抗SARS-CoV抗原的人源Fab段噬菌体抗体库的构建

目的 :利用抗SARS冠状病毒IgG抗体阳性的SARS康复患者外周血淋巴细胞 ,构建人源Fab段抗体文库。方法 :制备外周血淋巴细胞总RNA ,逆转录成cDNA。以其为模板 ,利用针对家族特异性Ig基因的引物扩增重链Fd段和轻链基因 ,并重组到噬菌粒载体pComb3中 ,将重组噬菌粒载体电转化大肠杆菌XL 1Blue,酶切鉴定抗体库的重组率 ,并测定噬菌体抗体库的库容量。结果 :构建了源于SARS康复患者血清中抗Fab段的抗体文库 ,轻链、重链Fd段基因的重组率分别为91%和 75 % ,库容量为 7.2 3× 10 7。结论 :成功地构建了抗SARS CoV抗原的人源Fab段噬菌体抗体库     

Stable Production of Recombinant Human Sperm Immobilizing Antibody Using cDNA Expression Vectors

PROBLEM: Sperm immobilizing antibodies present in sterile women may be one of the principal causes of immunological infertility. We already established cell lines that secrete recombinant human IgG sperm immobilizing antibody using class-switched (from IgM to IgG) genomic immunoglobulin genes. However, these transfectants produced a small quantity of antibody and required continuous use of a medium with selective reagents. We have now constructed cell lines that stably secrete the antibody in large quantities using immunoglobulin cDNAs and cDNA expression vectors. METHOD: The immunoglobulin heavy chain cDNA was cloned from transfectants that secrete the class-switched human IgG sperm immobilizing antibody. The light chain cDNA, which had already been cloned, and the heavy chain cDNA were inserted into the modified bovine papilloma virus-based cDNA expression vectors BCMGSNeo and BCMGSHyg, respectively. These constructs were sequentially transfected into a mouse myeloma cell line by electroporation. RESULTS: The established transfectants produced recombinant antibody that retained human sperm immobilizing activity in nonselective medium for at least 30 days. Moreover, the production of the antibody was increased three times over that of the previous cell lines. CONCLUSION: We have established an unique method that improves the production of sperm immobilizing antibody stably and in large quantities.     

人源性抗HBsAg抗体Fab段噬菌体呈现文库的构建、筛选及阳性克隆核苷酸序列测定

目的:构建一个经HBsAg主动免疫的人lgG1抗体噬菌体展示文库,通过特异的淘筛(panning)获得与HBsAg有较高亲和力的Fab抗体,并测定其编码序列。方法:从一经乙肝疫苗免疫的自愿者的外周血分离淋巴细胞提取RNA,逆转录合成cDNA。PCR扩增重链Fd和κ轻链cDNA。依次将PCR产物克隆进载体pComb3H相应的位点,构建成噬菌体呈现库,并以HBsAg包被的96孔板对抗体库进行3轮淘筛,将所获克隆分别与HBsAg进行ELISA反应,挑取显色大于对照20倍以上的阳性克隆进行DNA测序。结果:lgG1的Fd段和κ轻链cDNA得到扩增,构建了一实际库容量为1.8×10⁵的噬菌体呈现文库。通过特异性淘筛,获得与HBsAg有较高新合力的3株Fab抗体并测定其编码序列。DNA序列测定结果表明,3株抗体中两株的Fd段完全一样,且3株的轻链完全一样。结论:成功构建了一个经HBsAg抗原免疫的人源抗体Fab段噬菌体抗体库,筛选得到了3株特异性抗体的Fab片段。     

抗LPS mAb Fab片段cDNA的克隆及其在CHO细胞中的表达

目的 构建抗LPS单克隆抗体Fab段的真核表达载体,并在CHO细胞中表达。方法 以逆转录PCR方法扩增抗LPU mAb重链Fd和轻链cDNA,分别克隆入载体pGEM-Teasy。经测序分析后,再分别亚克隆入载体pcDNA3,构建成重组载体pcDNA3-Fd和pcDNA3-LC,并以PCR方法扩增pcDNA3-LC中包括CMV启动子、轻链序列和BGH Poly(A)在内的轻链表达序列。经适当的限制性内切酶酶切后,插入到质粒pcDNA3－Fd中Fd表达序列的前部,构建成表达载体pcDNA3-Fab,以脂质体包裹转染CHO细胞。经G418筛选,用ELISA、免疫荧光和Western blot鉴定阳性克隆。结果 用ELISA和免疫荧光法,筛选出4株高表达Fab的细胞株。Western blot显示,表达产物的Mr为50000。同时,ELISA结果显示,该Fab具有良好的结合LP5的活性。结论 成功地在CHO细胞中表达抗LPS mAb的Fab段,为进一步鉴定其抗LPS的作用,使之成为诊断和治疗革兰氏阴性细菌感染的工具奠定了基础。     

人源性抗乙酰胆碱受体自身抗体的构建及特异性鉴定

目的 用人抗乙酰胆碱受体(AchR)自身抗体的抗原结合片段(Fab)构建完整的抗人AchR自身抗体,为重症肌无力(MG)的实验研究提供人源性抗体材料. 方法 应用PCR技术从分离自MG患者胸腺的Fab637扩增重链Fd片段基因和轻链基因,并克隆至含有免疫球蛋白(IgG1)基因的载体pIgG1上,构建人源性抗AchR自身抗体哺乳类细胞表达的重组载体pIgG1-637.将pIgG1-637转染CHO-k1细胞,细胞培养上清液经斑点杂交试验检测抗体的表达,以放射免疫测定法测定表达的抗体与特异性人AchR结合的活性.结果 细胞培养24 h后能检测到抗体的分泌,表达的抗体能特异性地与抗原结合,且结合活性很高.结论 已成功地构建了具有与抗原特异性结合的人源性IgG1类抗人AchR自身抗体IgG1-637.     

人源腺病毒伴随病毒Ⅱ型单克隆抗体Fab段及其全抗体的研制

目的 运用噬菌体表面表达技术，获得基因工程抗腺病毒伴随病毒抗体Fab、IgG。方法 从酶联免疫吸附试验阳性的人外周血中分离淋巴细胞。提取总RNA，逆转录cDNA，聚合酶链反应扩增人IgG Fab轻、重链基因，利用pComb3载体构建噬菌体抗体库。用纯化的腺病毒伴随病毒为固相抗原筛选抗体Fab段，并在E.coli中进行分泌性表达。通过ELISA和间接免疫荧光试验、筛选和鉴定Fab抗体，并进行序列测定。然后将其重链和轻链基因克隆到全抗体表达载体pAC-L-Fc上。转染昆虫sf-9细胞，利用杆状病毒，昆虫细胞系统实现全抗体的分泌型表达，免疫沉淀试验鉴定它的抗蛋白。结果 分离到一株Fab克隆AAVs-31，腺病毒伴随病毒抗原和抗Fab抗体直接ELISA检测阳性，间接免疫荧光试验呈阳性，序列分析结果表明是一新的序列，所获得的基因为人源IgG Fab基因。由Gamma链和Kappa链组成。表达的全抗体ELISA反应、免疫荧光反应和间接免疫荧光试验均为阳性，免疫沉淀试验结果显示它结合病毒颗粒，不结合任一VP1、VP2、VP3单独衣鞘蛋白。结论 用噬菌体表面表达技术首次获得了抗腺病毒伴随病毒Ⅱ型单克隆抗体Fab段，并在真核系统中表达了其全抗体，它们识别的可能是衣鞘蛋白组装时形成的表位。     

Both VH and VL chains of polyreactive IgM antibody are required for polyreactivity: expression of Fab in Escherichia coli.

Monoclonal polyreactive antibodies can bind to many structurally dissimilar self and non-self antigens. Neither the precise antigen-binding site on the polyreactive antibody molecule nor the molecular basis of polyreactivity has been elucidated. The present study was initiated to see whether antibody genes encoding the Fab fragment of a human monoclonal polyreactive IgM antibody (MoAb 67) could be efficiently expressed in Escherichia coli, and whether the bacterially expressed Fab fragments possessed biological activity. cDNA encoding the variable domains of the heavy and light chains of MoAb 67 were cloned, amplified by polymerase chain reaction (PCR) and expressed in E. coli. Neither the recombinant heavy nor light chain showed antigen-binding activity. In contrast, the recombinant Fab 67 fragment showed the same antigen-binding reactivity profile as the native IgM antibody. It is concluded that the antigen-binding activity of polyreactive antibodies resides in the Fab fragment, and that both the heavy and light chains are required for activity.     

单克隆抗-D细胞株的建立及抗-D抗体Fab段的基因序列分析

目的:建立分泌抗-D抗体的人B淋巴细胞株,分析编码一株人单克隆抗-D抗体Fab段的基因序列。方法:以EB病毒(EBV)转化分泌抗-D抗体的人B淋巴细胞,建立分泌抗-D抗体的淋巴细胞株。设计扩增人lgM重链Fd段及κ轻链的引物,以聚合酶链反应进行扩增,对扩增产物进行分子克隆及测序。结果:分别用轻链引物和重链引物从一分泌lgM抗-D的单克隆细胞株扩增出一约650bp和700bp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lgFab段的序列特征。结论:获得了抗-D抗体Fab段基因,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。     

Generation of genetic engineering monoclonal antibodies against prion protein

Two strains of Fab monoclonal antibodies (mAbs) against prion protein, designated as IV-66 and IV-78, were selected from the phage display libraries. The gene sequences encoding the light κ chain and heavy Fd chain of IV-78 were inserted into a baculovirus expression cassette vector for mouse IgG expression. Western blot, Dot-ELISA and immunoprecipitation confirmed that these Fab and IgG mAbs reacted well with the recombinant hamster and human PrP proteins expressed in prokaryotic and in mammalian cells and PrP^Sc from scrapie-infected hamsters. It demonstrates that mAbs against prion protein are successfully generated by phage-display technique.     

抗角蛋白Fab抗体在大肠杆菌中的表达与复性研究

目的 :用大肠杆菌分别表达抗角蛋白抗体的轻链和Fd片段 ,体外复性得到Fab抗体。方法 :从已构建的质粒p3MH/Fab中 ,亚克隆抗角蛋白抗体的轻链和Fd片段基因 ,并分别插入载体pET32a中 ,构建重组质粒。以重组质粒分别转化大肠杆菌BL2 1(DE3) ,在IPTG诱导下进行表达。SDS PAGE分析发现 ,在相对分子量 (Mr)为 2 5 0 0 0处有外源蛋白表达。轻链和Fd片段变性后等量混合于折叠液中 ,复性后形成Mr 约为4 5 0 0 0的蛋白。结果 :成功地表达抗角蛋白抗体的轻链和Fd片段 ,ELISA和Westernblot证实 ,复性产物具有与角蛋白结合的能力。结论 :获得了具有活性的抗角蛋白的Fab抗体 ,为其应用研究打下了基础 ,也表明包涵体表达基因工程抗体技术是可行的。     

Isolation and amplification of human IgE Fd encoding mRNA from human peripheral blood lymphocytes.

In order to establish the feasibility of applying recombinatorial library technologies to investigate human in vivo IgE responses, and as a pre-requisite of recombinatorial library construction, we have attempted to determine workable peripheral blood sample volumes required for isolation of mRNA for polymerase chain reaction (PCR) amplification of human IgE Fd encoding sequences. Cells secreting chimeric human IgE monoclonal antibody specific for the hapten NIP were used to establish the conditions for specific amplification of C epsilon 1 domain and Fd encoding sequences, as determined by Southern hybridisation. Amplification of C epsilon 1 domain sequences could be achieved using as few as ten cultured cells as the source of RNA. Specific IgE+ B cell enrichment using immuno-magnetic particles prior to RNA extraction was, however, required to obtain amplification of IgE C epsilon 1 and Fd fragments from lymphocytes prepared from 40 ml human peripheral blood. IgG1+ B cell enrichment from similar samples was not required for detectable amplification of human C gamma 1 cDNA sequences. However, this procedure improved amplification efficiency. Optimisation of methods to separate specific B cell populations, or specific RNA/cDNA sequences, will facilitate in vitro generation of human IgE Fab fragments from peripheral blood.     

噬菌体呈现抗体库的构建及抗Fas—Fab抗体的研究

目的构建噬菌体抗体库,获得具有功能的抗Fas-Fab噬菌体抗体。方法以Fas重组蛋白为抗原免疫Balb/c小鼠。取其脾细胞提取mRNA,采用RT-PCR方法扩增抗体基因,构建重链和κ链基因库,用重组Fas抗原对所构建的抗体库进行4轮筛选,并以ELISA法鉴定其功能。结果获得抗体重链Fd基因和κ链基因长度约700bp。构建的重链Fd基因为3.5×106的抗体重链基因库。构建的重链和κ链基因库的容量均为3.1×106。经VCSM13感染得到噬菌体的滴度为8.9×1016cFu/L的噬菌体抗体库,含有抗体重链和κ链基因的噬菌体占27％。用重组人Fas抗原进行4轮筛选,得到100％的富集,说明Fas重组抗原富集了抗Fas-Fab噬菌体抗体,经ELISA检测均有抗Fas抗体的特异性。结论制备的可溶性抗Fas-Fab抗体具有抗Fas抗体的特异性,为进一步的研究奠定了基础。     

人源性噬菌体抗体库的构建及抗amylin抗体的初步筛选

目的:构建人源性噬菌体抗体库,从中筛选胰淀素(amylin)单克隆抗体(mAb),测定其特异性及抗原结合活性。方法:从正常健康人的外周血淋巴细胞中提取总RNA,用RT-PCR方法扩增人免疫球蛋白Fd段和轻链基因,构建噬菌体抗体库。酶切和PCR鉴定后,阳性克隆进行DNA测序分析。用人amylin抗原对抗体库进行筛选富集,将得到的阳性克隆进行Phage-ELISA鉴定,结果进行统计学分析。结果:最终构建的抗体库库容约为0.8×108,酶切鉴定显示有插入片段,抗体库重组率为70%。阳性克隆进行DNA测序证实所获基因为人免疫球蛋白可变区基因。以amylin抗原进行3轮筛选,抗体库得到特异性富集。阳性克隆进行Phage-ELISA检测证实有良好的抗胰淀素抗原的特异性。结论:成功构建了一个人源性噬菌体抗体库,为从中获得人源抗amylin的mAb奠定了实验基础。     

以鼠源重链Fd片段为模板导向筛选人源性抗γ-精浆蛋白抗体轻链

目的:以鼠源抗γ-sm抗体重链Fd片段为模板,筛选人源性抗人γ-精浆蛋白抗体轻链。方法:利用RT-PCR从前列腺癌患者外周血淋巴细胞扩增出全套的人抗体轻链基因,克隆入含鼠源性抗γ-sm抗体重链Fd片段的噬粒载体pComb3X/mFd中,电转化大肠杆菌XL1-Blue后建立人-鼠杂合的Fab抗体库。通过稀释滴定、限制性酶切和随机测序,对所建杂合库的库容、重组率和多样性分别进行鉴定,以M13K07辅助噬菌体超感染,利用亲和纯化的γ-sm为抗原,对挽救展示的噬菌体抗体库进行3轮淘筛和克隆鉴定。最后对筛选出的杂合抗体进行初步的功能检测。结果:构建成功1.2×107CFU、重组率为90%、多样性好的杂合人-鼠噬菌体抗体库。利用纯化的γ-sm3轮亲和淘筛后,5个克隆成功诱导表达特异性的pIII融合抗体。ELISA进一步检测表明,5个克隆均呈特异性阳性反应,其中2个克隆亲和力较高,测序显示其所含轻链基因序列完全相同,可变区来源于IGKV4-101胚系基因家族。结论:利用鼠源Fd片段为模板,导向筛选杂合噬菌体库,成功筛选到人源性抗人γ-精浆蛋白抗体轻链。     

人源性抗角蛋白抗体Fab段在巴氏毕赤酵母菌中的表达及表达条件的优化

目的:用巴氏毕赤酵母表达抗角蛋白抗体Fab段并优化表达条件。方法:将抗角蛋白人Fab原核表达载体p3MH/Fab中的κ链和Fd段基因,分别亚克隆到酵母菌表达载体pPIC9K和pPICZαA中,构建成重组质粒pPIC9K/L和pPICZαA/Fd并测序鉴定。将重组质粒pPIC9K/L和pPICZαA/Fd分步整合到酵母菌GS115的染色体上,经G418和Zeocin筛选得到高拷贝的转化子。对Mut表型鉴定后,优化pH及甲醇浓度等表达条件。结果:优化表达条件后,成功地表达抗角蛋白Fab段抗体。Westernblot分析证实,表达产物具有良好的角蛋白结合活性和特异性。结论:人源性抗角蛋白Fab段抗体在巴氏毕赤酵母菌中获得成功表达,为其进一步的应用研究打下了基础。     

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，克隆入载体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к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pSW1-2Fab/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；在大肠杆菌中的表达量约为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。     

半套式PCR扩增人抗HFRS病毒抗体基因及Fd段基因的克隆和序列分析

从肾综合征出血热恢复期患者外周血淋巴细胞（ＰＢＬ） 中提取细胞总ＲＮＡ, 反转录成ｃＤＮＡ 。以之为模板,用半套式聚合酶链反应（ＰＣＲ） 扩增人Ｉｇ Ｇ 抗体Ｆｄ 段及轻链基因,并将Ｆｄ 段基因克隆入噬菌体载体ｐｃｏ ｍ ｂ３ ;经酶切鉴定后,再亚克隆入ｐＧＥＭ７ｚｆ ,筛选阳性克隆测序。经计算机分析,Ｆｄ 段基因的全长为６３９ｂｐ ,编码２１３ 个氨基酸,属于人Ｉｇ Ｇ２ 亚类。