全人抗体高效真核表达载体的构建与表面重塑抗体rCAb1

发布时间：2018-05-03 01:18

本文选题：大肠癌 + 嵌合抗体　；参考：《第四军医大学》2008年博士论文

【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。
[Abstract]:On the basis of the HAb18G/CD147- HAb18 antigen antibody system, the high effective eukaryotic vector used for the expression of human antibody was constructed and screened. After that, the anti colon cancer antibody CAb1 light, the heavy chain variable region gene VH and VL were cloned, and the reliability and accuracy of the genes obtained by the preparation of complex chimeric cFab were verified, and then the results were obtained. Based on the VH and VL of CAb1, the human derived surface remodeling antibody rCAb1 was designed. Finally, the expressed eukaryotic expression vector was selected to realize the expression of recombinant rCAb1 in mammalian cells.
The first part is about the construction and screening of eukaryotic expression vector of high effective human antibody.
Objective: to obtain a high efficient eukaryotic expression vector with independent property rights and full human antibody expression.
Methods: according to the standard DNA recombination operation, the following different eukaryotic expression vectors are constructed, including the weakening of the polycis pIRES1-18L, pIRES2-18H, the fixed-point recombinant vector 18-pDHL-FRT, the dual carrier system 18H-pDHA, 18L-pCI, the reverse dual carrier system 18H-PCI, 18L-p105, the polycis anti subexpression vector 18H-L-pCI-FRT, and the weakening screening marker carrier 18H-L-pCII; And pAH/pAG dual carrier pAH4604-18, pAG4622-18, these carriers all contain the light heavy chain gene of mAb HAb18 and the light, heavy chain constant region gene of human IgG1 kappa. After that, these vectors are transfected to COS-7 cells respectively, and dot blot hybridization is used to detect the culture of supernatant, and the expression of the inlay antibody cHAb18 is observed, and the sandwich ELISA method is used to detect the recombination resistance. Then, the CHO cells with different phenotypes were transfected steadily with electroporation, and the positive clones were screened with limited dilution to screen the positive clones, and the transfected cells were screened. RT-PCR was used to detect the stable cell lines, to observe whether the antibody genes were lost, and to evaluate the cell stability of the transfer carriers. The expression of the product was purified, the expression of the antibody was detected by SDS-PAGE gel electrophoresis and its Western blot, and the specificity of the expression products was detected by cell immunofluorescence staining and flow cytometry.
Results: the different eukaryotic vectors containing the antibody genes were constructed, and the inserted sequences were identified by the necessary PCR or enzyme digestion. All the vectors were successfully constructed. All the vectors were successfully constructed. All the transfected COS-7 cells were transiently transfected. The dot blot hybridization showed that the carrier 18H-L-pCII was compared with the Unconverted cells. The expression of the antibody was detected by the other carriers without detection of the expression of IgG, while the ELISA results of the sandwich HAb18G/CD147 in the extracellular domain of the antigen HAb18G/CD147 confirmed that all the recombinant expression vectors could successfully detect the expression of the chimeric antibody cHAb18 after the transformation of all the recombinant vectors, and all the carriers were expressed in high water after transfection of 48h. After 120 h, the expression reached the highest; the amount of pIRES1-18L/pIRES2-18H and pDHL-18FRT reached about 1.4 mg/L., and three different carriers, pIRES1-18L/pIRES2-18H, pDHL-18FRT and 18H-L-pCII were used to transfect the CHO cells with different phenotypes. The sandwich ELISA results showed that all the three different carriers were not transformed. The expression of IgG, the expression of pIRES1-18L/pIRES2-18H in the double carrier system is about 0.3-16mg/L, and the expression of carrier pDHL-18FRT can reach 10mg/L, but the expression of the antibody of carrier 18H-L-pCII is not much changed under the condition of cloning and pressure screening, and the expression amount is less than 1.5 mg/L. to pIRES1-18L/pIRE. The transfected cell line, 1F6, was successfully cultured for 30 generations, and the expression of the antibody gene was still detected by RT-PCR. Further expanded culture, the affinity chromatography column was purified from the culture supernatant of about 500ml, and the expression product of 7.5mg was obtained. The purity of the product was high.SDS-PAGE electrophoresis and Western blot detection results: the molecular weight of the target protein was about 150k Da, after reduction, is two bands, with a molecular weight of about 50kDa and 25kDa, which conforms to the characteristics of human IgG antibody and can be combined with the anti human IgG of sheep. The immunofluorescence results show that the purified antibody can specifically express HAb18G/CD147 cells.
Conclusion: by optimizing the design and modification of the carrier, a high effective eukaryotic vector can be obtained for the expression of the whole human antibody. The high expression of the weak polycis pIRES1-18L, pIRES2-18H and the fixed-point recombinant vector 18-pDHL-FRT can be further used for the expression of other antibodies. The anti HAb18G/CD147 chimeric IgG antibody is successfully screened. The stable cell line of cHAb18 is highly expressed in mammalian cells, and the expression product maintains good specificity and affinity. The full IgG product of the recombinant expression is purified, that is, human chimeric antibody cHAb18, which further verifies that the obtained expression vector is correct and can be used to express the full length human IgG..
The second part is about cloning and identification of light and heavy chain genes of monoclonal antibody CAb1 against human colorectal cancer.
Objective: to obtain the light and heavy chain variable region genes of the monoclonal antibody CAb1 and their chimeric Fd or chimeric light chain genes, and to prepare the corresponding small molecular antibody cFab to identify the obtained genes.
Methods: first, the total RNA was extracted from the hybridoma cell CAb1, and the synthetic primers were designed and selected. The monoclonal antibody CAb1 Fd and the light chain full length gene were amplified by reverse transcription PCR, and the T vector was sequenced and analyzed. Then the full length Fd and light chain gene carrier pMD18-T/Fd and pMD1 were obtained by the homologous comparison of the complete Fd of the monoclonal antibody CAb1. 8-T/L, pMD18-T/Fd and pMD18-T/L were used as templates to amplify MAbCAb1 weight, VH and VL with corresponding primers B4, B4for and A8, A8for in light chain variable region gene, and the vector containing the expression vector containing human CH1 and CL were inserted respectively, and the vector was used as the template to amplify the chimeric or chimeric light chain base with the corresponding primers. The plasmid pET32a (+) and purified PCR products were digested with the corresponding restriction endonuclease, and the recombinant plasmid containing the recombinant expression plasmid was induced by the connection transformation, and the recombinant plasmid containing the recombinant expression plasmid was expressed by pET-CAbH and pET-CAbL., and the chimeric cFd and cL were expressed and purified respectively. The compound.SDS-PAGE and Westernblot were used to detect the chimeric Fab and observe the refolding of the products. After purifying the products by the ProteinG column, indirect ELISA, immunofluorescence staining, and flow cytometry were used to detect the target antigen binding activity of the complex products. Finally, the competitive ELISA was used to detect the small molecular antibody and the rat monoclonal antibody CAb1. Whether the same antigen epitopes are competing.
Results: the total RNA was obtained from 1 * 107 hybridoma cells CAb-1 cells, and the total RNA was about 62.5 Mu g. formaldehyde denaturation. The total RNA was more complete, and the sharp bands of 5S, 18S and 28S could be accurately and clearly observed. The mAbCAb1 gene sequence was obtained, the VH gene was long 351bp, 117 amino acids were encoded, VL gene long 336bp and 112 amino acids were encoded. L belongs to kappa type. Optimize the two level structural free energy of its mRNA, construct a non fusion expression vector pET-CAbH, pET-CAbL, successfully realized the high expression of the chimeric Fd or chimeric light chain in Escherichia coli. The protein expression reached 23.6% of the total protein of the bacterial body after the induction of 6h by 30 degree C and the expression of the protein was refolding in vitro, and the SDS-PAGE result was shown. When the initial total protein concentration was 100 g/ml, the recovery of the complex cFab was up to 70.2%. immunofluorescence and FACS. The complex cFab could be more specifically binding to the colorectal cancer cell line SW480 and Hce-8693, but did not combine with the normal cells. Competitive ELISA detection showed that the refolding cFab and the parent CAb-1 antibody fragment F (ab') 2 each other. Conclusion: the variable region gene of hybridoma cell CAb1 was obtained. The primers designed to amplify the IgG1 antibody gene of rat were effective. The expression and preparation of chimeric cFd and cL were expressed and prepared, and the two level structural free energy of mRNA was important for the non fusion expression of the protein, and the small molecule cFab was prepared in vitro. The molecules can specifically bind colorectal cancer cells, the molecules obtained by the molecules have binding activity, and the complex cFab can compete with the parent CAb-1, suggesting that the antigen epitopes identified by them are the same. Because of the binding activity of the prepared cFab antigen, it is suggested that the hybridoma CAb1 gene is correct and reliable, and is also proved to be the same. The protocol was applied to identify the CAb1 variable region gene cloned.
The third part of the study: anti human colorectal cancer surface remodeling antibody rCAb1 expression and purification purpose: to prepare the anti colon cancer surface remodeling antibody rCAb1 with the constructed eukaryotic expression vector, and to purify and identify the expressed antibody.
Methods: first, by analyzing the antibody variable region sequence of the monoclonal antibody CAb1 of colorectal cancer, and using the NR Library of Genbank as BlastP, the local antibody structure database was constructed from all the antibody variable region amino acid sequence information. Then the antibody species belonged to the light, heavy chain type and the anti body sequence was divided into four classes, in which the antibody sequence sources were respectively derived. Homo sapiens and Mus musculus two were selected. The difference residues and abnormal residues of the mAb CAb1 were obtained by the 200 strips of the human source of the mouse antibody variable region, and then the VH of CAb1, the intramolecular and intermolecular hydrogen bond interaction of the VL were obtained by the exact three-dimensional model of the variable region of the model antibody. Candidate mutant loci for the transformation of pedestrians; based on the identified candidate loci, the mutant antibody humanized variable region sequence was synthesized by overlapping PCR, and the T vector (pMD18-T) was constructed into a clone vector, pMD18-T/VH and pMD18-T/VL, respectively. The light chain expression vector pIR was constructed by using the previous variable region sequence. ES1-CAbL and heavy chain expression vector pIRES2-CAbH (weakening polycis anti subsystem), identified by PCR and corresponding enzyme digestion. The transfected COS-7 cells were transiently expressed and the ELISA method was used to detect the expression of antibodies. Finally, the two vectors were converted to CHO-dhfr- fine cell and the ELISA method of sandwich was used to detect the expression of the products. The dilution method was used to screen the positive clones, the antibody expression products were purified, the purified samples were analyzed by SDS-PAGE gel electrophoresis and Western blot detection. Cell immunofluorescence staining and flow cytometry were used to observe the binding activity of the purified products to colorectal cancer cells. Finally, competitive ELISA was selected to detect the rCAb1. The relative affinity of the human IgG was estimated at the same epitopes with the parent mouse CAb1, and the ratio of the human antibody to the parent mouse antibody concentration at the 50% inhibition rate was estimated.
Results: according to the rules of Kabat, Abm, Chothia and Contact, the different CDR, SDR region of the large intestine cancer McAb CAb1 was identified. The selected antibody sequence was analyzed to obtain the amino acid species and percentage of the existing antibody molecules, and the result was compared with the variable region amino acid of the monoclonal antibody CAb1, and the difference residues of the sequence were obtained. CAb1 VH, VL intermolecular and intermolecular hydrogen bond interaction and the accessibility of amino acid surface were obtained by the three-dimensional reconstruction model. Finally, the candidate mutation sites for the preliminary humanized transformation, namely, the heavy chain T018S, N086S and the Q018P. overlap of the light chain, were determined to be connected with T vectors respectively. PMD18-T/VH and pMD18-T/VL were cloned, and the sequencing results showed that the variable region gene was successfully synthesized. The constructed light chain expression vector pIRES1-CAbL and the pIRES2-CAbH of heavy chain expression vector were transfected to COS-7 cells, and the sandwich ELISA showed the expression of human IgG; the two vectors were converted to CHO-dhfr- cells, and the expression products were purified by cloning and the purity of the products was pure. The results of SDS-PAGE electrophoresis and Western blot detection showed that the molecular weight of the target protein was about 150 kDa in the non reductive state; two bands were reduced after reduction and the molecular weight was about 52kDa and 27kDa., respectively. The competitive ELISA detected the product that rCAb1 could compete with the parent rat CAb1 in the same epitope and affinity. 55%., which is about the antibody of the parent rat
Conclusion: the monoclonal antibody CAb1 of colorectal cancer was obtained by analyzing the variable region sequence of monoclonal antibody CAb1 of colorectal cancer.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392;Q78

【参考文献】