全人抗体高效真核表达载体的构建与表面重塑抗体rCAb1
详细信息    本馆镜像全文|  推荐本文 |  |   获取CNKI官网全文
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。
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-lengthhumanantibody
     Objective:Toobtaineukaryoticexpressionvectorsforantibodyexpression
     Methods: 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 CAb1againsthumancolorectalcancer
     Objective: To obtain the variable region genes and mouse-human chimeric Fd orchimeric light chain genes of mAb CAb1, then prepare the corresponding small moleculeantibodycFab
     Methods: 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 againsthumancolorectalcancer
     Objective:To obtain resurfacing antibodyrCAb1againsthuman colorectalcancerwiththe screened vectors, and then preliminary verify the binding activity of the expressedantibody
     Methods: 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.
引文
[1] Li M., Gu J. Changing patterns of colorectal cancer in China over a period of20 years[J]. World J Gastroenterol, 2005, 11(30): 4685-4688.
    [2] Wilkes G.M. Therapeutic options in the management of colon cancer: 2005update[J].Clin J Oncol Nurs, 2005, 9(1): 31-44.
    [3] http://www.centerwatch.com/; http://www.fda.gov/.
    [4] Sioud M. Antibodies guide the way[J]. Gene Therapy, 2006, 13(3): 194-195<110>.
    [5] Li L., Xu H.Y., Mi L., et al. Radioimmunotherapy of human colon cancerxenografts by using (131)I labeled-CAb(1) F(ab')(2)[J]. Int J Radiat Oncol BiolPhys, 2006, 66(4): 1238-1244.
    [6] Bregenholt S., Jensen A., Lantto J., et al. Recombinant human polyclonalantibodies: A new class of therapeutic antibodies against viral infections[J]. CurrPharm Des, 2006, 12(16): 2007-2015.
    [7] Chapman A.P., Antoniw P., Spitali M., et al. Therapeutic antibody fragmentswith prolonged in vivo half-lives[J]. Nat Biotechnol, 1999, 17(8): 780-783.
    [8] Brekke O.H., Loset G.A. New technologies in therapeutic antibodydevelopment[J]. Curr Opin Pharmacol, 2003, 3(5): 544-550.
    [9] Baker M. Upping the ante on antibodies[J]. Nat Biotechnol, 2005, 23(9):1065-1072.
    [10]Adams G.P., Weiner L.M. Monoclonal antibody therapy of cancer[J]. NatBiotechnol, 2005, 23(9): 1147-1157.
    [11]Liu X.Y., Pop L.M., Vitetta E.S. Engineering therapeutic monoclonalantibodies[J]. Immunol Rev, 2008, 222: 9-27.
    [12]Almagro J.C., Fransson J. Humanization of antibodies[J]. Front Biosci, 2008,13: 1619-1633.
    [13]Humphreys D.P., Glover D.J. Therapeutic antibody production technologies:molecules, applications, expression and purification[J]. Curr Opin Drug DiscovDevel, 2001, 4(2): 172-185.
    [14]Jain M., Kamal N., Batra S.K. Engineering antibodies for clinicalapplications[J]. Trends Biotechnol, 2007, 25(7): 307-316.
    [15]Lonberg N. Human antibodies from transgenic animals[J]. Nat Biotechnol,2005, 23(9): 1117-1125.
    [16]Reichert J.M., Valge-Archer V.E. Development trends for monoclonalantibody cancer therapeutics[J].Nat Rev Drug Discov, 2007, 6(5): 349-356.
    [17]Reff M.E., Heard C. A review of modifications to recombinant antibodies:attempt to increase efficacy in oncology applications[J]. Crit Rev Oncol Hematol,2001, 40(1): 25-35.
    [18]Wu A.M., Senter P.D. Arming antibodies: prospects and challenges forimmunoconjugates[J]. Nat Biotechnol, 2005, 23(9): 1137-1146.
    [19]Carson W.E., 3rd, Liang M.I. Current immunotherapeutic strategies in breastcancer[J]. Surg Oncol Clin N Am, 2007, 16(4): 841-860, ix.
    [20]Nissim A., Chernajovsky Y. Historical development of monoclonal antibodytherapeutics[J]. Handb Exp Pharmacol, 2008, (181): 3-18.
    [21]Creput C., Durrbach A., Deroure B., et al. New therapeutic targets forantibodies and recombinant proteins in organ transplantation[J]. Curr Opin MolTher, 2007, 9(2): 153-159.
    [22]Sheridan C. Pharma consolidates its grip on post-antibody landscape[J]. NatBiotechnol, 2007, 25(4): 365-366.
    [23]McCormack P.L., Keam S.J. Bevacizumab: a review of its use in metastaticcolorectal cancer[J]. Drugs, 2008, 68(4): 487-506.
    [24]Jonker D.J., O'Callaghan C.J., Karapetis C.S., et al. Cetuximab for theTreatment of Colorectal Cancer[J]. N Engl J Med, 2007, 357(20): 2040-2048.
    [25]Secord A.A., Blessing J.A., Armstrong D.K., et al. Phase II trial of cetuximaband carboplatin in relapsed platinum-sensitive ovarian cancer and evaluation ofepidermal growth factor receptor expression: a Gynecologic Oncology Groupstudy[J]. Gynecol Oncol, 2008, 108(3): 493-499.
    [26]Wu M., Rivkin A., Pham T. Panitumumab: Human monoclonal antibodyagainst epidermal growth factor receptors for the treatment of metastatic colorectalcancer[J]. Clin Ther, 2008, 30(1): 14-30.
    [27]Hoogenboom H.R. Selecting and screening recombinant antibody libraries[J].Nat Biotechnol, 2005, 23(9): 1105-1116.
    [28]Wurm F., Bernard A. Large-scale transient expression in mammalian cells forrecombinant protein production[J]. Curr Opin Biotechnol, 1999, 10(2): 156-159.
    [29]Birch J.R., Racher A.J. Antibody production[J]. Adv Drug Deliv Rev, 2006,58(5-6): 671-685.
    [30]King in the kingdom of uncertainty[J]. Nat Biotechnol, 2005, 23(9): 1025.
    [31]Ma J.K., Drake P.M., Chargelegue D., et al. Antibody processing andengineering in plants, and new strategies for vaccine production[J]. Vaccine, 2005,23(15): 1814-1818.
    [32]Walls M.A., Hsiao K.C., Harris L.J. Vectors for the expression ofPCR-amplified immunoglobulin variable domains with human constant regions[J].Nucleic Acids Res, 1993, 21(12): 2921-2929.
    [33]Colosimo A., Goncz K.K., Holmes A.R., et al. Transfer and expression offoreign genes in mammalian cells[J]. Biotechniques, 2000, 29(2): 314-318,320-312, 324 passim.
    [34]Hardy R.R., Hayakawa K. B cell development pathways[J]. Annu Rev Immunol,2001, 19: 595-621.
    [35]Schmitz F., Heit A. Protective cancer immunotherapy: what can the innateimmune system contribute?[J]. Expert Opin Biol Ther, 2008, 8(1): 31-43.
    [36]McLean G.R., Nakouzi A., Casadevall A., et al. Human and murineimmunoglobulin expression vector cassettes[J]. Mol Immunol, 2000, 37(14):837-845.
    [37]Sun Z., Zhou R., Liang S., et al. Hyperosmotic stress in murine hybridomacells: effects on antibody transcription, translation, posttranslational processing,and the cell cycle[J]. Biotechnol Prog, 2004, 20(2): 576-589.
    [38]Makrides S.C. Components of vectors for gene transfer and expression inmammalian cells[J]. Protein Expr Purif, 1999, 17(2): 183-202.
    [39]Fang J., Qian J.J., Yi S., et al. Stable antibody expression at therapeutic levelsusing the 2A peptide[J]. Nat Biotechnol, 2005, 23(5): 584-590.
    [40]Li J., Menzel C., Meier D., et al. A comparative study of different vectordesigns for the mammalian expression of recombinant IgG antibodies[J]. JImmunol Methods, 2007, 318(1-2): 113-124.
    [41]Eissenberg J.C., Elgin S.C. Boundary functions in the control of geneexpression[J]. Trends Genet, 1991, 7(10): 335-340.
    [42]Hinrich Boeger D.A.B., Ralph Davis, Joachim Griesenbeck, Yahli Lorch.Structural basisof eukaryotic gene transcription[J]. FEBS, 2005, 579: 899-903.
    [43]朱迎春, 王琰. 不同启动子驱动免疫球蛋白在CHO细胞中表达活性的比较[J].16, 2000, 4: 356-359.
    [44]来大志, 翁少洁, 于长明, et al. 应用人延伸因子 1α亚基启动子和人工转录激活因子提高外源基因在 Cho 细胞中的表达[J]. 生物化学与生物物理进展,2004, (02): 118-127.
    [45]Pacchia A.L., Adelson M.E., Kaul M., et al. An inducible packaging cellsystem for safe, efficient lentiviral vector production in the absence of HIV-1accessory proteins[J]. Virology, 2001, 282(1): 77-86.
    [46]Gillies S.D., Lo K.M., Wesolowski J. High-level expression of chimericantibodies using adapted cDNA variable region cassettes[J]. J Immunol Methods,1989, 125(1-2): 191-202.
    [47]张可伟 王.郑. 核基质结合序列(Mar)与基因表达调控[J]. 生物工程学报,2004, (01).
    [48]Thoger Andersen A.S., Jensen A.W., Grant P., et al. Concomitantdownregulation of IgH 3' enhancer activity and c-myc expression in aplasmacytoma x fibroblast environment: implications for dysregulation oftranslocated c-myc[J]. Mol Immunol, 1997, 34(2): 97-107.
    [49]Pan Q., Petit-Frere C., Stavnezer J., et al. Regulation of the promoter forhuman immunoglobulin gamma3 germ-line transcription and its interaction withthe 3'alpha enhancer[J]. Eur J Immunol, 2000, 30(4): 1019-1029.
    [50]Kozak M. Some thoughts about translational regulation: forward andbackward glances[J]. J Cell Biochem, 2007, 102(2): 280-290.
    [51]Wakiyama M., Hirao I., Kumagai I., et al. Effect of tandem repeated AUGcodons on translation efficiency of eukaryotic mRNA carrying a short leadersequence[J]. Mol Gen Genet, 1993, 238(1-2): 59-64.
    [52]Vivinus S., Baulande S., van Zanten M., et al. An element within the 5'untranslated region of human Hsp70 mRNA which acts as a general enhancer ofmRNA translation[J]. Eur J Biochem, 2001, 268(7): 1908-1917.
    [53]Mundt C.A., Nicholson I.C., Zou X., et al. Novel control motif cluster in theIgH delta-gamma 3 interval exhibits B cell-specific enhancer function in earlydevelopment[J]. J Immunol, 2001, 166(5): 3315-3323.
    [54]Levine C.G., Mitra D., Sharma A., et al. The efficiency of proteincompartmentalization into the secretory pathway[J]. Mol Biol Cell, 2005, 16(1):279-291.
    [55]阮承迈. 抗 HBsAg 抗体在 CHO 细胞中表达量提高研究[J]. 中国博士学位论文全文数据库, 2002: 1-60.
    [56]Skerra A., Pluckthun A. Secretion and in vivo folding of the Fab fragment ofthe antibody McPC603 in Escherichia coli: influence of disulphides andcis-prolines[J]. Protein Eng, 1991, 4(8): 971-979.
    [57]Wiersma E.J., Ronai D., Berru M., et al. Role of the intronic elements in theendogenous immunoglobulin heavy chain locus. Either the matrix attachmentregions or the core enhancer is sufficient to maintain expression[J]. J Biol Chem,1999, 274(8): 4858-4862.
    [58]Fouser L.A., Swanberg S.L., Lin B.Y., et al. High level expression on achimeric anti-ganglioside GD2 antibody: genomic kappa sequences improveexpression in COS and CHO cells[J]. Biotechnology (N Y), 1992, 10(10):1121-1127.
    [59]吕祁峰, 夏家辉. S/Mar 与基因表达[J]. 生命科学研究, 2000, (01).
    [60]de Poorter J.J., Lipinski K.S., Nelissen R.G., et al. Optimization of short-termtransgene expression by sodium butyrate and ubiquitous chromatin openingelements (UCOEs)[J]. J Gene Med, 2007, 9(8): 639-648.
    [61]Fujimoto T., Yoshimatsu K., Watanabe K., et al. Overexpression of humanX-box binding protein 1 (XBP-1) in colorectal adenomas and adenocarcinomas[J].Anticancer Res, 2007, 27(1A): 127-131.
    [62]Leitzgen K., Knittler M.R., Haas I.G. Assembly of Immunoglobulin LightChains as a Prerequisite for Secretion [J]. J Biol Chem, 1997, 272(31): 3117-3123.
    [63]Schubart K., Massa S., Schubart D., et al. B cell development andimmunoglobulin gene transcription in the absence of Oct-2 and OBF-1[J]. NatImmunol, 2001, 2(1): 69-74.
    [64]Schweitzer B.L., Huang K.J., Kamath M.B., et al. Spi-C has opposing effectsto PU.1 on gene expression in progenitor B cells[J]. J Immunol, 2006, 177(4):2195-2207.
    [65]Uematsu S., Kaisho T., Tanaka T., et al. The C/EBP beta isoform 34-kDa LAPis responsible for NF-IL-6-mediated gene induction in activated macrophages, butis not essential for intracellular bacteria killing[J]. J Immunol, 2007, 179(8):5378-5386.
    [66]Greenbaum S., Zhuang Y. Regulation of early lymphocyte development byE2A family proteins[J]. Semin Immunol, 2002, 14(6): 405-414.
    [67]Salas M., Eckhardt L.A. Critical role for the Oct-2/OCA-B partnership inIg-secreting cells[J]. J Immunol, 2003, 171(12): 6589-6598.
    [68]Schlatter S., Stansfield S.H., Dinnis D.M., et al. On the optimal ratio of heavyto light chain genes for efficient recombinant antibody production by CHO cells[J].Biotechnol Prog, 2005, 21(1): 122-133.
    [69]Crowley C.W. Method for selecting high-expressing host cells (P) UnitedStateshttp://www.freepatentsonline.com/5561053.html
    [70]Dirks W., Wirth M., Hauser H., et al. Multicistronic expression units and theiruse (P) United States
    [71]Martínez-Salas E. Internal ribosome entry site biology and its use inexpression vectors[J]. Current Opinion in Biotechnology, 1999, 10: 458-464.
    [72]Huang Y., Li Y., Wang Y.G., et al. An efficient and targeted gene integrationsystem for high-level antibody expression[J]. J Immunol Methods, 2007, 322(1-2):28-39.
    [73]Koduri R.K., Miller J.T., Thammana P. An efficient homologousrecombination vector pTV(I) contains a hot spot for increased recombinant proteinexpression in Chinese hamster ovary cells[J]. Gene, 2001, 280(1-2): 87-95.
    [74]Kobayashi N., Noguchi H., Westerman K.A., et al. Efficient Cre/loxPsite-specific recombination in a HepG2 human liver cell line[J]. Cell Transplant,2000, 9(5): 737-742.
    [75]Kingston R.E., Kaufman R.J., Bebbington C.R., et al. Amplification usingCHO cell expression vectors[J]. Curr Protoc Mol Biol, 2002, Chapter 16: Unit 1623.
    [76]Bianchi A.A., McGrew J.T. High-level expression of full-length antibodiesusing trans-complementing expression vectors[J]. Biotechnol Bioeng, 2003, 84(4):439-444.
    [77]Werner R.G., Noe W., Kopp K., et al. Appropriate mammalian expressionsystems for biopharmaceuticals[J]. Arzneimittelforschung, 1998, 48(8): 870-880.
    [78]Yenofsky R.L., Fine M., Pellow J.W. A mutant neomycin phosphotransferaseII gene reduces the resistance of transformants to antibiotic selection pressure[J].Proc Natl Acad Sci U S A, 1990, 87(9): 3435-3439.
    [79]高川, 朱旭东, 周晓巍, et al. 以新霉素抗性基因突变体为筛选标志的真核表达载体的构建[J]. 生物工程学报, 2002, 18(03): 308-313.
    [80]Houdebine L.M., Attal J. Internal ribosome entry sites (IRESs): reality anduse[J]. Transgenic Res, 1999, 8(3): 157-177.
    [81]Phi-Van L., von Kries J.P., Ostertag W., et al. The chicken lysozyme 5' matrixattachment region increases transcription from a heterologous promoter inheterologous cells and dampens position effects on the expression of transfectedgenes[J]. Mol Cell Biol, 1990, 10(5): 2302-2307.
    [82]Aldrich T.L., Viaje A., Morris A.E. EASE vectors for rapid stable expressionof recombinant antibodies[J]. Biotechnol Prog, 2003, 19(5): 1433-1438.
    [83]Hwang W.Y., Foote J. Immunogenicity of engineered antibodies[J]. Methods,2005, 36(1): 3-10.
    [84]林芸, 阎锡蕴. 人源化抗体研究历程及发展趋势[J]. 生物工程学报, 2004,20(1): 1-4.
    [85]Lai C.J., Goncalvez A.P., Men R., et al. Epitope determinants of a chimpanzeedengue virus type 4 (DENV-4)-neutralizing antibody and protection againstDENV-4 challenge in mice and rhesus monkeys by passively transferredhumanized antibody[J]. J Virol, 2007, 81(23): 12766-12774.
    [86]Kohler G., Milstein C. Continuous cultures of fused cells secreting antibody ofpredefined specificity. 1975[J]. J Immunol, 2005, 174(5): 2453-2455.
    [87]Lonberg N., Taylor L.D., Harding F.A., et al. Antigen-specific humanantibodies from mice comprising four distinct genetic modifications[J]. Nature,1994, 368(6474): 856-859.
    [88]Green L.L., Hardy M.C., Maynard-Currie C.E., et al. Antigen-specific humanmonoclonal antibodies from mice engineered with human Ig heavy and light chainYACs[J]. Nat Genet, 1994, 7(1): 13-21.
    [89]Booy E.P., Johar D., Maddika S., et al. Monoclonal and bispecific antibodiesas novel therapeutics[J]. Arch Immunol Ther Exp (Warsz), 2006, 54(2): 85-101.
    [90]Schellekens H. Immunogenicity of therapeutic proteins: clinical implicationsand future prospects[J].Clin Ther, 2002, 24(11): 1720-1740; discussion 1719.
    [91]Tanner J.E. Designing antibodies for oncology[J]. Cancer Metastasis Rev,2005, 24(4): 585-598.
    [92]Morea V., Lesk A.M., Tramontano A. Antibody modeling: implications forengineering and design[J]. Methods, 2000, 20(3): 267-279.
    [93]Damschroder M.M., Widjaja L., Gill P.S., et al. Framework shuffling ofantibodies to reduce immunogenicity and manipulate functional and biophysicalproperties[J]. Mol Immunol, 2007, 44(11): 3049-3060.
    [94]Lazar G.A., Desjarlais J.R., Jacinto J., et al. A molecular immunologyapproach to antibody humanization and functional optimization[J]. Mol Immunol,2007, 44(8): 1986-1998.
    [95]Reichert J.M., Rosensweig C.J., Faden L.B., et al. Monoclonal antibodysuccesses in the clinic[J]. Nat Biotechnol, 2005, 23(9): 1073-1078.
    [96]Gonzales N.R., Padlan E.A., De Pascalis R., et al. SDR grafting of a murineantibody using multiple human germline templates to minimize itsimmunogenicity[J]. Mol Immunol, 2004, 41(9): 863-872.
    [97]Jones P.T., Dear P.H., Foote J., et al. Replacing thecomplementarity-determining regions in a human antibody with those from amouse[J]. Nature, 1986, 321(6069): 522-525.
    [98]Riechmann L., Clark M., Waldmann H., et al. Reshaping human antibodies fortherapy[J]. Nature, 1988, 332(6162): 323-327.
    [99]Roguska M.A., Pedersen J.T., Henry A.H., et al. A comparison of two murinemonoclonal antibodies humanized by CDR-grafting and variable domainresurfacing[J]. Protein Eng, 1996, 9(10): 895-904.
    [100] Roguska M.A., Pedersen J.T., Keddy C.A., et al. Humanization of murinemonoclonal antibodies through variable domain resurfacing[J]. Proc Natl Acad SciU S A, 1994, 91(3): 969-973.
    [101] Fontayne A., Vanhoorelbeke K., Pareyn I., et al. Rational humanization ofthe powerful antithrombotic anti-GPIbalpha antibody: 6B4[J]. Thromb Haemost,2006, 96(5): 671-684.
    [102] Gora M., Gardas A., Watson P.F., et al. Key residues contributing todominant conformational autoantigenic epitopes on thyroid peroxidase identifiedby mutagenesis[J].Biochem Biophys Res Commun, 2004, 320(3): 795-801.
    [103] Clark L.A., Boriack-Sjodin P.A., Eldredge J., et al. Affinity enhancementof an in vivo matured therapeutic antibody using structure-based computationaldesign[J]. Protein Sci, 2006, 15(5): 949-960.
    [104] Staelens S., Hadders M.A., Vauterin S., et al. Paratope determination ofthe antithrombotic antibody 82D6A3 based on the crystal structure of its complexwith the von Willebrand factor A3-domain[J]. J Biol Chem, 2006, 281(4):2225-2231.
    [105] Schmiegel W., Schmielau J., Henne-Bruns D., et al. Cytokine-mediatedenhancement of epidermal growth factor receptor expression provides animmunological approach to the therapy of pancreatic cancer[J]. Proc Natl Acad SciU S A, 1997, 94(23): 12622-12626.
    [106] 梁瑞安, 杨蕾, 陈俊新, et al. 应用抗体“框架重塑”技术构建人源化抗体 fSM03[J]. 中国新药杂志, 2006, (21).
    [107] De Groot A.S., Knopp P.M., Martin W. De-immunization of therapeuticproteins by T-cell epitope modification[J]. Dev Biol (Basel), 2005, 122: 171-194.
    [108] Kipriyanov S.M., Le Gall F. Generation and production of engineeredantibodies[J]. Mol Biotechnol, 2004, 26(1): 39-60.
    [109] Dall'Acqua W.F., Damschroder M.M., Zhang J., et al. Antibodyhumanization by framework shuffling[J]. Methods, 2005, 36(1): 43-60.
    [110] Marks J.D., Griffiths A.D., Malmqvist M., et al. By-passingimmunization: building high affinity human antibodies by chain shuffling[J].Biotechnology (N Y), 1992, 10(7): 779-783.
    [111] Leung S.O. Reducing immunogenicities of immunoglobulins byframework-patching (P) Hongkong, 2002: 1-10.
    [112] Lv M., Li Y., Yu M., et al. Structured to reduce the mitogenicity ofanti-CD3 antibody based on computer-guided molecular design[J]. Int J BiochemCell Biol, 2007, 39(6): 1142-1155.
    [113] Tan P., Mitchell D.A., Buss T.N., et al. "Superhumanized" antibodies:reduction of immunogenic potential by complementarity-determining regiongrafting with human germline sequences: application to an anti-CD28[J]. JImmunol, 2002, 169(2): 1119-1125.
    [114] Hwang W.Y., Almagro J.C., Buss T.N., et al. Use of human germlinegenes in a CDR homology-based approach to antibody humanization[J]. Methods,2005, 36(1): 35-42.
    [115] Benhar I. Design of synthetic antibody libraries[J]. Expert Opin Biol Ther,2007, 7(5): 763-779.
    [116] Naziruddin B., Shiroki R., Shishido S., et al. Biochemical and functionalcharacterization of xenoreactive natural antibodies in hu-PBL-SCID mice[J]. JClin Invest, 1996, 97(5): 1267-1275.
    [117] Chua Y.J., Cunningham D. Panitumumab[J]. Drugs Today (Barc), 2006,42(11): 711-719.
    [118] Koenig R. Veterinary medicine. 'Camelized' antibodies make waves[J].Science, 2007, 318(5855): 1373.
    [119] Rothbauer U., Zolghadr K., Tillib S., et al. Targeting and tracing antigensin live cells with fluorescent nanobodies[J]. Nat Methods, 2006, 3(11): 887-889.
    [120] Lonberg N. Human monoclonal antibodies from transgenic mice[J].Handb Exp Pharmacol, 2008, (181): 69-97.
    [121] Eren R., Landstein D., Terkieltaub D., et al. Preclinical evaluation of twoneutralizing human monoclonal antibodies against hepatitis C virus (HCV): apotential treatment to prevent HCV reinfection in liver transplant patients[J]. JVirol, 2006, 80(6): 2654-2664.
    [122] Kellermann S.A., Green L.L. Antibody discovery: the use of transgenicmice to generate human monoclonal antibodies for therapeutics[J]. Curr OpinBiotechnol, 2002, 13(6): 593-597.
    [123] Xu J., Shen Z.Y., Chen X.G., et al. A randomized controlled trial ofLicartin for preventing hepatoma recurrence after liver transplantation[J].Hepatology, 2007, 45(2): 269-276.
    [124] Chen Z.N., Mi L., Xu J., et al. Targeting radioimmunotherapy ofhepatocellular carcinoma with iodine (131I) metuximab injection: clinical phaseI/II trials[J]. Int J Radiat Oncol Biol Phys, 2006, 65(2): 435-444.
    [125] Zhang S.H., Yang X.M., Xing J.L., et al. [Comparison of polyclonalanti-sera against extracellular domain of hepatoma associated antigenHAb18G/CD147 prepared by different immunization schemes][J]. Xi Bao Yu FenZi Mian Yi Xue Za Zhi, 2005, 21(1): 65-68.
    [126] Jiang J.L., Chan H.C., Zhou Q., et al. HAb18G/CD147-mediated calciummobilization and hepatoma metastasis require both C-terminal and N-terminaldomains[J].Cell Mol Life Sci, 2004, 61(16): 2083-2091.
    [127] Jin-Liang Xing X.-M.Y., Si-He Zhang, Xi-Ying Yao, Rui-An Liang,Zhi-Nan Chen. Construction of a universal expression vector for human-mousechimeric Fab antibody and expression of himeric Fab antibody against humanhepatoma associated antigen HAb18G[J]. World Chin J Digestol, 2004, 12(2):271-275.
    [128] Sambrook J., Fritsch E., Maniatis T. Molecular cloning [M ], 2nd ed[M].1989: 1-298.
    [129] Mittl P.R., Deillon C., Sargent D., et al. The retro-GCN4 leucine zippersequence forms a stable three-dimensional structure[J]. Proc Natl Acad Sci U S A,2000, 97(6): 2562-2566.
    [130] Slatko B.E., Albright L.M., Tabor S., et al. DNA sequencing by thedideoxy method[J]. Curr Protoc Mol Biol, 2001, Chapter 7: Unit7 4A.
    [131] Morrison S.L., Scharff M.D. Heavy chain-producing variants of a mousemyeloma cell line[J]. J Immunol, 1975, 114(2 Pt 1): 655-659.
    [132] Montano R.F., Morrison S.L. Influence of the isotype of the light chain onthe properties of IgG[J]. J Immunol, 2002, 168(1): 224-231.
    [133] 陈晓穗, 朱迎春, 王欲晓, et al. 抗 HBsAg人 IgG表达载体的构建及其在 CHO 细胞中的表达[J]. 中国免疫学杂志, 2000, 16(5): 248-251.
    [134] Zhang H., Zhang G.P., Zhang J.G., et al. [Secretable eukaryotic expressionof recombinant chicken Iglambda and preparation of monoclonal antibodiesagainst chicken Iglambda.][J]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2008, 24(3):260-262.
    [135] Horlick R.A., Schilling A.E., Samama P., et al. Combinatorial geneexpression using multiple episomal vectors[J]. Gene, 2000, 243(1-2): 187-194.
    [136] Kim S.J., Kim N.S., Ryu C.J., et al. Characterization of chimeric antibodyproducing CHO cells in the course of dihydrofolate reductase-mediated geneamplification and their stability in the absence of selective pressure[J]. BiotechnolBioeng, 1998, 58(1): 73-84.
    [137] Pelletier J.N., Campbell-Valois F.X., Michnick S.W. Oligomerizationdomain-directed reassembly of active dihydrofolate reductase from rationallydesigned fragments[J]. Proc Natl Acad Sci U S A, 1998, 95(21): 12141-12146.
    [138] M.Gorman M.T.F.H.a.C. Intervening sequences increase efficiency ofRNA 3' processing and accumulation of cytoplasmic RNA[J]. Nucleic AcidsResearch,, 1990, 18(4): 937-947.
    [139] Fang J., Yi S., Simmons A., et al. An antibody delivery system forregulated expression of therapeutic levels of monoclonal antibodies in vivo[J]. MolTher, 2007, 15(6): 1153-1159.
    [140] Toleikis L., Broders O., Dubel S. Cloning single-chain antibodyfragments (scFv) from hybridoma cells[J]. Methods Mol Med, 2004, 94: 447-458.
    [141] Wang Z., Raifu M., Howard M., et al. Universal PCR amplification ofmouse immunoglobulin gene variable regions: the design of degenerate primersand an assessment of the effect of DNApolymerase 3' to 5' exonuclease activity[J].J Immunol Methods, 2000, 233(1-2): 167-177.
    [142] Essono S., Frobert Y., Grassi J., et al. A general method allowing thedesign of oligonucleotide primers to amplify the variable regions fromimmunoglobulin cDNA[J]. J Immunol Methods, 2003, 279(1-2): 251-266.
    [143] Wang Y., Chen W., Li X., et al. Degenerated primer design to amplify theheavy chain variable region from immunoglobulin cDNA[J]. BMC Bioinformatics,2006, 7 Suppl 4: S9.
    [144] http://www.imgt.cines.fr:8104;http://www.ncbi.nlm.nih.gov/BLAST/.
    [145] Lefranc M.P. IMGT, the international ImMunoGeneTics informationsystem: a standardized approach for immunogenetics and immunoinformatics[J].Immunome Res, 2005, 1: 3.
    [146] Baum T.P., Hierle V., Pasqual N., et al. IMGT/GeneInfo: T cell receptorgamma TRG and delta TRD genes in database give access to all TR potentialV(D)J recombinations[J]. BMC Bioinformatics, 2006, 7: 224.
    [147] Jones L., Chalmers A.H., Dunlop D. A Fab' way to resolve diagnosticdilemmas[J]. Nuclear Medicine Communications, 2001, 22(3): 346 <392>.
    [148] Dattamajumdar A.K., Jacobson D.P., Hood L.E., et al. Rapid cloning ofany rearranged mouse immunoglobulin variable genes[J]. Immunogenetics, 1996,43(3): 141-151.
    [149] Leung S.O., Dion A.S., Pellegrini M.C., et al. An extended primer set forPCR amplification of murine kappa variable regions[J]. Biotechniques, 1993, 15(2):286-292.
    [150] Sodoyer R., Peubez I., Pion C., et al. Full-scale 'naive' human antibodyrepertoires assembled from VH and VL variable regions[J]. Hum Antibodies, 1997,8(1): 37-42.
    [151] 李竞, 王琰, 王卓智, et al. 抗胃癌鼠单抗 3H11 Fab 段载体的构建、表达及抗体活性检测[J]. 中华微生物学和免疫学杂志, 1999, 19(2): 158-162.
    [152] Ruberti F., Cattaneo A., Bradbury A. The use of the RACE method toclone hybridoma cDNA when V region primers fail[J]. J Immunol Methods, 1994,173(1): 33-39.
    [153] Stillman C.A., Linton P.J., Koutz P.J., et al. Specific immunoglobulincDNA clones produced from hybridoma cell lines and murine spleen fragmentcultures by 3SR amplification[J]. PCR Methods Appl, 1994, 3(6): 320-331.
    [154] Spira G., Yuan R., Paizi M., et al. Simultaneous expression of kappa andlambda light chains in a murine IgG3 anti-Cryptococcus neoformans hybridomacell line[J]. Hybridoma, 1994, 13(6): 531-535.
    [155] Cabilly S., Riggs A.D. Immunoglobulin transcripts and molecular historyof a hybridoma that produces antibody to carcinoembryonic antigen[J]. Gene, 1985,40(1): 157-161.
    [156] Burioni R., Plaisant P., Bugli F., et al. A vector for the expression ofrecombinant monoclonal Fab fragments in bacteria[J]. J Immunol Methods, 1998,217(1-2): 195-199.
    [157] Kozak M. Regulation of translation via mRNA structure in prokaryotesand eukaryotes[J]. Gene, 2005, 361: 13-37.
    [158] Forstner M., Leder L., Mayr L.M. Optimization of protein expressionsystems for modern drug discovery[J]. Expert Rev Proteomics, 2007, 4(1): 67-78.
    [159] Si-He Zhang J.-L.X., Xi-Ying Yao, Zhi-Nan Chen. Non-fused expressionof HAb18GEF by reducing stability of translational initiation region in mRNA[J].Sheng Wu Gong Cheng Xue Bao, 2004, 20: 175-180.
    [160] Lee M.H., Kwak J.W. Expression and functional reconstitution of arecombinant antibody (Fab') specific for human apolipoprotein B-100[J]. JBiotechnol, 2003, 101(2): 189-198.
    [161] Jeon Y.-E., Seo C.-W., Yu E.S., et al. Characterization of humanmonoclonal autoantibody Fab fragments against oxidized LDL[J]. MolecularImmunology, 2007, 44(5): 827-836.
    [162] Staelens S., Desmet J., Ngo T.H., et al. Humanization by variable domainresurfacing and grafting on a human IgG4, using a new approach for determinationof non-human like surface accessible framework residues based on homologymodelling of variable domains[J].Mol Immunol, 2006, 43(8): 1243-1257.
    [163] Al-Lazikani B., Lesk A.M., Chothia C. Standard conformations for thecanonical structures of immunoglobulins[J]. J Mol Biol, 1997, 273(4): 927-948.
    [164] Sohn J.H., Han K.L., Lee S.H., et al. Protective effects of panduratin Aagainst oxidative damage of tert-butylhydroperoxide in human HepG2 cells[J].Biol Pharm Bull, 2005, 28(6): 1083-1086.
    [165] Gonzales N.R., De Pascalis R., Schlom J., et al. Minimizing theimmunogenicity of antibodies for clinical application[J]. Tumour Biol, 2005, 26(1):31-43.
    [166] Haruyama H., Ito S., Miyadai K., et al. Humanization of the mouseanti-Fas antibody HFE7A and crystal structure of the humanized HFE7A Fabfragment[J]. Biol Pharm Bull, 2002, 25(12): 1537-1545.