CLONING AND SEQUENCING OF IMMUNOGLOBULINVARIABLE－REGION GENE OF A MONOCLONALANTIBODY SPECIFIC FOR HUMANHEPATOCARCINOMA

A murine monoclonal antibody HAb27 specific for human hepatocarcinoma has been developed for radioimmunolocalization in animal models. The isotype of this antibody was IgGl, k. In the present study, we used a set of oligonucleotide primers to amplify the cDNA of mouse immunoglobulin heavy and light chain variable region genes by the polymerase chain reaction. Sequence analysis of the heavy variable region indicated that the VH region was highly homologous to the plasmacytoma cell line MOPC21 gene, and closely related to germline genes of the VHⅢ family. The JH region was encoded by the JH3 gene. For the light chain, the VK segment of the antibody showed the highest homology to the germline VKOXl gene,and the JK region was JK5.

Cloning and sequencing of the variable region genes of anti-human melanoma monoclonal antibody

Reverse transcription-PCR (RT PCR) was applied to amplify the variable region genes of the heavy and light chains of a monoclonal antibody(mAb) from a mouse anti-human melanoma hybridoma cell line HB8759. The two genes were sequenced, analyzed by computer and expressed in E. colt. No genes in EMBL Gene Bank 1995were found identical to the VH and VL genes. VH and VL genes consisted of 366 and 324 base pairs respectively.Both VH and VL genes were open reading frames and had antibody characteristics. The two genes were insertedinto expression vector pGEX-4T-1. The results showed that GST-VH and-VL fusion proteins were of anticipated sizes. VH and VL genes were confirmed as functionally rearranged mouse lmmunoglobulin variable region genes.

Cloning and Sequence Analysis of Light Variable Region Gene of Anti-human Retinoblastoma Monoclonal Antibody

Purpose: To clone the variable region gene of light chain of monoclonal antibody against human retinoblastoma and to analyze the characterization of its nucleotide sequence as well as amino acid sequence.Methods: Total RNA was extracted from 3C6 hybridoma cells secreting specific monoclonal antibody(McAb)against human retinoblastoma(RB), then transcripted reversely into cDNA with olig-dT primers.The variable region of the light chain (VL) gene fragments was amplified using polymeerase chain reaction(PCR) and further cloned into pGEM(R) -T Easy vector. Then, 3C6 VL cDNA was sequenced by Sanger's method.Homologous analysis was done by NCBI BLAST.Results: The complete nucleotide sequence of 3C6 VL cDNA consisted of 321 bp encoding 107 amino acid residues, containing four workframe regions(FRs)and three complementarity-determining regions (CDRs) as well as the typical structure of two cys residues. The sequence is most homological to a member of the Vk9 gene family, and its chain utilizes the Jkl gene segment.Conclusion: The light chain variable region gene of the McAb against human RB was amplified successfully , which belongs to the Vk9 gene family and utilizes Vk-Jk1 gene rearrangement. This study lays a good basis for constructing a recombinant antibody and for making a new targeted therapeutic agents against retinoblastoma.

Expression and identification of recombinant soluble single-chain variable fragment of monoclonal antibody MC3

AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E.coli TG1.The transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect E.coli HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced. RESULTS: The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected from 30 preselected phage clones by ELISA. All the soluble ScFvs derived from the 4 out of the 18 antigen-positive phage clones were about M(r)32000 and concentrated in periplasmatic space under the given culture condition. The soluble ScFvs could bind the antigen, and they shared the same binding site with MC3. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup,kappa-type. CONCLUSION: The soluble ScFv of MC3 is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the antibody a stable genetic source.

Cloning, sequencing and analyzing of the heavy chain V region genes of human polyreactive antibodies

The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction (PCR) technique. We found that 2 and 3 mAbs utilized genes of the VHIV and VHIII families, respectively. The former 2 VH segments were in germline configuration. A common VH segment, with the best similarity of 90.1 % to the published VHIII germline genes, was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs. This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHIII gene. All these polyreactive mAbs displayed a large NDN region (VH-D-JH junction). The entire H chain V regions of these polyreactive mAbs are unusually basic. The analysis of the charge properties of these mAbs as well as those of other poly- and mono- reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites (CDRs), may be an important structural feature involved in antibody polyreactivity.

Preparation of single chain variable fragment of MG_7 mAb by phage display technology

AIM: To develop the single chain variable fragment of MG MG(7)murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS: mRNA was isolated from MG MG(7) producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG MG(7) recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATO III of highly expressing MG(7)-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG(7) ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG(7) ScFv. Finally, the relative molecular mass of soluble MG(7) ScFv was measured by SDS-PAGE. RESULTS: The V(H), V(L) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 X 10(6) and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG(7) antibody for binding to the antigen expressed on KATO III cells. Within 2 strong positive phage clones, the soluble MG(7) ScFv from one clone was found to have the binding activity with KATO III cells. SDS-PAGE showed that the relative molecular weight of soluble MG(7) ScFv was 32. CONCLUSION: The MG(7) ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.

抗HFRSV人单抗可变区基因克隆及其序列测定

从杂交瘤细胞株87-2提取细胞总RNA,反转录合成cDNA链,进行PCR反应。其中扩增重链可变区一对引物分别与人免疫球蛋白重链V区基因5＇端和J区基因3＇端互补;扩增人λ轻链可变区引物与人免疫球蛋白λ轻链V区基因5＇端和J区基因3＇端互补。将重、轻链可变区基因的PCR扩增产物分别插入M13噬菌体,经转化筛选分别获得重组克隆。双脱氧法测定其序列,所得核苷酸序列经计算机分析,轻、重链可变区基因长度分别为309bp和405pb,编码103个氨基酸和135个氨基酸,有明显抗体可变区特征,具有骨架区和抗原互补区。

9．1C3单克隆抗体轻、重链可变区基因克隆及序列分析

９．１Ｃ３分子是一种参与ＮＫ、巨噬细胞及ＬＡＫ细胞杀伤过程的重要细胞表面分子，主要表达于外周血单个核细胞表面。本文运用分子生物学技术，从分泌９．１Ｃ３ＭＣＡｂ的杂交瘤细胞中提取总ＲＮＡ，经反转录、ＰＣＲ分另１１分离了９．１Ｃ３ＭｃＡｂ轻链可变区（ＶＬ）基因及重链可变区（ＶＨ）基因，并用全自动荧光ＤＮＡ序列分析仪对９．１Ｃ３ＶＨ、ＶＬ基因序列进行了分析。结果表明：９．１Ｃ３ＶＨ基因为３５１ｂＰ，编码１１７ａａ残基，９．１３ＶＬ为３２４ｂＰ，编码１０８ａａ残基；从推导出的氨基酸序列分析证实９．１Ｃ３ＶＨ、ＶＬ序列均符合小鼠ｌｇ可变区的氨基酸序列特征；根据Ｋａｂｂａｔ分类体系，９．１Ｃ３ＶＨ隶属Ｉｇ重链第Ⅱ（Ｃ）亚组，９．１Ｃ３ＶＬ隶属小鼠ＩｇＫ链第Ⅴ亚组。９．１Ｃ３ＶＨ、ＶＬ基因的克隆成功为其单链抗体的构建、表达奠定了良好的物质基础。

抗膀胱癌单抗Fab段基因的克隆及表达

目的:克隆抗膀胱癌单抗BDI的Fab段基因并在大肠杆菌中表达。方法:用逆转录-聚合酶链反应技术(RT-PCR),从分泌抗人膀胱癌的鼠单抗杂交瘤细胞系中克隆k链和Fd段基因,克隆到Fab表达载体中,在大肠杆菌表达噬菌体抗体和可溶Fab;运用PCR介导的定位点突变改造V_H氨基端序列;用ELISA、免疫组化法等进行特异性鉴定。结果:从分泌抗膀胱癌的鼠单抗杂交瘤细胞系中克隆了重链Fd段和k链基因,在大肠杆菌中获得有抗原结合活性的噬菌体抗体和可溶性Fab的表达,但活性很弱,将V_H氨基端序列矫正为亲本单抗原始序列后,明显改善了其活性,通过ELISA、免疫组化及模拟抗体库筛选证实了所获抗体片段的特异性结合及在抗体库技术中的可用性。结论:获得了功能性抗膀胱癌小分子抗体,并再次提示抗体氨基端序列对抗体活性的影响的重要性。

抗人肝癌单克隆抗体重链可变区基因克隆和序列测定

应用反转录ＰＣＲ方法从分泌抗人肝癌单克隆抗体鼠杂交瘤细胞系ＨＡｂ２７中成功地克隆了单抗重链可变区基因，将此基因重组入Ｍ１３噬菌体中，双脱氧链终止法测定了核苷酸序列。经计算机分析，基因全长４０８ｂｐ，编码１３６个氨基酸，并进行了基因同源性分析。结果表明获得的重链可变区基因是具有功能性的，两端引物自带起始码和终止码，可直接插入原核载体中进行表达。

HIV-1 CRF01_AE重组型gp120 V1/V2结构域单克隆抗体的制备与鉴定

目的真核表达人类免疫缺陷病毒1型(HIV-1)CRF01_AE重组型gp120蛋白的V1/V2结构域,制备其单克隆抗体并鉴定其抗原反应性。方法构建pTriEx-3-V1/V2CNE55真核表达载体,在HEK293F细胞中表达V1/V2-His融合蛋白,纯化后作为抗原免疫BALB/c小鼠,取免疫后的小鼠脾细胞与Sp2/0骨髓瘤细胞融合,ELISA筛选分泌抗V1/V2重组蛋白单克隆抗体的阳性杂交瘤,对单克隆抗体的特异性、型别以及效价进行鉴定。结果制备并获得一株稳定分泌抗HIV-1 AE亚型V1/V2结构域单克隆抗体的杂交瘤细胞株,ELISA测定腹水效价高达1∶81 000,单克隆抗体类型属于IgG1/κ型。Western blot结果表明该单克隆抗体同样能够识别不同亚型的HIV gp120蛋白。结论成功制备出抗HIV-1 AE重组型gp120蛋白V1/V2结构域的单克隆抗体。

抗乙型脑炎病毒单克隆抗体轻链可变区基因的分离和鉴别

为了构建抗乙型脑炎病毒的人—鼠嵌合抗体,以分泌抗乙型脑炎病毒单克隆体的51-8杂交瘤细胞株为材料,以J_κ和V_κ为探针,从λEMBL-3为载体构建的基因文库中分离有功能性的轻链可变区基因。经3次复筛得到19个阳性噬菌斑。将阳性噬菌斑的DNA重组体和小鼠肝DNA分别用BamH I酶切后进行分析,在分析的8个重组体中,其中5个含有2.8kb片段,而肝DNA含有8.9kb、5.6kb、3.6kb及3.1kb4个片段,没有2.8kb片段。由于肝DNA的4个片段是未经重排的种系基因,因此可以推断重组体中的2.8kb片段是经过重排的轻链可变区基因。为了进一步鉴定,又从中选出3个含有2.8kb片段的重组噬菌斑,以V_κ和J_κ作探针分别进行点杂交,结果都为阳性,进一步说明了2.8kb片段是经过重排的轻链可变区基因。将这一2.8kb片段克隆到pUC19中,得一重组子,命名为pVLD24。利用pCR扩增技术从pVLD24中扩增出预期的334 bp轻链V基因。综合证实了所鉴别得到的功能性轻链可变区基因是完整的,并且用不同的限制性内切酶绘制了这一功能性基因的酶切图谱。

抗体与抗体药物的前世今生

抗体药物是生物技术制药领域的一个重要方面。抗体具有识别抗原的特异性,因而利用抗体诊断与治疗疾病是医药研究者长期以来追求的目标。抗体的发现与应用离不开免疫学。从免疫系统入手,详细介绍了抗体物质发现的历史进程与临床应用。近年来,以细胞工程技术和基因工程技术为主体的抗体工程技术已成为生物技术制药领域的热点。抗体药物的研制与产业化正以迅速的发展势头进入新的时代。

抗口蹄疫病毒单克隆抗体1C7重轻链可变区基因的克隆及序列分析

应用分子生物学技术 ,从分泌抗O型口蹄疫病毒单克隆抗体的杂交瘤细胞 1C7中提取总RNA ,经反转录 ,PCR扩增及克隆 ,分别得到VH 基因及VL 基因。序列测定结果表明 :1C7的VH 基因为 36 8bp ,VL 基因为 32 3bp。用NCBIGenBank分析表明 ,VH 和VL 均符合小鼠抗体可变区特征 ,为功能性重排的抗体可变区基因。根据Kabat分类体系 ,1C7的VH 基因中的VH基因片段隶属于抗体重链第 7183家族 ,其VL 基因中的VL基因片段隶属于抗体K轻链 2 0家族 ,VH 基因由VH76_1BG_DFL16 .1_JH4重排而形成 ,VL 基因由VKbw2 0_JK2重排而形成。 1C7的VH 和VL

带有引导序列的抗-D抗体可变区基因的扩增和序列分析

目的 :从引导区扩增获得高度特异性和亲和力的抗 -D抗体的可变区基因 ,并进行序列分析。方法 :提取 1株分泌抗 -D抗体的人B淋巴细胞株的总RNA ,从引导区设计引物 ,RT -PCR扩增抗 -D抗体的可变区基因 ,分别对扩增产物进行分子克隆和测序。结果 :用重链引物和轻链引物分别扩增出 4 4 0bp和 4 10bp的特异带。序列分析表明 ,其核苷酸及其所推导的氨基酸 (AA)序列符合人Ig可变区基因的序列特征。结论 :获得了抗 -D抗体可变区基因 ,为基因工程抗 -D的研制及Rh新生儿溶血病的预防提供物质基础。

人-鼠嵌合抗血小板单抗SZ-2 Fab片段基因的构建和表达研究

目的:降低鼠源性抗血小板单克隆抗体SZ-2的免疫原性,为其进一步研究、应用奠定基础。方法:用Trizol试剂从SZ-2杂交瘤细胞抽提RNA,应用RT-PCR技术,扩增出SZ-2重链、轻链可变区基因,克隆人载体pUCm-T,测序分析。应用基因重组技术,将SZ-2轻、重链可变区基因与人免疫球蛋白γ1重链CH1和κ轻链恒区基因进行拼接,构建人-鼠嵌合Fab片段基 因表达质粒pSW1-2 Fab/Hu,并导入大肠杆菌XL1-blue诱导表达。以ELISA、Western blot和瑞斯托霉素诱导血小板聚集抑制试验,对表达产物进行检测、验证。结果:克隆的基因序列符合小鼠轻、重链可变区基因的特征;表达质粒pSW1-2Fab/Hu拼接正确;在大肠肝菌中的表达量约为180μg/L;表达产物具有与血小板GPIb结合的特性并可抑制瑞斯托霉素诱导血小板聚集。结论:成功地克隆了SZ-2可变区基因;并表达了可溶性人-鼠嵌合Fab基因工程抗体。

抗乙型脑炎病毒单抗重链可变区基因的筛选和鉴别

为了制备抗乙型脑炎病毒的人-鼠嵌合抗体,以分泌抗乙型脑炎病毒单抗的51-8杂交瘤细胞株为材料,分离这一单抗的重链可变区基因。杂交瘤细胞的大分子量DNA经Bam HI部分酶切后,以λEMBL-3为载体,构建了总数为2×10~7pfu的基因文库。以抗乙脑病毒单抗重链可变区cDNA为探针,从360000个噬菌斑中筛选出9个阳性斑,经点杂交及Southern杂交证明它们都含有重链可变区基因的片段。进一步以J_(11)探针(含J_3、J_4和重链增强子)对其中4个重组体进行鉴别。经EcoRI酶切后,有3个重组体含有与肝细胞和Sp2/0细胞相同的3.8kb片段,而第4个重组体(λ8a4)没有这一片段,却有一个4.5kb片段,这是在肝细胞和Sp2/0细胞中不存在的。从而证明前3个重组体的插入片段是未经重排的重链可变区基因片段,而λ8a4中的插入片段含有经过重排的功能性可变区基因。这一4.5kb片段不能与含有J_1—J_2的探针杂交,却同时含有V_H、J,或/和J_4和增强子。进一步证明,这是一个功能性的可变区基因。因此,将这一4.5kb片段分离出来之后,在pUC19中亚克隆并作酶切图。为构建抗乙脑病毒的人-鼠嵌合重链基因奠定了基础。

鼠抗CD71单抗轻链及重链可变区基因的克隆和序列分析

目的 获得鼠抗人CD71分子单抗的轻链及重链可变区基因。方法 用一步法提取总RNA,逆转录形成cDNA,设计并合成一套扩增小鼠重、轻链可变区的通用引物,通过PCR扩增基因．分别克隆人pMD18－T载体中,作核苷酸序列分析。结果用重链引物扩增获得长约350bp的片段,用轻链引物扩增获得长约330bp的片段,序列测定获得它们的DNA序列。结论克隆的重链可变区基因长度为348bp,属于小鼠H链可变区IA亚组;克隆的轻链可变区基因长度为336bp,属于小鼠K轻链可变区Ⅱ亚组。

抗人黑色素瘤单抗HB8759轻、重链可变区基因的克隆和序列分析

目的：获得新的鼠抗人黑色素瘤单抗ＨＢ８７５９轻、重链可变区基因，以便对其进行基因工程改造．方法：采用反转录－ＰＣＲ法（ＲＴ－ＰＣＲ）从杂交瘤细胞中扩增抗体轻、重链可变区基因，测定ＤＮＡ序列，输入计算机分析，并在大肠杆菌中表达．结果：在ＥＭＢＬＧｅｎｅＢａｎｋ１９９５中未发现有与轻、重链可变区（ＶＨ，ＶＬ）基因序列相同的基因．ＶＨ，ＶＬ基因均为单一开放读框，并具有抗体可变区结构特征，ＶＨ，ＶＬ基因分别长３６６ｂｐ，３２４ｂｐ．将ＶＨ，ＶＬ基因重组入融合蛋白表达载体ｐＧＥＸ－４Ｔ－１，诱导后可见ＶＨ，ＶＬ分别在４０ｋｕ和３８ｋｕ处有深染的新蛋白带出现，与预计的ＶＨ，ＶＬ与ＧＳＴ的融合蛋白的分子质量相符．结论：ＶＨ，ＶＬ基因均为新发现的基因序列，符合功能重排的鼠抗体可变区基因特征．

肝癌单克隆抗体可变区基因的克隆及序列分析

目的：获取抗人原发性肝癌单克隆抗体重、轻链可变区基因。方法：从分泌高亲和力的抗人原发性肝癌单克隆抗体的杂交瘤细胞株ＳＭ８．１１中提取细胞的总ＲＮＡ，以随机引物反转录合成ｃＤＮＡ，以特异引物进行ＰＣＲ，扩增了单抗的重链可变区（ＶＨ）及轻链可变区（ＶＬ）基因，并进行了序列分析。结果：ＶＨ基因片段长度为３５１ｂｐ，编码１１７个氨基酸残基；ＶＬ基因片段长度为３２４ｂｐ，编码１０７个氨基酸残基。两者均有４个框架区（ＦＲｓ）及３个抗原互补决定区（ＣＤＲｓ），并含有抗体特征性的两个半胱氨酸残基。