【作者】 杨向民；

【导师】 陈志南； 邢金良； 李郁；

【作者基本信息】 第四军医大学 ， 细胞生物学， 2008， 博士

【摘要】 本实验以HAb18G/CD147- HAb18抗原抗体系统为基础,构建并筛选了用于全人抗体表达的高效真核载体;之后,克隆了抗人大肠癌抗体CAb1轻、重链可变区基因VH和VL,通过制备复性的嵌合cFab对获得的基因的可靠性和准确性进行验证;进而以获得的CAb1的VH和VL为基础,对其进行人源化表面重塑抗体rCAb1设计;最终,选用鉴定的抗体真核表达载体,实现了重组rCAb1在哺乳动物细胞中的表达。第一部分研究内容:高效全人抗体真核表达载体的构建及筛选目的:获得具有自主产权,且能表达全人抗体的高效真核表达载体方法:按照标准DNA重组操作,构建以下不同的真核表达载体:包括弱化多顺反子pIRES1-18L;pIRES2-18H;定点重组载体18-pDHL-FRT;双载体系统18H-pDHA,18L-pCI;反向双载体系统18H-PCI,18L-p105;多顺反子表达载体18H-L-pCI-FRT;弱化筛选标记载体18H-L-pCII;以及pAH/pAG双载体pAH4604-18,pAG4622-18,这些载体均含有单抗HAb18轻重链可变区基因和人IgG1κ的轻、重链恒定区基因。之后,将这些载体分别转染COS-7细胞,斑点杂交检测培养上清的情况,观测是否有嵌合抗体cHAb18的表达;用夹心ELISA法检测重组抗体的表达量。随后,选用部分构建载体,用电穿孔稳定转染不同表型的CHO细胞,有限稀释进行阳性克隆筛选,完成转染细胞的筛选;RT-PCR检测稳定传代的细胞系,观察抗体基因是否丢失,评估转染载体的细胞稳定性。进行阳性克隆的扩大培养,表达产物的纯化;SDS-PAGE凝胶电泳及其Western blot检测抗体的表达情况;选用细胞免疫荧光染色以及流式细胞检测表达产物的特异性。结果:构建了含有抗体基因的不同的真核载体,通过必要的PCR或酶切鉴定插入序列,均获得了和理论上大小一致的片段,提示所有的载体均成功构建。将其全部瞬时转染COS-7细胞,斑点杂交结果显示,与未转化的细胞相比,除载体18H-L-pCII与双载体pAH/pAG没有检测到表达的IgG外,其余的载体均能检测到抗体的表达;而用抗原HAb18G/CD147胞外区进行的夹心ELISA结果证实:所有重组表达载体转化后均能成功检测到嵌合抗体cHAb18的表达,所有的载体均在转染48h后表达量达到较高水平,120 h后表达量达到最高;其中双载体pIRES1-18L/pIRES2-18H和pDHL-18FRT的量约达到1.4 mg/L。进一步选用三种不同的载体pIRES1-18L/pIRES2-18H,pDHL-18FRT和18H-L-pCII进行稳定转染不同表型的CHO细胞。夹心ELISA结果显示上述不同的三种载体均较未转化的细胞有人IgG的表达,其中双载体系统pIRES1-18L/pIRES2-18H的表达量约介于0.3-16mg/L;载体pDHL-18FRT的表达量可达到10mg/L;但是载体18H-L-pCII的抗体表达量,在进行克隆化并加压筛选的情况下却没有太多的变化,表达量均少于1.5 mg/L。对pIRES1-18L/pIRES2-18H转染后的细胞株1F6,稳定传代培养30代,RT-PCR仍能检测到抗体基因的表达;进一步扩大培养,亲合层析柱从约500ml的培养上清中纯化,获得约了7.5mg的表达产物,产物的纯度较高。SDS-PAGE电泳和Western blot检测结果:目的蛋白分子量约为150kDa,还原后为两条带,分子量约为50kDa和25kDa,符合人IgG抗体的特征,且可以和羊抗人IgG结合。免疫荧光结果显示表达纯化的抗体可特异结合表达有HAb18G/CD147的细胞。结论:通过对载体的优化设计和改造,获得了能用于全人抗体表达的高效真核载体;其中以弱化多顺反子pIRES1-18L; pIRES2-18H以及定点重组载体18-pDHL-FRT表达量较高,均可进一步用于其他抗体的表达。成功的筛选了抗HAb18G/CD147的嵌合IgG抗体cHAb18的稳定细胞株,并在哺乳动物细胞中获得高效表达,表达产物保持了良好的特异性和亲合力。纯化了重组表达的目标全长IgG产物即人鼠嵌合抗体cHAb18,从而也进一步验证了所获得的表达载体是正确的且能用于表达全长人IgG。第二部分研究内容:抗人大肠癌单抗CAb1轻、重链基因克隆与鉴定目的:获得单抗CAb1的轻、重链可变区基因,及其嵌合Fd或嵌合轻链基因,制备相应的小分子抗体cFab对所获得的基因进行鉴定。方法:首先,从杂交瘤细胞CAb1提取总RNA,设计并选用合成引物,通过反转录PCR扩增单抗CAb1 Fd和轻链全长基因,连接人T载体后进行序列测定和分析;然后用同源比较对单抗CAb1完整Fd的扩增,获得含有全长的Fd和轻链基因的载体pMD18-T/Fd和pMD18-T/L;分别以pMD18-T/Fd和pMD18-T/L为模版,用对应的引物B4、B4for和A8、A8for扩增MAbCAb1重、轻链可变区基因VH和VL,分别插入含有人CH1和CL的表达载体pComb3C后获得载体pComb3C/cFab;以载体pComb3C/cFab为模板,再次采用相应的引物扩增嵌合Fd或嵌合轻链基因,用相应的限制性内切酶分别消化质粒pET32a(+)及纯化的PCR产物,经过连接转化鉴定获得载体pET-CAbH和pET-CAbL。诱导表达含有重组表达质粒的菌株,分别表达嵌合cFd和cL并进行纯化。将其分别溶解后,按绝对量等摩尔量比混合,用梯度透析的方法进行复性。SDS-PAGE和Westernblot对嵌合Fab进行检测,观察产物的复性情况;用ProteinG的柱子纯化产物后,选用间接ELISA,免疫荧光染色,流式细胞术检测复性产物的靶抗原结合活性,最后采用竞争性ELISA检测该小分子抗体和鼠源单抗CAb1是否竞争同一个抗原表位。结果:从1×107的杂交瘤细胞CAb-1细胞中获得总RNA量约62.5μg。甲醛变性电泳显示总RNA比较完整,能够准确清晰的观察到5S,18S和28S的锐利条带。获得mAbCAb1基因序列,VH基因长351bp,编码117个氨基酸;VL基因长336bp,编码112个氨基酸;CAb-1CH1属于IgG1,CL属于κ型。优化其mRNA的二级结构自由能,构建了非融合表达载体pET-CAbH,pET-CAbL,成功实现了抗体的嵌合Fd或嵌合轻链在大肠杆菌中的高效表达,蛋白表达量在30°C诱导6h后分别达到菌体总蛋白的23.6%和29.2%。通过体外复性,SDS-PAGE结果显示在起始总蛋白浓度100μg/ml时,复性的cFab的回收率高达70.2%。免疫荧光和FACS的结果显示复性的cFab能够比较特异的结合到大肠癌细胞系SW480和Hce-8693,但是却不和正常的细胞结合。竞争ELISA检测显示复性的cFab和亲本CAb-1抗体片段F(ab’)2互相竞争相同表位。结论:获得了杂交瘤细胞CAb1的轻重链可变区基因,所设计的引物对鼠IgG1类抗体基因的扩增是有效的。表达并制备了嵌合cFd和cL,优化mRNA的二级结构自由能对蛋白的非融合表达具有重要作用。实现体外复性制备小分子cFab,该分子能特异的结合大肠癌细胞,所获得的分子具有结合活性;复性的cFab能和亲本的CAb-1相互竞争,提示它们识别的抗原表位一致;由于制备的cFab抗原有结合活性,进一步提示所获得杂交瘤细胞CAb1基因是正确和可靠的,同时也印证了该方案对鉴定所克隆的CAb1可变区基因的有效性。第三部分研究内容:抗人大肠癌表面重塑抗体rCAb1表达和纯化目的:用构建的真核表达载体制备抗人大肠癌表面重塑抗体rCAb1,对表达的抗体进行初步纯化和鉴定方法:首先通过对大肠癌单抗CAb1的抗体可变区序列分析,并同Genbank的nr库做BlastP,从中抽取所有抗体可变区氨基酸序列信息构建本地抗体结构数据库。进而将抗体种属来源结合轻、重链类型将抗体序列分为四类,其中抗体序列来源分别选择Homo sapiens和Mus musculus两类。对相似性得分最高的人源、鼠源抗体可变区序列各200条进行统计,获得单抗CAb1的差异残基和异常残基;之后通过模建抗体可变区的精确三维模型,获得CAb1的VH、VL分子内和分子间氢键相互作用,最终确定进行人源化改造的候选突变位点;依据确定的候选位点,对突变后的抗体人源化可变区序列用重叠PCR的方法合成,分别与T载体(pMD18-T)连接构建成克隆载体pMD18-T/VH和pMD18-T/VL进行序列测定。利用上述的可变区序列,分别构建轻链表达载体pIRES1-CAbL和重链表达载体的pIRES2-CAbH(弱化多顺反子系统),用PCR和相应的酶切鉴定。将获得的双载体转染COS-7细胞进行瞬时表达,夹心ELISA法检测抗体的表达情况。最后,将这两个载体共转CHO-dhfr-细胞,夹心ELISA法检测产物的表达情况;用有限稀释法进行阳性克隆的筛选,扩大培养后进行抗体表达产物的纯化,纯化样品进行SDS-PAGE凝胶电泳分析和Western blot检测。细胞免疫荧光染色以及流式细胞检测观察纯化产物对大肠癌细胞的结合活性。最后选用竞争性ELISA检测该rCAb1是否与亲本鼠CAb1竞争同一表位,同时根据50%抑制率时的人源抗体与亲本鼠抗体浓度的比值,估计该人源化IgG的相对亲和力。结果:按照Kabat、Abm、Chothia和Contact的规则标出了大肠癌单抗CAb1的不同的CDR,SDR区;经过筛选的抗体序列经过分析,获得了已有抗体分子不同位点上氨基酸种类和百分比,将该结果和单抗CAb1的可变区氨基酸比对后,获得了该序列的差异残基和异常残基;结合三维重建模型,获得了CAb1 VH、VL分子内和分子间氢键相互作用和氨基酸表面可及性,并最终确定了初步进行人源化改造的候选突变位点即重链的T018S,N086S和轻链的Q018P。重叠PCR合成突变的基因后,分别与T载体连接,获得了克隆载体pMD18-T/VH和pMD18-T/VL,测序结果显示可变区基因成功合成。构建的轻链表达载体pIRES1-CAbL和重链表达载体的pIRES2-CAbH经转染COS-7细胞后,夹心ELISA证明存在人IgG的表达;将这两个载体共转CHO-dhfr-细胞,经过克隆筛选,表达产物的纯化,SDS-PAGE电泳和Western blot检测结果表明:在非还原状态下,目标蛋白的分子量约为150 kDa;还原后为两条带,分子量分别约为52kDa和27kDa。该产物可特异的和大肠癌细胞结合。竞争性ELISA检测该产物即rCAb1可与亲本鼠CAb1竞争同一表位,且亲和力约为亲本鼠抗体的55%。结论:通过对大肠癌单抗CAb1的抗体可变区序列分析,获得了对大肠癌单抗CAb1进行表面重塑改造的候选突变位点;成功的用Over-lapping PCR的方法合成了突变的CAb1轻、重链可变区基因;用弱化筛选标记的双载体pIRES1-CAbL和pIRES2-CAbH制备了表面重塑抗体rCAb1;所制备的抗体能特异的结合大肠癌细胞;保持了和亲本抗体相似的结合活性和特异性。更多还原

【Abstract】 In this experiment, a series of eukaryotic expression vectors were constructed andscreened for the expression of full-length humanized antibody using the well-establishedantigen-antibodysystemHAb18G/CD147-HAb18;Then, thevariableregiongenesVHandVL of mAb CAb1 against human colorectal cancer were cloned, and their reliability andaccuracy were validated through reconstitution of a human-mouse chimeric Fab of CAb-1;Furthermore, the mutated variable regions of the surface reshaping antibody rCAb1 weredesigned and obtained; Finally, the recombinant rCAb1 were expressed and purified inmammaliancellsbytheconstructedeukaryoticexpressionvector.Experimentalstudyonthecontentsabovecanbedividedintothreeparts:Part I: Constructing and screening high efficient eukaryotic vectors with our ownintellectualpropertyfortheexpressionoffull-lengthhumanantibodyObjective:ToobtaineukaryoticexpressionvectorsforantibodyexpressionMethods: According to standard recombinant DNA operations, the followingeukaryotic vectors were constructed: 1Weakened screening system using“crippled”DHFR(pIRES1-18L; pIRES2-18H); 2 Site integration system (pDHL-18FRT); 3 Binary vectorsystem (18H-pDHA , 18L-pCI); 4 Reversed binary vector system (18H-PCI, 18L-p105); 5Multicistron expression vector (18H-L-pCI-FRT); 6 Weakened multicistron expressionvector(18H-L-pCII);and 7TraditionalpAH/pAGsystem(pAH4604-18, pAG4622-18).Allthe vectors contain the variable region genes of mAb HAb18 and the constant regions ofhuman IgG1γ1 andκchain. After transfected them into COS-7 cells respectively, wedetected the chimeric antibody cHAb18 in the culture supernatant by dot blot analysis. Atthe same time, we measured the expression levels of each system by sandwich ELISA.Subsequently, some of the constructs were transfected into different phenotypes of CHOcellsbyelectroporation andpositive cloneswere selected with limited dilution method.The antibody genes in continuous daughter cells were assessed by RT-PCR. For the scale-upculture, the products in the supernatant were purified. SDS-PAGE and Western blot wereemployed for the detection of antibody expression; And finally immunofluorescencestaining and flow cytometry were also used for the specificity analysis of the expressionproducts.Results: All the vectors containing antibody genes were constructed. PCR or enzymerestriction analysis both showed the corresponding segments as expected size, whichsuggested that all the constructs were successfully obtained. After transfected into COS-7cells, dot-blot hybridization showed that almost all transfectants could detect the expressedhuman IgG compared with those of non-transfectants, except for vectors 18 H-L-pCII anddual-vector pAH/pAG. However, when using the sandwich ELISA coated with theextracellular domain of the HAb18G/CD147, the results showed that all transfectants coulddetect the expressed chimeric antibody cHAb18, and the expression level reached to ahigher level at 48 h and the maximum expression level at 120 h after transfection. Theproductivity of transfectants with plasmids pIRES1-18L/pIRES2-18H was about 1.4milligram per liter. Further, we used pIRES1-18L/pIRES2-18H,pDHL-18FRT and18H-L-pCII to transfect different phenotypes of CHO cells. The Sandwich ELISA showedthat all transfectants could detect the expressed chimeric antibody cHAb18 compared withthose of non-transfectants. Among all the transfectants with plasmidspIRES1-18L/pIRES2-18H, the productivity varies from 0.3 to 16 milligram per liter; andtransfectantswith plasmids pDHL-18FRTwith pOG44 could reachto 10 milligramperliter;However,thetransfectantswithplasmid18H-L-pCIIwasratherlowerevenunderscreeningpressure and productivity was less than 1.5 milligram per liter. RT-PCR could detectantibody gene expression to the stable transfectants 1F6 with 30 continuous passages. Inlarge scale culture, we obtained 7.5 mg of expression products from about 500 ml culturesupernatant through affinity chromatographycolumn. SDS-PAGE andWestern blot showedthe purified products were approximately 150 kDa under nonreducing condition, and itbecame two newbandsaftertreatmentwith reduction reagent, with the molecularweightof50 kDa and 25 kDa, respectively. And the interested protein could bind with goatanti-human IgG, this indicated that the molecular was human IgG. Immunofluorescence showedthepurifiedIgGcouldbindwithcellsbearingHAb18G/CD147specifically.Conclusion: Through designing and reconstruction, a series of eukaryotic expressionvector for full-length human antibody expression were constructed. Among these vectors,weakened screening system (pIRES1-18L; pIRES2-18H) and site integration system(pDHL-18FRT) showed a high efficiency expression. And they could be further used forother antibody expression. What’s more, a high expression levels and stable cell linesexpressed cHAb18 was obtained, and the expression products maintained good specificityand affinity with that of parental antibody. Finally, recombinant IgG product cHAb18 waspurified, and this further corroborated the correctness of the constructed vectors and theirfitnessfortheexpressionoffull-lengthhumanIgG.Part II: Cloning and identification of the light and heavy chain gene of mAb CAb1againsthumancolorectalcancerObjective: To obtain the variable region genes and mouse-human chimeric Fd orchimeric light chain genes of mAb CAb1, then prepare the corresponding small moleculeantibodycFabMethods: First ofall, we extracted totalRNAfromhybridoma cellCAb-1. The Fd andlight chain genes were amplified by RT-PCR using the synthesized primers. The amplifiedproducts were inserted into T vector to obtain pMD18-T/Fd and pMD18-T/L for analysisandsequencing.ThechimericcFdorchimericlightchainwasobtainedbyPCRfromvectorpComb3C/cFab, in which the amplified VH and VL from pMD18-T/Fd and pMD18-T/Lwith the corresponding primers B4, B4 for and A8, A8, were ligated with human CH1 andCL in pComb3C, respectively. cFd and cL of CAb-1 were then amplified by usingpComb3C/cFd-cLastemplateand joined with pET32a(+)through Nde I/Sal I(cFd)orNdeI/Xho I (cL) digestion, respectively. The resultant constructs pET-CAbH and pET-CAbLwere transformed into E.coli. BL21-DE3 and checked for the presence of the genes bycolony PCR and restriction digestion. Then cFd and cL were expressed and purified,respectively. After cFd and cL proteins were mixed at the equal molar concentration, weused a modified stepwise dialysis in vitro refolding system to obtain cFab. SDS-PAGE andWestern blotwere employed for the detection of the refolding products; After purified withProtein G column, we further used indirect ELISA, immunofluorescence staining and flow cytometry to detect the binding activity of the products. Finally, competition ELISA wasusedtodeterminewhethertheobtainedcFabcouldcompetewiththemurineCAb1.Results: About 62.5μg total RNA was obtained from 1×107 hybridoma cells, andclearly 5S, 18S and 28S of bands can be observed by RNAelectrophoresis. The amplifiedVH and VLgenes of CAb-1 have 351 bp (117 amino acids) and 336 bp (112 amino acids),respectively.AndtheCH1ofCAb-1 belongs to mouse IgG1, and the CL belongs toκchain. By optimizing their free energy of mRNAsecondary structures, two non-fusion expressionvector pET-CAbH and pET-CAbL were successfully constructed. The cFd and cL can beexpressed efficiently in E.coli with expression 29.2% and 23.6% of total bacteria proteinsafter6 h inductionat30°C, respectively. Byreconstitution in vitro, SDS-PAGEshowed thatcFd and cLwere refolded into cFab with 70.2%ofprotein recoveryrate at100μg/ml initialtotal proteins. Immunofluorescence and FACS results showed that the refolded cFab couldbind with colon cancer cells SW480 and Hce-8693, but not normal cells. Finally,competitive ELISAshowed the purified cFab can compete with murine CAb-1 F(ab’)2, andviceversa.Conclusions: The variable region genes of CAb1 were cloned successfully usingdesigned primers of mouse IgG1 antibody. The cFd and cL of CAb1 were expressed andprepared through optimization of the free energy of the mRNA secondary structure. Weobtained functional refolded cFab successfully in vitro, and the products could bind withcolon cancer cells specifically. Refolded cFab could compete with murine CAb-1, whichindicated both of them identifying the same epitope; Finally, The functional cFab alsoindicatedthatthevariableregiongenesofCAb-1arecorrectandreliable,andthestrategyisalsoeffectiveincheckingtheobtainedvariablegenes.Part III: The expression and purification of the resurfacing antibody rCAb1 againsthumancolorectalcancerObjective:To obtain resurfacing antibodyrCAb1againsthuman colorectalcancerwiththe screened vectors, and then preliminary verify the binding activity of the expressedantibodyMethods: First, the variable region genes of CAb1 were analyzed and blastP withsequences in non-redundant human immunoglobulin VL and VH sequences of Genbank, local antibody structure databases was established. All the chosen sequences were dividedinto four categories, which were selected from two species (Mus musculus and Homosapiens)sinceeachcontaininglightandheavychaintypes.Weselected200sequencesformeach category for analysis patterns of surface exposed residues that most closely matchedthe patterns found on VL and VH of CAb1 according to the scores of similarity. After wegot the differential and abnormal residues of the variable region sequences, thethree-dimensional of Fv structure of CAb1 was modeled and the intermolecular hydrogenbonding was calculated. According to the results, the candidate sites for mutation weredetermined. After that, the variable region sequences were obtained by overlapping PCR.Then, theywere inserted into Tvector respectively to get cloning vector pMD18-T/VH andpMD18-T/VL for sequencing. After that, the VH and VL were cloned into the weakenedscreeningsystemtogetexpressionvectorpIRES1-CAbLandpIRES2-CAbH.Afteranalysisby PCR and restriction enzyme digestion, they were transfected into COS-7 cells; theculture supernatantwasdetected bysandwich ELISA. Forthe scale-up culture,the productsin the supernatant were purified. SDS-PAGE and Western blot were employed for thedetection of antibody expression; And also, immunofluorescence staining and flowcytometry were used for the specificity analysis of the expression products to colon cancercells. Finally,competitionELISAwasemployedforthebindingactivitywithmurine CAb1,and the relative affinity was calculated in accordance with the antibody concentration ratiowheninhibitionratewas50%.Results: The different CDR and SDR of CAb1 sequences was marked according toKabat, Abm, Chothia and Contact rules, after alignment and screened antibody sequences,the surface exposed positions in a set of heavy and light chain variable region frameworkwere determined and the mostcloselyidentical to the set ofMusmusculusorhomo sapienssurface residues wasalso labeled.We finallydetermined the initial candidate mutation siteswere T018S, N086S and Q018P according to the intermolecular hydrogen bondinginteractions and amino acids surface accessibility (A residue was defined as beingaccessible when its relative accessibility was greater than 30%). Using overlapping PCR,the mutated variable regions of VH and VLwere obtained and cloned into pMD18-T. AftertheconstructiontheexpressionvectorpIRES1-CAbLandpIRES2-CAbH,SandwichELISA results showed the transfectants with the two plasmids expressed human IgG. By selectingwith MTX, the expression product was finally purified. SDS-PAGE and Western blotshowed that the protein molecular weight of about 150 kDa, after reduction for the twobands, the molecular weight of about 52 kDa and 27 kDa. The products could bind withcolon cancer cells specifically. Competitive ELISA showed the purified rCAb1 couldcompete with murine CAb1, the relative affinity was about 55% of murine parentalantibody.Conclusion: The candidate mutation sites of the CAb1 for surface remodeling wereobtained by analysis the variable region sequences of CAb1 against human colorectalcancer; the mutation variable region genes of CAb1 were obtained successfully usingover-lapping PCR. Dual-vector pIRES1-CAbL and pIRES2-CAbH were constructed, andthey couldbe used for the expression of resurfacing antibody rCAb1. The prepared rCAb1could bind with colon cancer cells specifically. Compared with that of the parental murineantibody CAb1, the expressed rCAb1 maintains desirable binding activity and specifity tocoloncancercells.更多还原