pTriEx-4neo载体多克隆位点的改造抗跨膜TNF-α单克隆抗体C1可变区基因测序及嵌合抗体的构建
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摘要
在现代生物医学对于某一基因的研究中,常常因为试验目的和试验系统的需要在不同的载体表达同一目的基因,如需要获得大量高纯度蛋白时往往要在有纯化标签的原核或昆虫表达载体中插入目的基因,而当需要研究其在细胞内部或表面的生物学功能时,又需要在真核表达载体中插入目的基因。而由于不同载体的多克隆位点迥异,若每更换一次载体,都需要从头构建,经历引物设计,在上下游引物中引入酶切位点,PCR扩增,酶切,连接,转化,筛选和测序比对等诸多分子克隆操作,过程繁琐复杂;且PCR扩增目的片段时,易出现碱基突变,导致分子克隆前功尽弃,需要重头进行,严重延缓实验进程。
为了解决这一问题,实现同一目的基因在多种不同载体间的快速切换,本课题组设计了系列载体的改造方案。其基本策略是:以pEGFP-C1的多克隆位点(multiple clone site,MCS)替换需改造系列质粒的原有MCS。由于pEGFP-C1的MCS仅有不到100bp,如此短的片段酶切后纯化回收困难,得率低,损失大,故在其EcoRI处,先插入一段238bp的无关辅助片段,再用此延长的MCS取代被改造载体的原有MCS,将延长的238bp的辅助片段切除后即构建成功。本课题主要完成pTriEx-4 neo载体的改造工作。
1. pTriEx-4 neo/MCS-238重组体的构建和克隆
以pEGFP-C1/238为模板,PCR扩增已加长238bp的pEGFP-C1的多克隆位点片段(命名为MCS-238),并在MCS-238片段的两端分别引入EcoR V和Bsu36 I酶切位点。对MCS-238片段的PCR产物和pTriEx-4 neo载体分别进行EcoR V和Bsu36 I双酶切,酶切回收PCR产物和载体,以T4 DNA连接酶进行连接,转化DH5α感受态细菌,挑选阳性克隆。
2.阳性克隆的鉴定
验证阳性克隆,结果显示:重组质粒经EcoR V和Bsu36 I双酶切得到约320bp的目的片断,与所连接的PCR片段大小一致。证明pTriEx-4 neo/MCS-238重组质粒构建成功,已将pEGFP-C1/238载体的MCS-238成功置换入pTriEx-4 neo载体中。
3. 238bp延长片段的切除和pTriEx-4 neo/C1重组体的构建
将pTriEx-4 neo/MCS-238重组体中的238bp延长辅助小片段经EcoRI单酶切去除。连接转化后通过菌落PCR初步鉴定后,经DNA测序分析显示改造后的pTriEx-4 neo载体中移植入的pEGFP-C1载体多克隆位点序列无突变,pTriEx-4 neo /C1载体构建成功(C1代表pEGFP-C1的MCS)。
结论:本课题将pEGFP-C1的多克隆位点成功置换入pTriEx-4 neo载体中,替换其原有的MCS,完成部分本课题组的载体改造工程:即包括pTriEx-4 neo在内的各种常用载体的多克隆位点的标准化和统一化。本工作为实现目的基因在pcDNA3.1(+),pTriEx-4 neo,pET-28a(+)和pEGFP-C1等各类本室常用载体之间方便快捷的转移,为本课题组开展以分子克隆为基础的各项科研提供了重要的实验材料。
TNF-α(tumor necrosis factor,TNF-α)是一种具有多种生物学效应的细胞因子,在免疫应答与炎症反应中起着重要作用,控制着细胞分化、增殖与凋亡。在体内以两种形式存在:跨膜型(Transmembrane TNF-α, TM-TNF-α)和分泌型(Secreted-TNF-α,S-TNF-α)。TM- TNF-α是S-TNF-α的前体,比S-TNF-α多一段76个氨基酸组成的的信号肽。TNF-α转化酶(TACE)可在跨膜TNF-α的胞外结构域切割TNF-α,释放出S-TNF-α。两型TNF-α具有不同的生物学功能,病理状态下,炎症病灶处S-TNF-α常常过量表达,如类风湿关节炎和Crohn’s病等自身免疫病,过量的S-TNF-α是该类疾病最显著的症状之一,也是临床治疗干预的主要切入点。
本室的前期工作获得了一株全新的抗跨膜TNF-α的单克隆抗体C1,它只能通过识别跨膜TNF-α的TACE酶切位点结合跨膜TNF-α,而对S-TNF-α无亲和力。该株单抗的优点是能选择性结合跨膜TNF-α,通过空间位阻阻止TACE对于跨膜TNF-α的酶切,从而减少S-TNF-α的生成,而同时又不封闭TM-TNF-α的功能结构域,因此具有独特的抗炎作用方式。
鼠源性抗体无法直接应用于人体,因此抗体的基因工程改造,如嵌合化和人源化是单抗用于人体试验和治疗的必经之路,本课题的目的是克隆并测序得到鼠源母本C1单克隆抗体可变区基因序列,并初步构建其人鼠嵌合抗体,为后续抗体人源化奠定基础。
一.单克隆抗体C1轻链可变区基因的克隆与测序
选用家族特异性的鼠免疫球蛋白轻链先导肽简并引物VL5’2与骨架四区简并引物VL3’(JK1/2)配对,PCR成功扩增出390bp大小的单克隆抗体C1轻链可变区基因
目的条带,对C1轻链可变区基因C1-VL5’2核苷酸序列进行测序,结果经NCBI IgBLAST和IMGT V-quest等免疫球蛋白生物信息学工具分析得到V/J框内重排的结果:即无终止密码子出现,确认为有功能的轻链可变区重排序列。
二.单克隆抗体C1重链可变区基因的克隆与测序
选用家族特异性的鼠免疫球蛋白重链先导肽简并引物VH5’1与骨架四区简并引物VH3’(JH1/3)配对,PCR成功扩增出396bp大小的单克隆抗体C1重链可变区基因目的条带,将C1重链可变区基因C1-VH5’1核苷酸序列测序结果经NCBI IgBLAST和IMGT V-quest等免疫球蛋白生物信息学工具分析,得到V/D/J框内重排的结果,即无终止密码子出现,确认为有功能的重链可变区重排序列。
三. C1人鼠嵌合抗体pIRES表达载体的构建与表达
应用重叠延伸PCR方法将C1轻链可变区基因与人kappa链恒定区基因融合构建C1嵌合轻链基因,插入pIRES的多克隆位点A;将C1重链可变区基因与人gamma 1链恒定区基因融合构建C1嵌合重链基因,插入pIRES的多克隆位点B。ELISA检测转染pIRES-C1表达载体的CHO细胞培养上清中的嵌合抗体表达,得到阳性结果,提示嵌合抗体表达载体构建成功,顺利表达并分泌人鼠嵌合抗体。
结论:成功克隆抗跨膜TNF-?单克隆抗体C1轻链及重链可变区基因,经序列分析证明为一对功能性免疫球蛋白可变区基因。利用重叠延伸PCR法成功构建pIRES单启动子双顺反子嵌合抗体表达载体,并在哺乳动物表达体系(CHO细胞)中表达嵌合抗体。
In modern scientific research of certain target gene, we often need to express the same gene within different expression vectors tailored to fulfill diverse experiment purposes and experiment systems. When large amouts of pure protein is needed, the gene of interest must be inserted into prokaryotic or insect expresson system with purification tag. When the function of gene in cells is under investigation, the gene of interest must be inserted into eukaryotic expression vector. However, MCS from different vectors differs from each other significantly, when expressed in different vectors, we have to conduct primer design, primer synthesis, PCR, digestion, ligation, transformation, screening and sequencing all over again, which is labor intensive and demanding; in addition, there may be mutations introduced into gene of interest during PCR, making all the procedures above futile.
To solve this problem, we design strategy for construction of series of vectors. The basic idea is to supplant all Multiple Cloning Site of different vectors with the MCS of pEGFP- C1. Because the MCS of pEGFP- C1 is too short to recycle, so we need to insert a small fragment about 238bp into it at the EcoRⅠsite. After removing the 238bp fragment, the vector was successfully constructed. The reconstruction of MCS of pTriEx-4 neo is a part of reconstruction of expression vectors project: Recostructionof the MCS of the pTriEx-4 neo
1. Construction and cloning of pTriEx-4 neo/MCS-238 recombinants:
Using the plasmid pEGFP-C1/238 as template, the MCS of pEGFP-C1 with 238bp in it was amplified by PCR and EcoR V and Bsu36 I restriction sites was introducted at the same time. After digestion with EcoR V and Bsu36 I, the the fragment of 320bp was inserted into pTriEx-4 neo plasmid by T4 DNA ligase. Then the recombinant pTriEx-4 neo/MCS-238 was transformed into E. coli DH5αand the positive clones were screened by Ampicillin resistance.
2. Identification of positive clones: The positive clones were confirmed by different way, the result showed that: the recombinants are digested by EcoR V and Bsu36 I digestion, showing the cleaved fragment from the recombinant with the molecule weights of 320bp, suggesting pTriEx-4 neo/MCS-238 plasmid was successfully constructed.
3. Digestion of 238 bp with EcoRI and construction of pTriEx-4 neo/C1 recombinant:
TriEx-4 neo/MCS-238 was digested by EcoR I, and then connected by T4 DNA ligase. The recombinant was transformed into E. coli DH5αand the positive clones were screened. The positive clones were confirmed firstly by colony PCR, one positive clones selected for sequencing. DNA sequence analysis showed that MCS of pTriEx-4 neo/ C1 have been successfully replaced by the MCS of pEGFP-C1 and no shift and mutation. Conclusion: This study had replaced its original MCS of pTriEx-4 neo with the MCS of pEGFP-C1 successfully. In order to standardize the MCS of pEGFP-C1,pET-28a(+), pcDNA3.1(+)and pTriEx-4, this study completed the work partly. The research of reconstruction of MCS provides an effective experiment tool and system for further study which based on the molecular cloning.
Tumor necrosis factor alpha (TNF-α), a pleiotropic cytokine with a wide range of biological activities, plays important roles in regulation of immune response and inflammation and also governs cell differentiation, proliferation and apoptosis. There are two forms of TNF-α, namely secreted TNF-α(S-TNF-α) and transmembrance TNF-α(TM-TNF-α). TM-TNF-αis the precursor of S-TNF-α. TNF-alpha converting enzyme (TACE) cleaves TNF- alpha within the extracellular domain of TM-TNF-α, releasing soluble TNF-αfrom cells. TM-TNF-αand S-TNF-αhave different bioactivities. Excessive S-TNF-αproduction is one of the most significant manifestations under pathological conditions such as rheumatoid arthritis and Crohn’s disease.
Our lab successfully obtained a new anti-TM-TNF-αmonoclonal antibody, which only binds TM-TNF-α, while displays no affinity to S-TNF-α. Actually, this new mAb recognizes the TM-TNF-αcleavage site selectively, blocking TACE enzymatic action through spatial hindrance. The beauty of this mAb lies in its potential to prevent TACE from shedding S-TNF-αinto extracellular fluid without interfering with the functional domain of TM-TNF-α.
One of the major impediments of murine mAb clinical application is its immunogenicity on human body, which makes chimerization and humanization necessary for modern therapeutic mAbs. This project is aimed at sequencing variable region gene of mAb C1 and construct chimeric antibody, paving the way for further humanization.
1. Cloning and sequencing of light chain variable region gene of mAb C1 Employing family specific mouse immunoglobulin light chain leader sequence degenerate primer combined with framework region 4 degenerate primer, we have successfully amplified light chain variable region gene of mAb C1 by polymerase chain reaction. The C1-VL5’2 sequence is confirmed to be a new functional productive light chain variable region gene that has undergone in frame rearrangement of V/J segments without stop codon by online immunoglobulin V-region analysis and alignment bioinformatics tools from NCBI IgBLAST and IMGT V-quest.
2. Cloning and sequencing of heavy chain variable region gene of mAb C1
Employing family specific mouse immunoglobulin heavy chain leader sequence degenerate primer combined with framework region 4 degenerate primer, we have successfully amplified heavy chain variable region gene of mAb C1 by polymerase chain reaction. The C1-VH5’1 sequence is confirmed to be a new functional productive heavy chain variable region gene that has undergone in frame rearrangement of V/D/J segments without stop codon by online immunoglobulin V-region analysis and alignment bioinformatics tools from NCBI IgBLAST and IMGT V-quest.
3. construction of C1 chimeric antibody expression vector and expression of C1 chimeric antibody
By overlapping extension polymerase chain reaction, we fuse light chain variable region gene of mAb C1 with human kappa constant region gene, which is inserted into multiple cloning site A of pIRES vector; we also fuse heavy chain variable region gene of mAb C1 with human gamma 1 constant region gene, which is inserted into multiple cloning site B of pIRES vector. Detection of chimeric antibody in supernatant of pIRES-C1 expression vector transfected CHO cell culture by sandwich ELISA leads to positive result, indicating successful construction of C1 chimeric antibody expression vector and secretion of chimeric antibody by pIRES-C1 transfected CHO cells. Conclusion: We have successfully cloned light chain and heavy chain variable region gene of anti- TM-TNF-αmonoclonal antibody C1, which is confirmed to be a new pair of functional rearranged variable region gene by immunoglobulin sequence analysis. By overlapping extension polymerase chain reaction, we successfully construct pIRES single promoter bicistronic chimeric antibody expression vector and express chimeric antibody in CHO mammalian expression system.
为了解决这一问题,实现同一目的基因在多种不同载体间的快速切换,本课题组设计了系列载体的改造方案。其基本策略是:以pEGFP-C1的多克隆位点(multiple clone site,MCS)替换需改造系列质粒的原有MCS。由于pEGFP-C1的MCS仅有不到100bp,如此短的片段酶切后纯化回收困难,得率低,损失大,故在其EcoRI处,先插入一段238bp的无关辅助片段,再用此延长的MCS取代被改造载体的原有MCS,将延长的238bp的辅助片段切除后即构建成功。本课题主要完成pTriEx-4 neo载体的改造工作。
1. pTriEx-4 neo/MCS-238重组体的构建和克隆
以pEGFP-C1/238为模板,PCR扩增已加长238bp的pEGFP-C1的多克隆位点片段(命名为MCS-238),并在MCS-238片段的两端分别引入EcoR V和Bsu36 I酶切位点。对MCS-238片段的PCR产物和pTriEx-4 neo载体分别进行EcoR V和Bsu36 I双酶切,酶切回收PCR产物和载体,以T4 DNA连接酶进行连接,转化DH5α感受态细菌,挑选阳性克隆。
2.阳性克隆的鉴定
验证阳性克隆,结果显示:重组质粒经EcoR V和Bsu36 I双酶切得到约320bp的目的片断,与所连接的PCR片段大小一致。证明pTriEx-4 neo/MCS-238重组质粒构建成功,已将pEGFP-C1/238载体的MCS-238成功置换入pTriEx-4 neo载体中。
3. 238bp延长片段的切除和pTriEx-4 neo/C1重组体的构建
将pTriEx-4 neo/MCS-238重组体中的238bp延长辅助小片段经EcoRI单酶切去除。连接转化后通过菌落PCR初步鉴定后,经DNA测序分析显示改造后的pTriEx-4 neo载体中移植入的pEGFP-C1载体多克隆位点序列无突变,pTriEx-4 neo /C1载体构建成功(C1代表pEGFP-C1的MCS)。
结论:本课题将pEGFP-C1的多克隆位点成功置换入pTriEx-4 neo载体中,替换其原有的MCS,完成部分本课题组的载体改造工程:即包括pTriEx-4 neo在内的各种常用载体的多克隆位点的标准化和统一化。本工作为实现目的基因在pcDNA3.1(+),pTriEx-4 neo,pET-28a(+)和pEGFP-C1等各类本室常用载体之间方便快捷的转移,为本课题组开展以分子克隆为基础的各项科研提供了重要的实验材料。
TNF-α(tumor necrosis factor,TNF-α)是一种具有多种生物学效应的细胞因子,在免疫应答与炎症反应中起着重要作用,控制着细胞分化、增殖与凋亡。在体内以两种形式存在:跨膜型(Transmembrane TNF-α, TM-TNF-α)和分泌型(Secreted-TNF-α,S-TNF-α)。TM- TNF-α是S-TNF-α的前体,比S-TNF-α多一段76个氨基酸组成的的信号肽。TNF-α转化酶(TACE)可在跨膜TNF-α的胞外结构域切割TNF-α,释放出S-TNF-α。两型TNF-α具有不同的生物学功能,病理状态下,炎症病灶处S-TNF-α常常过量表达,如类风湿关节炎和Crohn’s病等自身免疫病,过量的S-TNF-α是该类疾病最显著的症状之一,也是临床治疗干预的主要切入点。
本室的前期工作获得了一株全新的抗跨膜TNF-α的单克隆抗体C1,它只能通过识别跨膜TNF-α的TACE酶切位点结合跨膜TNF-α,而对S-TNF-α无亲和力。该株单抗的优点是能选择性结合跨膜TNF-α,通过空间位阻阻止TACE对于跨膜TNF-α的酶切,从而减少S-TNF-α的生成,而同时又不封闭TM-TNF-α的功能结构域,因此具有独特的抗炎作用方式。
鼠源性抗体无法直接应用于人体,因此抗体的基因工程改造,如嵌合化和人源化是单抗用于人体试验和治疗的必经之路,本课题的目的是克隆并测序得到鼠源母本C1单克隆抗体可变区基因序列,并初步构建其人鼠嵌合抗体,为后续抗体人源化奠定基础。
一.单克隆抗体C1轻链可变区基因的克隆与测序
选用家族特异性的鼠免疫球蛋白轻链先导肽简并引物VL5’2与骨架四区简并引物VL3’(JK1/2)配对,PCR成功扩增出390bp大小的单克隆抗体C1轻链可变区基因
目的条带,对C1轻链可变区基因C1-VL5’2核苷酸序列进行测序,结果经NCBI IgBLAST和IMGT V-quest等免疫球蛋白生物信息学工具分析得到V/J框内重排的结果:即无终止密码子出现,确认为有功能的轻链可变区重排序列。
二.单克隆抗体C1重链可变区基因的克隆与测序
选用家族特异性的鼠免疫球蛋白重链先导肽简并引物VH5’1与骨架四区简并引物VH3’(JH1/3)配对,PCR成功扩增出396bp大小的单克隆抗体C1重链可变区基因目的条带,将C1重链可变区基因C1-VH5’1核苷酸序列测序结果经NCBI IgBLAST和IMGT V-quest等免疫球蛋白生物信息学工具分析,得到V/D/J框内重排的结果,即无终止密码子出现,确认为有功能的重链可变区重排序列。
三. C1人鼠嵌合抗体pIRES表达载体的构建与表达
应用重叠延伸PCR方法将C1轻链可变区基因与人kappa链恒定区基因融合构建C1嵌合轻链基因,插入pIRES的多克隆位点A;将C1重链可变区基因与人gamma 1链恒定区基因融合构建C1嵌合重链基因,插入pIRES的多克隆位点B。ELISA检测转染pIRES-C1表达载体的CHO细胞培养上清中的嵌合抗体表达,得到阳性结果,提示嵌合抗体表达载体构建成功,顺利表达并分泌人鼠嵌合抗体。
结论:成功克隆抗跨膜TNF-?单克隆抗体C1轻链及重链可变区基因,经序列分析证明为一对功能性免疫球蛋白可变区基因。利用重叠延伸PCR法成功构建pIRES单启动子双顺反子嵌合抗体表达载体,并在哺乳动物表达体系(CHO细胞)中表达嵌合抗体。
In modern scientific research of certain target gene, we often need to express the same gene within different expression vectors tailored to fulfill diverse experiment purposes and experiment systems. When large amouts of pure protein is needed, the gene of interest must be inserted into prokaryotic or insect expresson system with purification tag. When the function of gene in cells is under investigation, the gene of interest must be inserted into eukaryotic expression vector. However, MCS from different vectors differs from each other significantly, when expressed in different vectors, we have to conduct primer design, primer synthesis, PCR, digestion, ligation, transformation, screening and sequencing all over again, which is labor intensive and demanding; in addition, there may be mutations introduced into gene of interest during PCR, making all the procedures above futile.
To solve this problem, we design strategy for construction of series of vectors. The basic idea is to supplant all Multiple Cloning Site of different vectors with the MCS of pEGFP- C1. Because the MCS of pEGFP- C1 is too short to recycle, so we need to insert a small fragment about 238bp into it at the EcoRⅠsite. After removing the 238bp fragment, the vector was successfully constructed. The reconstruction of MCS of pTriEx-4 neo is a part of reconstruction of expression vectors project: Recostructionof the MCS of the pTriEx-4 neo
1. Construction and cloning of pTriEx-4 neo/MCS-238 recombinants:
Using the plasmid pEGFP-C1/238 as template, the MCS of pEGFP-C1 with 238bp in it was amplified by PCR and EcoR V and Bsu36 I restriction sites was introducted at the same time. After digestion with EcoR V and Bsu36 I, the the fragment of 320bp was inserted into pTriEx-4 neo plasmid by T4 DNA ligase. Then the recombinant pTriEx-4 neo/MCS-238 was transformed into E. coli DH5αand the positive clones were screened by Ampicillin resistance.
2. Identification of positive clones: The positive clones were confirmed by different way, the result showed that: the recombinants are digested by EcoR V and Bsu36 I digestion, showing the cleaved fragment from the recombinant with the molecule weights of 320bp, suggesting pTriEx-4 neo/MCS-238 plasmid was successfully constructed.
3. Digestion of 238 bp with EcoRI and construction of pTriEx-4 neo/C1 recombinant:
TriEx-4 neo/MCS-238 was digested by EcoR I, and then connected by T4 DNA ligase. The recombinant was transformed into E. coli DH5αand the positive clones were screened. The positive clones were confirmed firstly by colony PCR, one positive clones selected for sequencing. DNA sequence analysis showed that MCS of pTriEx-4 neo/ C1 have been successfully replaced by the MCS of pEGFP-C1 and no shift and mutation. Conclusion: This study had replaced its original MCS of pTriEx-4 neo with the MCS of pEGFP-C1 successfully. In order to standardize the MCS of pEGFP-C1,pET-28a(+), pcDNA3.1(+)and pTriEx-4, this study completed the work partly. The research of reconstruction of MCS provides an effective experiment tool and system for further study which based on the molecular cloning.
Tumor necrosis factor alpha (TNF-α), a pleiotropic cytokine with a wide range of biological activities, plays important roles in regulation of immune response and inflammation and also governs cell differentiation, proliferation and apoptosis. There are two forms of TNF-α, namely secreted TNF-α(S-TNF-α) and transmembrance TNF-α(TM-TNF-α). TM-TNF-αis the precursor of S-TNF-α. TNF-alpha converting enzyme (TACE) cleaves TNF- alpha within the extracellular domain of TM-TNF-α, releasing soluble TNF-αfrom cells. TM-TNF-αand S-TNF-αhave different bioactivities. Excessive S-TNF-αproduction is one of the most significant manifestations under pathological conditions such as rheumatoid arthritis and Crohn’s disease.
Our lab successfully obtained a new anti-TM-TNF-αmonoclonal antibody, which only binds TM-TNF-α, while displays no affinity to S-TNF-α. Actually, this new mAb recognizes the TM-TNF-αcleavage site selectively, blocking TACE enzymatic action through spatial hindrance. The beauty of this mAb lies in its potential to prevent TACE from shedding S-TNF-αinto extracellular fluid without interfering with the functional domain of TM-TNF-α.
One of the major impediments of murine mAb clinical application is its immunogenicity on human body, which makes chimerization and humanization necessary for modern therapeutic mAbs. This project is aimed at sequencing variable region gene of mAb C1 and construct chimeric antibody, paving the way for further humanization.
1. Cloning and sequencing of light chain variable region gene of mAb C1 Employing family specific mouse immunoglobulin light chain leader sequence degenerate primer combined with framework region 4 degenerate primer, we have successfully amplified light chain variable region gene of mAb C1 by polymerase chain reaction. The C1-VL5’2 sequence is confirmed to be a new functional productive light chain variable region gene that has undergone in frame rearrangement of V/J segments without stop codon by online immunoglobulin V-region analysis and alignment bioinformatics tools from NCBI IgBLAST and IMGT V-quest.
2. Cloning and sequencing of heavy chain variable region gene of mAb C1
Employing family specific mouse immunoglobulin heavy chain leader sequence degenerate primer combined with framework region 4 degenerate primer, we have successfully amplified heavy chain variable region gene of mAb C1 by polymerase chain reaction. The C1-VH5’1 sequence is confirmed to be a new functional productive heavy chain variable region gene that has undergone in frame rearrangement of V/D/J segments without stop codon by online immunoglobulin V-region analysis and alignment bioinformatics tools from NCBI IgBLAST and IMGT V-quest.
3. construction of C1 chimeric antibody expression vector and expression of C1 chimeric antibody
By overlapping extension polymerase chain reaction, we fuse light chain variable region gene of mAb C1 with human kappa constant region gene, which is inserted into multiple cloning site A of pIRES vector; we also fuse heavy chain variable region gene of mAb C1 with human gamma 1 constant region gene, which is inserted into multiple cloning site B of pIRES vector. Detection of chimeric antibody in supernatant of pIRES-C1 expression vector transfected CHO cell culture by sandwich ELISA leads to positive result, indicating successful construction of C1 chimeric antibody expression vector and secretion of chimeric antibody by pIRES-C1 transfected CHO cells. Conclusion: We have successfully cloned light chain and heavy chain variable region gene of anti- TM-TNF-αmonoclonal antibody C1, which is confirmed to be a new pair of functional rearranged variable region gene by immunoglobulin sequence analysis. By overlapping extension polymerase chain reaction, we successfully construct pIRES single promoter bicistronic chimeric antibody expression vector and express chimeric antibody in CHO mammalian expression system.
引文
1. Esposito, D., L.A. Garvey, and C.S. Chakiath, Gateway cloning for protein expression. Methods Mol Biol, 2009. 498: p. 31-54.
2. Picaud, S., M.E. Olsson, and P.E. Brodelius, Improved conditions for production of recombinant plant sesquiterpene synthases in Escherichia coli. Protein Expr Purif, 2007. 51(1): p. 71-9.
3. Malheiro, A., et al., pcDNA-IL-12 vaccination blocks eosinophilic inflammation but not airway hyperresponsiveness following murine Toxocara canis infection. Vaccine, 2008. 26(3): p. 305-15.
4. You, Y., et al., Inhibition effect of pcDNA-tum-5 on the growth of S180 tumor. Cytotechnology, 2008. 56(2): p. 97-104.
5. Gong, L., et al., Cloning, expression and purification of human S-adenosylmethio- nine decarboxylase gene alpha subunit. Chin J Physiol, 2007. 50(1): p. 29-33.
6. Fu, H.L., et al., Dendrimer/DNA complexes encapsulated functional biodegradable polymer for substrate-mediated gene delivery. J Gene Med, 2008. 10(12): p. 1334-42.
7. Li, S., et al., Transgene expression of enhanced green fluorescent protein in cloned rabbits generated from in vitro-transfected adult fibroblasts. Transgenic Res, 2009. 18(2): p. 227-35.
8. Zhou, C.H., et al., [Inhibition of proliferation in MCF-7 breast cancer cells by plasmid-based siRNA targeting to oncogene c-myc]. Sichuan Da Xue Xue Bao Yi Xue Ban, 2008. 39(3): p. 373-7.
9. Li, C., et al., [Helicobacter pylori VacA up-regulates secretion of macrophages by transfection of VacA eukaryotic expression vector]. Wei Sheng Wu Xue Bao, 2008. 48(3): p. 349-54.
10. Wang, Y., et al., [Construction and tumorigenic study on a novel fusion gene AML1-MTG16]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi, 2002. 19(4): p. 271-5.
11. Diegelmann, S., M. Bate, and M. Landgraf, Gateway cloning vectors for the LexA-based binary expression system in Drosophila. Fly (Austin), 2008. 2(4).
12. Magnani, E., L. Bartling, and S. Hake, From Gateway to MultiSite Gateway in one recombination event. BMC Mol Biol, 2006. 7: p. 46.
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2. Black, R.A., et al., A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature, 1997. 385(6618): p. 729-33.
3. Huang, A., et al., Structure-based design of TACE selective inhibitors: manipulations in the S1'-S3' pocket. Bioorg Med Chem, 2007. 15(18): p. 6170-81.
4. Govinda Rao, B., et al., Novel thiol-based TACE inhibitors: rational design, synthesis, and SAR of thiol-containing aryl sulfonamides. Bioorg Med Chem Lett, 2007. 17(8): p. 2250-3.
5. Zhang, H., et al., Transmembrane TNF-{alpha} mediates "forward" and "reverse" signaling, inducing cell death or survival via the NF-{kappa}B pathway in Raji Burkitt lymphoma cells. J Leukoc Biol, 2008. 84(3): p. 789-797.
6. Lin, J., et al., TNF[alpha] blockade in human diseases: An overview of efficacy and safety. Clinical Immunology, 2008. 126(1): p. 13-30.
7. Carroll, W.L., E. Mendel, and S. Levy, Hybridoma fusion cell lines contain an aberrant kappa transcript. Molecular Immunology, 1988. 25(10): p. 991-995.
8. Grell, M., et al., The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor. Cell, 1995. 83(5): p. 793-802.
9. Amin, A.R., Regulation of tumor necrosis factor-alpha and tumor necrosis factor converting enzyme in human osteoarthritis. Osteoarthritis Cartilage, 1999. 7(4): p. 392-4.
10. Waetzig, G.H., et al., Soluble tumor necrosis factor (TNF) receptor-1 induces apoptosis via reverse TNF signaling and autocrine transforming growth factor-beta1. FASEB J, 2005. 19(1): p. 91-3.
11. Mitoma, H., et al., Infliximab induces potent anti-inflammatory responses by outside-to-inside signals through transmembrane TNF-alpha. Gastroenterology, 2005. 128(2): p. 376-92.
12. Norman, P., New drug targets in inflammation and immunomodulation. Drug News Perspect, 1998. 11(7): p. 442-7.
13. Olleros, M.L., et al., Transmembrane TNF induces an efficient cell-mediated immunity and resistance to Mycobacterium bovis bacillus Calmette-Guerin infection in the absence of secreted TNF and lymphotoxin-alpha. J Immunol, 2002. 168(7): p. 3394-401.
14. Newton, R.C., et al., Biology of TACE inhibition. Ann Rheum Dis, 2001. 60 Suppl 3: p. iii25-32.
15. Tang, P., M.C. Hung, and J. Klostergaard, Length of the linking domain of human pro-tumornecrosis factor determines the cleavage processing. Biochemistry, 1996. 35(25): p. 8226-33.
16. Peterson, N.C., Advances in monoclonal antibody technology: genetic engineering of mice, cells, and immunoglobulins. ILAR J, 2005. 46(3): p. 314-9.
17. Chen, H.T., et al., Nucleotide and translated amino acid sequences of cDNA coding for the variable regions of the light and heavy chains of mouse hybridoma antibodies to blood group A and B substances. J. Biol. Chem., 1987. 262(28): p. 13579-13583.
18. Songsivilai, S., et al., Cloning and sequencing of human lambda immunoglobulin genes by the polymerase chain reaction. Eur J Immunol, 1990. 20(12): p. 2661-6.
19. Essono, S., et al., A general method allowing the design of oligonucleotide primers to amplify the variable regions from immunoglobulin cDNA. Journal of Immunological Methods, 2003. 279(1-2): p. 251-266.
20. Dattamajumdar, A.K., et al., Rapid cloning of any rearranged mouse immunoglobulin variable genes. Immunogenetics, 1996. 43(3): p. 141-51.
21. LeBoeuf, R.D., et al., Cloning and sequencing of imniunoglobulin variable-region genes using degenerate oligodeoxy-ribonucleotides and polymerase chain reaction. Gene, 1989. 82(2): p. 371-377.
22. Welschof, M., et al., Amino acid sequence based PCR primers for amplification of rearranged human heavy and light chain immunoglobulin variable region genes. Journal of Immunological Methods, 1995. 179(2): p. 203-214.
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30. Zhou Jiang, Y.H.S.T.S., Regulation of Recombinant Monoclonal Antibody Production in Chinese Hamster Ovary Cells: A Comparative Study of Gene Copy Number, mRNA Level, and Protein Expression. Biotechnology Progress, 2006. 22(1): p. 313-318.
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2. Picaud, S., M.E. Olsson, and P.E. Brodelius, Improved conditions for production of recombinant plant sesquiterpene synthases in Escherichia coli. Protein Expr Purif, 2007. 51(1): p. 71-9.
3. Malheiro, A., et al., pcDNA-IL-12 vaccination blocks eosinophilic inflammation but not airway hyperresponsiveness following murine Toxocara canis infection. Vaccine, 2008. 26(3): p. 305-15.
4. You, Y., et al., Inhibition effect of pcDNA-tum-5 on the growth of S180 tumor. Cytotechnology, 2008. 56(2): p. 97-104.
5. Gong, L., et al., Cloning, expression and purification of human S-adenosylmethio- nine decarboxylase gene alpha subunit. Chin J Physiol, 2007. 50(1): p. 29-33.
6. Fu, H.L., et al., Dendrimer/DNA complexes encapsulated functional biodegradable polymer for substrate-mediated gene delivery. J Gene Med, 2008. 10(12): p. 1334-42.
7. Li, S., et al., Transgene expression of enhanced green fluorescent protein in cloned rabbits generated from in vitro-transfected adult fibroblasts. Transgenic Res, 2009. 18(2): p. 227-35.
8. Zhou, C.H., et al., [Inhibition of proliferation in MCF-7 breast cancer cells by plasmid-based siRNA targeting to oncogene c-myc]. Sichuan Da Xue Xue Bao Yi Xue Ban, 2008. 39(3): p. 373-7.
9. Li, C., et al., [Helicobacter pylori VacA up-regulates secretion of macrophages by transfection of VacA eukaryotic expression vector]. Wei Sheng Wu Xue Bao, 2008. 48(3): p. 349-54.
10. Wang, Y., et al., [Construction and tumorigenic study on a novel fusion gene AML1-MTG16]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi, 2002. 19(4): p. 271-5.
11. Diegelmann, S., M. Bate, and M. Landgraf, Gateway cloning vectors for the LexA-based binary expression system in Drosophila. Fly (Austin), 2008. 2(4).
12. Magnani, E., L. Bartling, and S. Hake, From Gateway to MultiSite Gateway in one recombination event. BMC Mol Biol, 2006. 7: p. 46.
1. Kriegler, M., et al., A novel form of TNF/cachectin is a cell surface cytotoxic transmembrane protein: ramifications for the complex physiology of TNF. Cell, 1988. 53(1): p. 45-53.
2. Black, R.A., et al., A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature, 1997. 385(6618): p. 729-33.
3. Huang, A., et al., Structure-based design of TACE selective inhibitors: manipulations in the S1'-S3' pocket. Bioorg Med Chem, 2007. 15(18): p. 6170-81.
4. Govinda Rao, B., et al., Novel thiol-based TACE inhibitors: rational design, synthesis, and SAR of thiol-containing aryl sulfonamides. Bioorg Med Chem Lett, 2007. 17(8): p. 2250-3.
5. Zhang, H., et al., Transmembrane TNF-{alpha} mediates "forward" and "reverse" signaling, inducing cell death or survival via the NF-{kappa}B pathway in Raji Burkitt lymphoma cells. J Leukoc Biol, 2008. 84(3): p. 789-797.
6. Lin, J., et al., TNF[alpha] blockade in human diseases: An overview of efficacy and safety. Clinical Immunology, 2008. 126(1): p. 13-30.
7. Carroll, W.L., E. Mendel, and S. Levy, Hybridoma fusion cell lines contain an aberrant kappa transcript. Molecular Immunology, 1988. 25(10): p. 991-995.
8. Grell, M., et al., The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor. Cell, 1995. 83(5): p. 793-802.
9. Amin, A.R., Regulation of tumor necrosis factor-alpha and tumor necrosis factor converting enzyme in human osteoarthritis. Osteoarthritis Cartilage, 1999. 7(4): p. 392-4.
10. Waetzig, G.H., et al., Soluble tumor necrosis factor (TNF) receptor-1 induces apoptosis via reverse TNF signaling and autocrine transforming growth factor-beta1. FASEB J, 2005. 19(1): p. 91-3.
11. Mitoma, H., et al., Infliximab induces potent anti-inflammatory responses by outside-to-inside signals through transmembrane TNF-alpha. Gastroenterology, 2005. 128(2): p. 376-92.
12. Norman, P., New drug targets in inflammation and immunomodulation. Drug News Perspect, 1998. 11(7): p. 442-7.
13. Olleros, M.L., et al., Transmembrane TNF induces an efficient cell-mediated immunity and resistance to Mycobacterium bovis bacillus Calmette-Guerin infection in the absence of secreted TNF and lymphotoxin-alpha. J Immunol, 2002. 168(7): p. 3394-401.
14. Newton, R.C., et al., Biology of TACE inhibition. Ann Rheum Dis, 2001. 60 Suppl 3: p. iii25-32.
15. Tang, P., M.C. Hung, and J. Klostergaard, Length of the linking domain of human pro-tumornecrosis factor determines the cleavage processing. Biochemistry, 1996. 35(25): p. 8226-33.
16. Peterson, N.C., Advances in monoclonal antibody technology: genetic engineering of mice, cells, and immunoglobulins. ILAR J, 2005. 46(3): p. 314-9.
17. Chen, H.T., et al., Nucleotide and translated amino acid sequences of cDNA coding for the variable regions of the light and heavy chains of mouse hybridoma antibodies to blood group A and B substances. J. Biol. Chem., 1987. 262(28): p. 13579-13583.
18. Songsivilai, S., et al., Cloning and sequencing of human lambda immunoglobulin genes by the polymerase chain reaction. Eur J Immunol, 1990. 20(12): p. 2661-6.
19. Essono, S., et al., A general method allowing the design of oligonucleotide primers to amplify the variable regions from immunoglobulin cDNA. Journal of Immunological Methods, 2003. 279(1-2): p. 251-266.
20. Dattamajumdar, A.K., et al., Rapid cloning of any rearranged mouse immunoglobulin variable genes. Immunogenetics, 1996. 43(3): p. 141-51.
21. LeBoeuf, R.D., et al., Cloning and sequencing of imniunoglobulin variable-region genes using degenerate oligodeoxy-ribonucleotides and polymerase chain reaction. Gene, 1989. 82(2): p. 371-377.
22. Welschof, M., et al., Amino acid sequence based PCR primers for amplification of rearranged human heavy and light chain immunoglobulin variable region genes. Journal of Immunological Methods, 1995. 179(2): p. 203-214.
23. Wang, Z., et al., Universal PCR amplification of mouse immunoglobulin gene variable regions: the design of degenerate primers and an assessment of the effect of DNA polymerase 3' to 5' exonuclease activity. Journal of Immunological Methods, 2000. 233(1-2): p. 167-177.
24. Coloma, M.J., et al., Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction. Journal of Immunological Methods, 1992. 152(1): p. 89-104.
25. Morrison, S.L., et al., Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains. Proc Natl Acad Sci U S A, 1984. 81(21): p. 6851-5.
26. Idusogie, E.E., et al., Engineered Antibodies with Increased Activity to Recruit Complement. J Immunol, 2001. 166(4): p. 2571-2575.
27. Walls, M.A., K.-c. Hsiao, and L.J. Harris, Vectors for the expression of PCR-amplified immunoglobulin variable domains with human constant regions. Nucl. Acids Res., 1993. 21(12): p. 2921-2929.
28. Cao, M., et al., Construction, Purification, and Characterization of Anti-BAFF scFv-Fc Fusion Antibody Expressed in CHO/dhfr(-) Cells. Appl Biochem Biotechnol, 2008.
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