现在同一个体的不同细胞克隆、甚至不同个体肿瘤来源的Ig序列之间高度同源,甚至完全相同,并且这些序列在B淋巴细胞表达的Ig数据库中均无记录,提示这些序列的确是肿瘤细胞产生的。更令人惊奇的是,所有肿瘤细胞均表达序列完全相同或极其相似的(个别碱基的替换)重排模式,即Vκ4—1/Jκ3型Igκ转录本(专利申请号为200510107833.9)。这一现象提示,这些序列特点与组织类型无关,它们可能在不同的组织中发挥着相似的作用。
根据上述发现,作为与北京大学邱晓彦教授的合作项目,本研究的主要目的是制备这种保守的κ链可变区抗体。我们根据其特有Vκ4—1/Jκ3氨基酸序列,对比胚系基因可变区序列,综合考虑氨基酸组成、疏水性、抗原性、表面暴露性等因素,利用生物信息学方法分析预测了Vκ4—1/Jκ3型Igκ两个较为特异的B细胞识别表位,设计并合成三个短肽AL13(序列ASINCKSSQRVSL)、QF20(QAEDVAVYYCQQYYDTPVTF),AL13B(TQSPDSLVVSLGERASINCKSSQRVSLG,即AL13的延长序列)。同时获得由邱晓彦教授课题组提供的原核表达Vκ4—1/Jκ3型Igκ纯化融合蛋白(GST-V_κ4-1-J_κ3)。本实验分别以合成肽AL13、QF20交联载体免疫家兔获得单表位特异性多克隆抗体;分别以合成肽AL13B-KLH、原核蛋白GST-V_κ4-1-J_κ3免疫小鼠获得系列单克隆抗体抗。再通ELISA、WesternBlot、流式细胞术等方法鉴定了抗体的效价和特异性,为后续研究提供了有效工具。
第一章Vκ4-1-Jκ3型IgκV区特异性单表位多抗的制备及鉴定
将合成肽AL13、QF20分别与载体蛋白交联制备免疫原,免疫新西兰大白兔获得相应抗血清,经ELISA检测其抗体滴度分别为0.3×10~(-5)和0.6×10~(-5)。运用亲和层析方法对兔血清进行纯化,得到anti-AL13和anti-QF20单表位特异性多抗。ELISA结果显示,所得抗体与合成肽特异性结合,但不与正常人IgG反应。为了进一步证明所得抗体的特异性,以原核表达蛋白GST-Vκ4-1-Jκ3(39kD)和V5-51-Vκ4-1-Jκ3-pRA(34kD)作为抗原进行Western Blot检测,结果显示anti-AL13和anti-QF20都能够正确识别含Vκ4-1-Jκ3序列的原核表达蛋白,在预期位置上都有明确的条带出现,但基本不与商品化的人IgG的轻链结合;用anti-AL13检测HT-29细胞中内源性Igκ的表达,结果显示anti-AL13的阳性信号主要在HT-29细胞不可溶成分中(仅少量的存在于可溶性组分中),这种与经典Ig分子完全不同的细胞内定位,提示其可能具有不同于经典Ig分子的生物学活性。
第二章Vκ4-1-Jκ3型IgκV区特异性单克隆抗体的制备及鉴定
以合成肽AL13B-KLH作为免疫原免疫Balb/c小鼠,取其脾细胞与NS-1细胞融合制备杂交瘤,经合成肽和原核表达蛋白(GST-Vκ4-1-Jκ3)筛选和三次克隆化,并从制备的腹水中纯化单克隆抗体,筛选后共获得3株(6G5、3H9.2、5D3)稳定分泌针对Vκ4-1-Jκ3型Igκ的抗体的杂交瘤细胞;同时以原核重组蛋白(GST-Vκ4-1-Jκ3)作为免疫原免疫Balb/c小鼠,取其脾细胞与NS-1细胞进行融合,经筛选、克隆化后获得1株(4E9)稳定分泌针对Vκ4-1-Jκ3型Igκ的抗体的杂交瘤细胞。这4株单抗的亚类分别为IgG1、IgG1、IgG1和IgG2b。将腹水单抗经辛酸—硫酸铵沉淀法纯化,并用改良过碘酸钠法对4株抗体标记了辣根过氧化物酶。ELISA鉴定结果显示所得单抗均与合成肽及原核表达蛋白(GST-Vκ4-1-Jκ3)发生特异性结合。对合成肽AL13B-KLH来源的3株单抗的近一步ELISA鉴定结果显示:这3株单抗与常用的载体蛋白KLH、BC、BSA、OVA均无交叉反应,同时与商品化的人IgG、羊IgG、兔IgG、鼠IgG均不反应;用流式细胞术鉴定上述单抗可与以人结肠癌细胞系HT-29和人肺癌细胞系A549的细胞内蛋白发生明显的特异性结合,验证了Vκ4-1-Jκ3型Igκ的胞内表达;与人末梢血淋巴细胞、单核细胞、粒细胞均有高比例的结合。鉴于人与小鼠Igκ的同源性高达70%,我们对靶抗原序列与小鼠IgκV区进行了比对,用流式细胞术证明上述4株单抗可与小鼠肿瘤细胞系的胞内蛋白有明显结合;与小鼠脾细胞的部分细胞膜结合。用上述4株单抗鉴定人和小鼠细胞的Vκ4-1-Jκ3型Igκ表达谱工作仍在进行中。
第三章抗蓝载体单克隆抗体的制备及特性鉴定
该部分是作为硕士研究生进入实验室初期进行免疫学技能训练时完成的一项工作,并得以发表。蓝载体(Blue Carrier,BC)是从软体动物Concholepasconcholepas中分离出的血蓝蛋白,是钥孔戚血蓝素(Keyhole limpethemocyanin,KLH)的一种类似物。BC的溶解性与均一性明显优于KLH,作为合成肽以及半抗原免疫时的载体蛋白已被成功用于多克隆抗体以及单克隆抗体的制备。但此前国内并无针对BC单抗的工作报道。本研究在制备抗短肽(以蓝载体为交联载体)单克隆抗体的同时,有目的地筛选了抗蓝载体单克隆抗体,所获单抗585只与蓝载体特异性结合,与钥孔戚血蓝素(KLH)、牛血清白蛋白(BSA)、鸡卵清白蛋白(OVA)等载体蛋白不发生交叉反应。该单抗可成为深入研究蓝载体抗原、以及短肽或半抗原交联抗原的相关研究的有效工具。
Immunoglobulin(Ig) is a kind of important molecule in immune response,which can be presented as antigen receptor on B lymphocytes(BCR),and secreted soluble phase as antibody into blood and body fluids to bind with antigen and mediate a variety of biologic function.According to classical immunological theory, immunoglobulin is a characteristic product of B lymphocyte or plasma cell.The rearrangement,transcription and expression of the Ig gene are considered as B lymphocyte-specific events;Ig was believed to be absent in other cell types because their Ig genes were in germline state,or rearrange insufficiently,which is no function as Ig produced by B cell.
However,the team of Dr.Xiaoyan Qiu of Beijing University published one paper in "Cancer Rearch" in 2003,they proved that Ig can be produced by human cancer cells of epithelial origin.Their work has been awarded as one of the top 10 news for 2004 in the medicine and hygienical fields of China.They found that some non-B cells such as human cancer cells of epithelial origin(the including carcinomas of lung,oophorus,nasopharynx,colon,breast,stomach,pancreas,etc) as well as some normal lung tissues,express Ig gene functional rearrangement and produce Ig in both cytoplasmic and secreted forms.Now we call this kind of Immunoglobulin as "Ig derived from non-B cell(non B-Ig)".Furthermore,investigation of function on tumor-derived IgG suggests that blockade of tumor-derived IgG by either antisense DNA or anti IgG antibody increased programmed cell death and inhibited growth of cancer cells in vitro.These finding might redefine the conception about source of immunoglobulin and significance of immunoglobulin in physiological and pathological state up to now.These results would also have an impact on the exploration of tumor biology and tumor biotherapy using tumor-derived Ig as target. It is encouraged by that non B-Ig has been confirmed by more and more scientists in China and in abroad.
Igs produced by B lymphocytes with specific structure and function can recognize specific antigen epitope clusters by their variable region(V region),and diversity of Ig in binding a varity of antigen depends on different variable region structure,and mediating the fixation of complement,signal transducers etc depends on Fc fragment in constant region(C region).Immunoglobulin can be classified into five kinds as IgG,IgM,IgA,IgD and IgE according to the C region of heavy chain, and two kinds of light chain asκandλaccording to C region of light chain.
The V region of Ig produced by B lymphocytes is hypervariable,which is the structural basis for antibody and BCR diversity.What is the situation in non-B? Dr. Qiu's team used the laser capture microdissection(LCM) technology and RT-PCR to research Ig derived from human epithelial cancers.After sequencing,and blast the sequences with that in normal B cells Ig,the results showed that all of genes of heavy chain and the light chain derived from these cancer cells undergo typical gene transcription and rearrangement as that in classical Ig.But the caner-derived Ig genes have their own characteristics in sequence variety,and somatic hypermutation and class switch are different from classic Ig.Moreover,all these sequence can not be found in Ig database of B cells,which suggests that these sequences are produced by tumor cell.It is more surprising to find the phemomena in which different types of cancer cells can express almost same or similar V region sequence.Especially,all of cancer cells detected showed the same rearrangement:Vk4-1-Jk3 group and extremely similar sequence(Patent No.200510107833.9),which suggests that the sequence seems to be not related with the tissue types,and may exert the same function in different tissue.
Based on above discoveries,our work was to prepare the antibody to this conservative structure of Vk as a part of collaboration with Dr.Qiu.According to the special sequence of cancer-derived Vk4-1-Jk3,three antigenic peptides were designed and systhesized by analysis and comparison with the V region sequence of germline gene,amino acids composition,hydrophobicity,antigenicity,accessibility of surface exposure,which are named as AL13(ASINCKSSQRVSL),QF20(QAEDVAVYYCQ QYYDTPVTF),and AL13B(TQSPDSLVVSLGERASINCKSSQRVSLG,prolonged AL13).In addition,we got prokaryotic recombinant Vk4-1-Jk3 fusion protein with GST tag from Dr.Xiaoyan Qiu.Polyclonal antibodies with single specificity to AL13 and QF20 were prepared in rabbits immunized with peptide-carrier conjugates.The monoclonal antibodies were prepared in mice immunized with AL13B-KLH and recombinant Vk4-1-Jk3 protein respectively.Then the monoclonal antibodies(mAbs) were identified by ELISA,western Blot,flow cytometry and immunohistochemistry. These antibodies are useful tools for further research on non-B Ig.
PartⅠPreparation and Characterization of polyclonal antibodies against V region of Vk4-1-Jk3 Igk
Polyclonal antibodies against V region sequence of Vk4-1-Jk3 Igk were prepared in rabbits immunized with synthetic peptide AL13,QF20 conjugates with carrier protein respectively.The titer of anti-AL13 and anti-QF20 were 0.3×10~(-5),and 0.6×10~(-5) detected by indirect ELISA.Then purified polyclonal antibodies were obtained by affinity chromatography.These antibodies could react with synthetic peptide, recombinant protein GST-Vk4-1-Jk3 and V5-51-Vk4-1-Jk3-pRA,not with normal human IgG and Igk.And the component recognized by anti QF20 was mainly presented in the insoluble fraction of HT-29 cell line(only a little in the soluble fraction).The different cellular localization between cancer derived Ig and classical Ig molecules suggests that cancer-derived Igk might have a different significance compared with classical Ig.
PartⅡPreparation and Characterization of monoclonal antibodies against V region of Vk4-1-Jk3 Igk
In this part,mAbs against V region sequence of Vk4-1-Jk3 Igk were prepared in mice immunized with antigenic peptide AL13B-KLH conjugate and recombinant GST-Vk4-1-Jk3 fusion protein.The hybridoma cells were generated by fusion of mouse spleen cells and myeloma cells NS-1.After successively subcloning,three strains named as 6G5,3H9.2,5D3 of hybridoma cell lines which secrete monoclonal antibodies against Vk4-1-Jk3 of Igk were obtained.And also a strain of hybridoma cell line named as 4E9 which secrete mAb to react with Vk4-1-Jk3 protein and AL13B peptide,mAb 6G5,3H9.2 and 5D3 belong to IgG1 isotype and mAb 4E9 belongs to IgG2b.mAbs were purified from ascites by methods of caprylic acid-ammonium sulphate precipitarion(CAASP),and made to be conjugates with horse radish peroxidase(HRP),mAb 6G5,3H9.2 and 5D3 could bind with synthetic peptide and recombinant protein,but not common carrier proteins such as GST,KLH, Blue Carrier(BC),BSA and OVA.In addition,these mAbs had no reaction with serum IgG from human,goat,rabbit,and mouse too.In the expression of Vk4-1-Jk3, these mAbs could react with intracellular Proteins of human colon cancer cell lines HT-29 and human lung cancer cell lines A549.Interestly,we found that the mAbs could react with nomal human Peripheral blood mononuclear cell(PBMC),and granulocyte obviously.As the sequence of human Igk has 70%homology with that of mice,we blasted our target sequences with murine Igk gene in Genebank,and tried to detect murine cell line and spleen cells by flow cytometry.The results showed that these mAbs can react with intracellular proteins of murine tumor cell lines obviously, and react with membrane surface proteins of mouse spleen cells slightly.The identifying and characterizing of these mAbs still were kept on going in different lab now.
PartⅢPreparation of monoclonal antibody against Blue Carrier
The work in this part was performed as skills training in our lab,in which a useful mAb was obtained.Blue Carrier(BC) is a novel formulation of the hemocyanin obtained from the mollusk Concholepas concholepas(CCH).Both BC and KLH as the largest proteins are used as carrier protein to prepare immunogen with peptide or hapten.As application of BC only began in recent years,it is necessary to prepare the mAb to BC for the research the epitopic of mollusk hemocyanins and antibody response to immunogenic conjugates with BC.In fact,we just got this anti-BC mAb simultaneously in preparation of a mAb againt an antigenic peptide presented in peptide-BC conjugate.This mAb named as 5B5 could react with BC specifically,not with KLH,BSA and OVA,which provides a useful tool for evaluation of BC conjugate immunogen,some new products in diagnosis and therapy.
2003年,北京大学邱晓彦教授课题组在Cancer Research发表论文证实上皮来源肿瘤细胞可产生Ig分子,该工作被评为2004年中国医药研究领域十大新闻之一。其工作证明了除B淋巴细胞及浆细胞外,在一些非免疫细胞,如上皮来源的肿瘤细胞(如肺癌、卵巢癌、肠癌、鼻咽癌、乳腺癌、胃癌、胰腺癌等)及部分正常的上皮细胞内Ig基因同样存在功能性基因重排,并可以产生Ig分子,因此现将其称为非B细胞来源Ig(non B-Ig);此外,更重要的是该课题组通过对non B-Ig分子功能的研究,表现在基因及蛋白质水平抑制癌细胞Ig分子的表达或封闭其活性,则癌细胞增殖能力明显减弱。上述现象将有可能使人们重新定义Ig分子的来源及其潜在的、有别于经典Ig的生物学活性,具有重要的学术价值。同时,也将对肿瘤发生与发展的生物学研究以及以肿瘤细胞表达的Ig分子做为靶点的肿瘤生物治疗研究产生重要的影响。可喜的是,非B细胞能够产生Ig的现象已逐渐被国内外学者所证实与认同。
已知,B细胞来源的Ig具独特的结构和功能,主要表现在:其可变区(V区)识别特定的抗原决定簇,不同的B淋巴细胞克隆产生不同的IgV区结构,从而构成了Ig分子的多样性,使得免疫系统能够应对自然界不同的抗原;其恒定区与一组特定的分子(如Fc受体、信号传导分子、补体的级联成分)相结合,行使Ig分子的效应功能。根据恒定区的不同,B淋巴细胞产生的Ig分子根据重链不同被分为五类,分别是IgG、IgM、IgA、IgD和IgE;其轻链则分为κ与λ型。
B细胞来源的Ig分子的可变区序列呈高度多样性,这是构成抗体及BCR多样性的结构基础。对非B细胞来源的Ig基因研究结果显示,与经典的Ig分子相似,这些non B-Ig分子的重链及轻链编码基因均发生了典型的基因重排及转录。但在其V-D-J或V-J序列、体细胞突变特点和类别转换等方面均呈现出与经典Ig分子不同的特征。邱晓彦教授课题组借助激光显微切割(LCM)及RT-PCR技术,证明所有肿瘤细胞来源的Ig分子V-D-J或V-J片段组合方式有明显的倾向性。表现在同一个体的不同细胞克隆、甚至不同个体肿瘤来源的Ig序列之间高度同源,甚至完全相同,并且这些序列在B淋巴细胞表达的Ig数据库中均无记录,提示这些序列的确是肿瘤细胞产生的。更令人惊奇的是,所有肿瘤细胞均表达序列完全相同或极其相似的(个别碱基的替换)重排模式,即Vκ4—1/Jκ3型Igκ转录本(专利申请号为200510107833.9)。这一现象提示,这些序列特点与组织类型无关,它们可能在不同的组织中发挥着相似的作用。
根据上述发现,作为与北京大学邱晓彦教授的合作项目,本研究的主要目的是制备这种保守的κ链可变区抗体。我们根据其特有Vκ4—1/Jκ3氨基酸序列,对比胚系基因可变区序列,综合考虑氨基酸组成、疏水性、抗原性、表面暴露性等因素,利用生物信息学方法分析预测了Vκ4—1/Jκ3型Igκ两个较为特异的B细胞识别表位,设计并合成三个短肽AL13(序列ASINCKSSQRVSL)、QF20(QAEDVAVYYCQQYYDTPVTF),AL13B(TQSPDSLVVSLGERASINCKSSQRVSLG,即AL13的延长序列)。同时获得由邱晓彦教授课题组提供的原核表达Vκ4—1/Jκ3型Igκ纯化融合蛋白(GST-V_κ4-1-J_κ3)。本实验分别以合成肽AL13、QF20交联载体免疫家兔获得单表位特异性多克隆抗体;分别以合成肽AL13B-KLH、原核蛋白GST-V_κ4-1-J_κ3免疫小鼠获得系列单克隆抗体抗。再通ELISA、WesternBlot、流式细胞术等方法鉴定了抗体的效价和特异性,为后续研究提供了有效工具。
第一章Vκ4-1-Jκ3型IgκV区特异性单表位多抗的制备及鉴定
将合成肽AL13、QF20分别与载体蛋白交联制备免疫原,免疫新西兰大白兔获得相应抗血清,经ELISA检测其抗体滴度分别为0.3×10~(-5)和0.6×10~(-5)。运用亲和层析方法对兔血清进行纯化,得到anti-AL13和anti-QF20单表位特异性多抗。ELISA结果显示,所得抗体与合成肽特异性结合,但不与正常人IgG反应。为了进一步证明所得抗体的特异性,以原核表达蛋白GST-Vκ4-1-Jκ3(39kD)和V5-51-Vκ4-1-Jκ3-pRA(34kD)作为抗原进行Western Blot检测,结果显示anti-AL13和anti-QF20都能够正确识别含Vκ4-1-Jκ3序列的原核表达蛋白,在预期位置上都有明确的条带出现,但基本不与商品化的人IgG的轻链结合;用anti-AL13检测HT-29细胞中内源性Igκ的表达,结果显示anti-AL13的阳性信号主要在HT-29细胞不可溶成分中(仅少量的存在于可溶性组分中),这种与经典Ig分子完全不同的细胞内定位,提示其可能具有不同于经典Ig分子的生物学活性。
第二章Vκ4-1-Jκ3型IgκV区特异性单克隆抗体的制备及鉴定
以合成肽AL13B-KLH作为免疫原免疫Balb/c小鼠,取其脾细胞与NS-1细胞融合制备杂交瘤,经合成肽和原核表达蛋白(GST-Vκ4-1-Jκ3)筛选和三次克隆化,并从制备的腹水中纯化单克隆抗体,筛选后共获得3株(6G5、3H9.2、5D3)稳定分泌针对Vκ4-1-Jκ3型Igκ的抗体的杂交瘤细胞;同时以原核重组蛋白(GST-Vκ4-1-Jκ3)作为免疫原免疫Balb/c小鼠,取其脾细胞与NS-1细胞进行融合,经筛选、克隆化后获得1株(4E9)稳定分泌针对Vκ4-1-Jκ3型Igκ的抗体的杂交瘤细胞。这4株单抗的亚类分别为IgG1、IgG1、IgG1和IgG2b。将腹水单抗经辛酸—硫酸铵沉淀法纯化,并用改良过碘酸钠法对4株抗体标记了辣根过氧化物酶。ELISA鉴定结果显示所得单抗均与合成肽及原核表达蛋白(GST-Vκ4-1-Jκ3)发生特异性结合。对合成肽AL13B-KLH来源的3株单抗的近一步ELISA鉴定结果显示:这3株单抗与常用的载体蛋白KLH、BC、BSA、OVA均无交叉反应,同时与商品化的人IgG、羊IgG、兔IgG、鼠IgG均不反应;用流式细胞术鉴定上述单抗可与以人结肠癌细胞系HT-29和人肺癌细胞系A549的细胞内蛋白发生明显的特异性结合,验证了Vκ4-1-Jκ3型Igκ的胞内表达;与人末梢血淋巴细胞、单核细胞、粒细胞均有高比例的结合。鉴于人与小鼠Igκ的同源性高达70%,我们对靶抗原序列与小鼠IgκV区进行了比对,用流式细胞术证明上述4株单抗可与小鼠肿瘤细胞系的胞内蛋白有明显结合;与小鼠脾细胞的部分细胞膜结合。用上述4株单抗鉴定人和小鼠细胞的Vκ4-1-Jκ3型Igκ表达谱工作仍在进行中。
第三章抗蓝载体单克隆抗体的制备及特性鉴定
该部分是作为硕士研究生进入实验室初期进行免疫学技能训练时完成的一项工作,并得以发表。蓝载体(Blue Carrier,BC)是从软体动物Concholepasconcholepas中分离出的血蓝蛋白,是钥孔戚血蓝素(Keyhole limpethemocyanin,KLH)的一种类似物。BC的溶解性与均一性明显优于KLH,作为合成肽以及半抗原免疫时的载体蛋白已被成功用于多克隆抗体以及单克隆抗体的制备。但此前国内并无针对BC单抗的工作报道。本研究在制备抗短肽(以蓝载体为交联载体)单克隆抗体的同时,有目的地筛选了抗蓝载体单克隆抗体,所获单抗585只与蓝载体特异性结合,与钥孔戚血蓝素(KLH)、牛血清白蛋白(BSA)、鸡卵清白蛋白(OVA)等载体蛋白不发生交叉反应。该单抗可成为深入研究蓝载体抗原、以及短肽或半抗原交联抗原的相关研究的有效工具。
Immunoglobulin(Ig) is a kind of important molecule in immune response,which can be presented as antigen receptor on B lymphocytes(BCR),and secreted soluble phase as antibody into blood and body fluids to bind with antigen and mediate a variety of biologic function.According to classical immunological theory, immunoglobulin is a characteristic product of B lymphocyte or plasma cell.The rearrangement,transcription and expression of the Ig gene are considered as B lymphocyte-specific events;Ig was believed to be absent in other cell types because their Ig genes were in germline state,or rearrange insufficiently,which is no function as Ig produced by B cell.
However,the team of Dr.Xiaoyan Qiu of Beijing University published one paper in "Cancer Rearch" in 2003,they proved that Ig can be produced by human cancer cells of epithelial origin.Their work has been awarded as one of the top 10 news for 2004 in the medicine and hygienical fields of China.They found that some non-B cells such as human cancer cells of epithelial origin(the including carcinomas of lung,oophorus,nasopharynx,colon,breast,stomach,pancreas,etc) as well as some normal lung tissues,express Ig gene functional rearrangement and produce Ig in both cytoplasmic and secreted forms.Now we call this kind of Immunoglobulin as "Ig derived from non-B cell(non B-Ig)".Furthermore,investigation of function on tumor-derived IgG suggests that blockade of tumor-derived IgG by either antisense DNA or anti IgG antibody increased programmed cell death and inhibited growth of cancer cells in vitro.These finding might redefine the conception about source of immunoglobulin and significance of immunoglobulin in physiological and pathological state up to now.These results would also have an impact on the exploration of tumor biology and tumor biotherapy using tumor-derived Ig as target. It is encouraged by that non B-Ig has been confirmed by more and more scientists in China and in abroad.
Igs produced by B lymphocytes with specific structure and function can recognize specific antigen epitope clusters by their variable region(V region),and diversity of Ig in binding a varity of antigen depends on different variable region structure,and mediating the fixation of complement,signal transducers etc depends on Fc fragment in constant region(C region).Immunoglobulin can be classified into five kinds as IgG,IgM,IgA,IgD and IgE according to the C region of heavy chain, and two kinds of light chain asκandλaccording to C region of light chain.
The V region of Ig produced by B lymphocytes is hypervariable,which is the structural basis for antibody and BCR diversity.What is the situation in non-B? Dr. Qiu's team used the laser capture microdissection(LCM) technology and RT-PCR to research Ig derived from human epithelial cancers.After sequencing,and blast the sequences with that in normal B cells Ig,the results showed that all of genes of heavy chain and the light chain derived from these cancer cells undergo typical gene transcription and rearrangement as that in classical Ig.But the caner-derived Ig genes have their own characteristics in sequence variety,and somatic hypermutation and class switch are different from classic Ig.Moreover,all these sequence can not be found in Ig database of B cells,which suggests that these sequences are produced by tumor cell.It is more surprising to find the phemomena in which different types of cancer cells can express almost same or similar V region sequence.Especially,all of cancer cells detected showed the same rearrangement:Vk4-1-Jk3 group and extremely similar sequence(Patent No.200510107833.9),which suggests that the sequence seems to be not related with the tissue types,and may exert the same function in different tissue.
Based on above discoveries,our work was to prepare the antibody to this conservative structure of Vk as a part of collaboration with Dr.Qiu.According to the special sequence of cancer-derived Vk4-1-Jk3,three antigenic peptides were designed and systhesized by analysis and comparison with the V region sequence of germline gene,amino acids composition,hydrophobicity,antigenicity,accessibility of surface exposure,which are named as AL13(ASINCKSSQRVSL),QF20(QAEDVAVYYCQ QYYDTPVTF),and AL13B(TQSPDSLVVSLGERASINCKSSQRVSLG,prolonged AL13).In addition,we got prokaryotic recombinant Vk4-1-Jk3 fusion protein with GST tag from Dr.Xiaoyan Qiu.Polyclonal antibodies with single specificity to AL13 and QF20 were prepared in rabbits immunized with peptide-carrier conjugates.The monoclonal antibodies were prepared in mice immunized with AL13B-KLH and recombinant Vk4-1-Jk3 protein respectively.Then the monoclonal antibodies(mAbs) were identified by ELISA,western Blot,flow cytometry and immunohistochemistry. These antibodies are useful tools for further research on non-B Ig.
PartⅠPreparation and Characterization of polyclonal antibodies against V region of Vk4-1-Jk3 Igk
Polyclonal antibodies against V region sequence of Vk4-1-Jk3 Igk were prepared in rabbits immunized with synthetic peptide AL13,QF20 conjugates with carrier protein respectively.The titer of anti-AL13 and anti-QF20 were 0.3×10~(-5),and 0.6×10~(-5) detected by indirect ELISA.Then purified polyclonal antibodies were obtained by affinity chromatography.These antibodies could react with synthetic peptide, recombinant protein GST-Vk4-1-Jk3 and V5-51-Vk4-1-Jk3-pRA,not with normal human IgG and Igk.And the component recognized by anti QF20 was mainly presented in the insoluble fraction of HT-29 cell line(only a little in the soluble fraction).The different cellular localization between cancer derived Ig and classical Ig molecules suggests that cancer-derived Igk might have a different significance compared with classical Ig.
PartⅡPreparation and Characterization of monoclonal antibodies against V region of Vk4-1-Jk3 Igk
In this part,mAbs against V region sequence of Vk4-1-Jk3 Igk were prepared in mice immunized with antigenic peptide AL13B-KLH conjugate and recombinant GST-Vk4-1-Jk3 fusion protein.The hybridoma cells were generated by fusion of mouse spleen cells and myeloma cells NS-1.After successively subcloning,three strains named as 6G5,3H9.2,5D3 of hybridoma cell lines which secrete monoclonal antibodies against Vk4-1-Jk3 of Igk were obtained.And also a strain of hybridoma cell line named as 4E9 which secrete mAb to react with Vk4-1-Jk3 protein and AL13B peptide,mAb 6G5,3H9.2 and 5D3 belong to IgG1 isotype and mAb 4E9 belongs to IgG2b.mAbs were purified from ascites by methods of caprylic acid-ammonium sulphate precipitarion(CAASP),and made to be conjugates with horse radish peroxidase(HRP),mAb 6G5,3H9.2 and 5D3 could bind with synthetic peptide and recombinant protein,but not common carrier proteins such as GST,KLH, Blue Carrier(BC),BSA and OVA.In addition,these mAbs had no reaction with serum IgG from human,goat,rabbit,and mouse too.In the expression of Vk4-1-Jk3, these mAbs could react with intracellular Proteins of human colon cancer cell lines HT-29 and human lung cancer cell lines A549.Interestly,we found that the mAbs could react with nomal human Peripheral blood mononuclear cell(PBMC),and granulocyte obviously.As the sequence of human Igk has 70%homology with that of mice,we blasted our target sequences with murine Igk gene in Genebank,and tried to detect murine cell line and spleen cells by flow cytometry.The results showed that these mAbs can react with intracellular proteins of murine tumor cell lines obviously, and react with membrane surface proteins of mouse spleen cells slightly.The identifying and characterizing of these mAbs still were kept on going in different lab now.
PartⅢPreparation of monoclonal antibody against Blue Carrier
The work in this part was performed as skills training in our lab,in which a useful mAb was obtained.Blue Carrier(BC) is a novel formulation of the hemocyanin obtained from the mollusk Concholepas concholepas(CCH).Both BC and KLH as the largest proteins are used as carrier protein to prepare immunogen with peptide or hapten.As application of BC only began in recent years,it is necessary to prepare the mAb to BC for the research the epitopic of mollusk hemocyanins and antibody response to immunogenic conjugates with BC.In fact,we just got this anti-BC mAb simultaneously in preparation of a mAb againt an antigenic peptide presented in peptide-BC conjugate.This mAb named as 5B5 could react with BC specifically,not with KLH,BSA and OVA,which provides a useful tool for evaluation of BC conjugate immunogen,some new products in diagnosis and therapy.
引文
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3.陈慰峰主编,《医学免疫学》.北京:人民卫生出版社,2001,230-231
4.Visser KE,Eichten A.Coussens LM Paradoxical roles of the immune system during cancer development[J].Nature Reviews Cancer,2006,6:24-37.
5.杨贵贞,等.恶性肿瘤细胞内呈现的Ig样蛋白特性及Ig基因结构分析[J].中国免疫学,1996,5,:296.
6.杨贵贞,等.上皮性恶性肿瘤细胞内Ig基因结构分析[J].中国肿瘤生物治疗,1997,4,:157-158.
7.邱晓彦,等.上皮性恶性肿瘤细胞内RAG1/RAG2基因的转录及胞浆内Ig样蛋白分析.北京大学基础医学院基础医学专业本科生毕业论文,2000.
8.邱晓彦,等.人宫颈癌传代细胞Ig样蛋白的纯化分析[J].北京医科大学学报,2000,32:310-312.
9.Jian Z,X-H Z,Peng H,et al.Immunoglobulin G expression in lung cancer cells[J].Acta Biochemica et Biophysica Sinica,2003,35:197-214.
10.毛宁,等.HL-60细胞Ig样蛋白的表达及IgH V-D-J序列分析[J].中华血液学1999,20:319.
11.Qiu X,Zhu X,Zhang L,et al.Human epithelial cancers secrete immunoglobulin G with unidentified specificity to promote growth and survival of tumor cells[J].Cancer Res,2003,63:6488-6495.
12.Kimoto,Y.Expression of heavy-chain constant region of immunoglobulin and T-cell receptor gene transcripts in human non-hematopoietic tumor cell lines[J].Genes Chromosomes Cancer,1998(22):83-86.
13.Chun JJ,Schatz DG,Oettinger MA,et al.The recombination activating gene-1(RAG-1) transcript is present in the murine central nervous system[J].Cell,1991,64:189-200.
14.Upender MB,Dunn JA,Wilson SM,Naegele JR.Immunoglobulin molecules are present in early-generated neuronal populations in the rat cerebral cortex and retina[J].J Comp Neurol,1997,384:271-282.
15.Yoshimi K,Woo M,Son Y,et al.IgG-immunostaining in the intact rabbit brain:variable but significant staining of hippocampal and cerebellar neurons with anti-IgG[J].Brain Res,2002,956:53-66.
16.Feng B,Bulchand S,Yaksi E,et al.The recombination activation gene 1(Rag1)is expressed in a subset of zebrafish olfactory neurons but is not essential for axon targeting or amino acid detection[J].BMC Neurosci,2005,6:46.
17.Jessen JR,Jessen TN,Vogel SS,et al.Concurrent expression of recombination activating genes 1 and 2 in zebrafish olfactory sensory neurons[J].Genesis,2001,29:156-162.
18.汪泱,邱晓彦,张友会.Ig样物质在上皮来源的恶性肿瘤中的表达[J].中国肿瘤临床,2001,28:169-171.
19.Gurevich A,Ben-Hur H,Moldavsky M,et al.Immunoprotection of gonads and genital tracts in human embryos and fetuses:immunohistochemical study[J].Am J Reprod Immuno1,2001,46:381-385.
20.Okabe H,Satoh S,Kato T,et al.Genome-wide analysis of gene expression in human hepatocellular carcinomas using cDNA microarray:identification of genes involved in viral carcinogenesis and tumor progression[J].Cancer Res,2001,61:2129-2137.
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