抗人TNF-α单抗基因的克隆及鉴定

目的克隆抗人ＴＮＦ－α鼠单抗得可变区基因以构建人－鼠嵌合抗体表达载体。方法采用ＲＴ－ＰＣＲ技术,以前导肽序列的引物从１个分泌抗人ＴＮＦ－α的鼠单抗杂交瘤细胞系中克隆抗体轻链、重链可变区基因（Ｖκ,ＶＨ）,在大肠杆菌中表达Ｆａｂ段核实其功能活性。结果分别得到了２个Ｖκ和２个ＶＨ基因。ＤＮＡ序列测定表明,其中１个轻链可变区基因为骨髓瘤细胞系中固有的无功能基因。１个重链可变区基因经原核系统表达测活表明无抗体活性。另一个轻链和重链可变区基因的成熟蛋白编码部分与从第一骨架区引物所克隆的、可在大肠杆菌表达出抗体活性的Ｖκ、ＶＨ序列相符。将该轻链、重链基因分别克隆到了人－鼠嵌合轻链、重链表达载体中。结论通过原核表达系统核实,获得了抗ＴＮＦ－α单抗的可变区基因     

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。     

两株人单抗重链可变区基因特征分析

对采用PCR方法克隆获得的两株抗出血热病毒人单抗重链可变区基因(V_H87—2,V_HC8)进行了基因特征分析。同源性比较显示:两基因来源于不同的人抗体重链可变区家族。V_H87-2与人免疫球蛋白重链可变区第三亚组同源性最高,达87％,而V_HC8与人免疫球蛋白重链可变区第一亚组同源性最高,达71％。空间结构分析表明两个可变区具有不同空间构象:V_H87—2属人抗体重链可变区1—1类空间结构,V_HC8属人抗体重链可变区1—2类空间结构。上述分析证明,此两株人单抗重链可变区存在着基因特征及蛋白空间构象的差异。通过基因分析掌握人单抗分子特征,对指导人源性基因工程抗体的研究具有重要意义。     

抗人ClqMcAb轻、重链可变区基因的克隆、序列分析及轻链可变区基因的表达（摘要）

抗人ＣｌｑＭｃＡｂ轻、重链可变区基因的克隆、序列分析及轻链可变区基因的表达（摘要）陈克敏，朱锡华应用ＰＣＲ技术，从一株抗人ＣｌｑＭｃＡｂ杂交瘤细胞基因组ＤＮＡ及反转录合成ｃＤＮＡ中，扩增、克隆分别获得抗人Ｃｌｑ轻、重链可变区基因，序列分析表明：抗人Ｃ...     

免疫球蛋白可变区基因的体外扩增、序列分析及人-鼠嵌合重链抗体的表达

本文用聚合酶链反应法(PCR)扩增了抗人小细胞肺癌单克隆抗体的轻、重链可变区基因(V_k、V_H基因)。逐步克隆将V_H基因与表达质粒重组,最终与载有人恒定区基因的载体拼接,得嵌合质粒的在哺乳类细胞中表达了人-鼠嵌合重链抗体。 1 免疫球蛋白可变区基因的体外扩增用酸性异硫氰酸胍-苯酚-氯仿抽提抗人小细胞肺癌单克隆抗体的细胞,得较高质量的总RNA,用oligo(dT)为引物合成第一链     

抗癌胚抗原鼠人嵌合抗体重、轻链基因在昆虫细胞中的表达

本文采用杆状病毒表达系统在昆虫细胞(Sf_9,秋粘虫细胞)中表达了抗癌胚抗原(CEA)鼠-人嵌合抗体重、轻链基因.将鼠源性单抗杂交瘤细胞中已克隆到的重、轻链可变区(V_H、Vk)基因分别与人免疫球蛋白恒定区基因Cr_3、Ck相拼接,构建鼠人嵌合抗体基因.含嵌合抗体基因的转移载体与线性化病毒DNA共转染Sf_9细胞,并通过点杂交、PCR扩增和Southern blot分析获得重组病毒.Western blot分析表明以重组病毒感染的Sf_9细胞分别表达了嵌合重链和轻链,放射免疫分析法(RIA)和间接ELISA测定的结果表明嵌合重、轻链基因表达产物具有与CEA结合的能力.     

免疫球蛋白重链可变区基因的结构及表达

在阐明真核基因组结构及功能的研究进程中,免疫球蛋白(Ig)基因十分引人注目,这不仅是由于它的结构比较复杂,而且由于它的表达牵涉体细胞分化过程中特有的DNA重组排列,突出反映了真核基因组织和表达的精巧。对Ig基因已有过不少综述,本文着重归纳重链(H)可变区(V)基因的结构和表达方面研究的进展。胚原型重链可变区基因的结构概貌重链V区(V_H)基因群与其3＇侧下游的恒定区(C_H)基因群相连成重链基因族(gene family),位于小鼠第12号染色体,     

HBV Pre S2 3B9单抗轻、重链可变区基因的分离,序列分析及单链可变区抗体基因的构建

3B9(杂交瘤细胞)是一株分泌HBV Pre S2抗原的单抗细胞,为从分子水平分析、了解3B9株进而为制备新一代基因工程抗体奠定基础,用分子生物学技术,提取3B9杂交瘤细胞总RNA,经反转录、PCR分离获得了3B9单抗的轻、重链可变区基因,并利用PCR及双脱氧核苷酸链末端终止法对3B9单抗的可变区基因的核苷酸序列进行了分析。结果表明,3B9单抗轻链隶属小鼠Ig Kappa轻链第Ⅱ亚组,JK_1基因重排;而3B9单抗重链隶属小鼠Ⅰg重链第Ⅱc亚组。在此基础上,作者用一编码疏水性多肽接头的DNA片断将3B9单抗轻、重链可变区基因连接,成功地构建了3B9单抗单链可变区抗体基因。为下一步克隆、表达有抗原结合活性的单链抗体打下了良好的物质基础。     

肝癌单抗重链可变区基因的克隆及序列测定

从分泌与肝细胞肝癌特异性强并具有良好人体内导向作用的鼠源性单克隆抗体的杂交瘤细胞ＨＡｂ２５中提取ＲＮＡ，逆转录成ｃＤＮＡ，用合成的寡核甘酸引物从中扩增出抗体重链可变区基因。将此基因克隆到ｐＵＣ１９质粒中，测定了此可变区基因的全序列。经计算机分析，结果表明基因长度为３６０ｂｐ，编码１２０个氨基酸，是具有功能性的抗体可变区基因，在核苷酸序列上与小鼠重链ＶＨ１８６．２家族同源性最高，属免疫球蛋白重链可变区基因９个家族中的第三族     

抗阿尔茨海默病Aβ人-鼠嵌合抗体基因的真核载体构建和表达

目的 通过基因工程抗体技术构建和表达抗β-淀粉样多肽(Aβ)人-鼠嵌合抗体,减低鼠源单克隆抗体在临床应用中引起的人体免疫排斥反应.方法从分泌抗Aβ1-42鼠单克隆抗体杂交瘤细胞株中提取总RNA,用逆转录-聚合酶链反应(RT-PCR)扩增鼠源性抗体全长基因,并通过Blast对其序列进行分析;利用重组PCR技术拼接重轻链可变区及人IgG1的恒定区基因,并对重链Fc段进行定点突变以降低排斥反应;分别构建人-鼠嵌合基因重轻链表达载体,用脂质体法将其同时导入COS-7细胞中表达,并利用ELISA和免疫组织化学(SP法)对分泌的抗体功能和性质进行初步鉴定.结果 Blast比对分析结果显示克隆的基因序列符合小鼠抗体基因序列,将可变区基因与人IgG1的恒定区基因拼接以及Fc定点突变后,成功构建了嵌合抗体的真核表达载体,并实现真核表达;ELISA和免疫组织化学方法证实了所分泌抗体的人源性和与Aβ的结合特异性.结论成功地构建和表达了抗阿尔茨海默病Aβ人-鼠嵌合抗体,为其在阿尔茨海默病的临床诊治中应用和进一步改造奠定了基础.     

β-肌球蛋白重链基因与肥厚型心肌病临床表型关系的研究

目的 研究肥厚型心肌病(hypertrophic cardiomyopathy,HCM)的主要致病基因β-肌球蛋白重链基因(beta-myosin heavy chin gene,MYH7)的突变位点,探寻基因型与表型的关系.方法 扩增3个HCM家系成员的MyH7基因第3、5、7～9、11～16、18～23外显子序列,进行直接测序分析,应用软件与标准序列比对,确定可能的突变位点.结果 发现其中1个家系MYH7基因第14外显子存在Thr441Met 突变,正常对照组相同位置未见异常.3个家系均发现多个同义突变位点.结论 在中国汉族人群中发现MYH2基因Thr441Met突变,该突变位于β-肌球蛋白重链头部肌动蛋白结合位点,可能是HCM的致病突变.HCM具有遗传异质性,多种因素可能参与其发生和发展过程.     

Analysis of the immunoglobulin heavy chain gene variable region of 101 cases with peripheral B cell neoplasms and B cell chronic lymphocytic leukemia in the Japanese population

We have analyzed the immunoglobulin heavy chain (VH) gene variable regions (CDR2 and FW3) of 101 Japanese cases with peripheral B cell neoplasms. When all except one case with a deletion were graphed by frequency of replacement mutation, the 100 cases could be separated into two groups: 24 cases with zero, one and two mutations (germline or low frequency of somatic mutation); and 76 cases with three or more mutations (medium to high frequency of somatic mutation). While most mantle cell lymphoma cases (11/13) showed germline or low frequency of somatic mutation, all cases of mucosa-associated lymphoid tissue (MALT) lymphoma (11/11), follicular lymphoma (three of three cases), plasma cell myeloma (seven of seven cases) and most cases of diffuse large B cell lymphoma (DLBCL; 42/47) belonged to the latter group. These 76 cases, therefore, may be considered to show somatic hypermutation. More than half of chronic lymphocytic leukemia/small lymphocytic lymphoma cases (CLL/SLL; eight of 13) showed a hypermutated VH gene and the ratio of replacement mutation: silent mutation in CDR2 of CLL/SLL was considerably higher compared with DLBCL and MALT lymphoma, showing somatic hypermutation. When comparing VH gene type of B cell-CLL (B-CLL) among our series and those in the literature, more cases of CD5+ B-CLL in the Western literature have the VH5 and VH6 family types, while more cases in Japan are reported to have VH4 family. The occurrence of VH families in B-CLL between Japanese and Western people seems to be comparable.     

中国人群慢性淋巴细胞白血病免疫球蛋白重链可变区基因组成及突变分析

目的 研究中国慢性淋巴细胞白血病(chronic lymphocytic leukemia,CLL)患者免疫球蛋白重链可变区(immunoglobulin variable heavy chain region,IGVH)基因各个片段的组成以及突变情况.方法 应用多重PCR技术扩增64例CLL患者的IGVH基因片段,纯化PCR扩增产物后直接测序,应用IMGT/V-QUEST及IGBlast数据库分析,明确VH基因有无突变和突变位置,以及VH、DH,JH家族各个成员组成情况.结果 64例CLL患者中,VH3家族31例(48%)、VH4家族26例(41%)、VH1家族4例(6%)、VH2家族2例(3%)、VH7家族1例(2%).44例患者发生体细胞突变,占CLL患者的69%;20例无突变,占CLL患者的31%,其中6例(9%)VH基因的同源性为100%.VH4家族中有9例无突变(9/26,35%);VH3家族中8例无突变(8/31,26%);VH1家族中1例无突变(1/4,25%).在所检测的CLL标本中,VH4-34是最常见的VH基因片段,检测出8例(13%),其中无突变3例;其次为VH4-59,检测出7例(11%),无突变者3例.DH基因中DH3家族最常见,有25例(39%);其中11例(11/20,55%)出现在无突变组中.无突变组中最常见的DH基因片段为DH3-10和DH3-22,各为4例(4/20,20%).JH基因中JH6家族最常见,检测出23例(36%),其中9例出现在无突变组中(9/20,45%).结论 中国CLL患者IGVH基因家族表达比例和突变情况与西方国家存在显著差异,推测可能与不同的种族和环境中的抗原选择有关,其预后意义有待进一步探讨.     

Inducible nuclear factors binding the IgM heavy chain pre-mRNA secretory poly(A) site

Two alternative forms of IgM heavy-chain mRNA are produced from a common precursor mRNA as a result of competition between cleavage/poly(A) addition at the upstream (secretory) poly(A) site and cleavage/poly(A) addition at the downstream (membrane) poly(A) site coupled with splicing. The efficiency of cleavage at the secretory poly(A) site is thought to play a crucial role in this alternative processing. We therefore examined RNA binding factors recognizing the secretory poly(A) site, in the absence of the splicing option, to look for transacting factors that may play a role in cleavage/polyadenylation efficiency at this site. Purified primary B cells produce the secretory form of μ mRNA when stimulated with lipopolysaccharide (LPS) and the membrane form of μ mRNA when their antigen receptors are ligated by anti-μ antibodies. We compared RNA binding factors in nuclear extracts from cells produced by these different stimulatory conditions and show that induction of the secretory form of μ mRNA by LPS correlates with the induction of a 28–32-kDa secretory poly(A) site-specific polypeptide which is also present in the plasmacytoma cell line J558L. Visualization of the 28–32-kDa polypeptide in UV cross-linking assays depends on a GU-rich element downstream of the secretory poly (A) site. We show that this GU-rich region enhances polyadenylation efficiency in vivo by transfection of luciferase reporter constructs into the plasmacytoma J558L. We also examined nuclear extracts from B cells doubly stimulated with LPS and anti-μ antibodies in which expression of the secretory form of μ mRNA is selectively inhibited. This inhibition may be due to a down-regulation of polyadenylation at the secretory poly(A) site or an up-regulation of the competitive splicing process. This form of stimulation does not lead to the disappearance of the 28–32-kDa polypeptide, but to an enhanced binding of a 50–55-kDa factor which binds both the secretory and membrane poly(A) site. We report the first detection of changes in RNA binding factors taking place at the secretory poly(A) site which correlate with the expression of different forms of μ mRNA produced by primary B cells under different stimulation conditions.     

抗癌胚抗原单链抗体与核心链霉亲和素基因的融合及在大肠杆菌的表达

制备抗癌胚抗原 (CEA )单克隆抗体的单链抗体 ,在保留抗原抗体结合位点的同时有效降低抗体的分子量不仅可减少人抗鼠抗体反应 (HAMA ) ,而且适合放射免疫显像。为纯化及核素标记抗体 ,将单链抗体基因与核心链霉亲和素基因融合并在大肠杆菌得到高效表达 ,表达量占菌体总蛋白的 2 4%。SDS PAGE和蛋白质印迹图谱显示表达产物分子量为 41kD ,与其基因编码蛋白质的理论推算值相符。表明融合蛋白能特异性地与生物素结合 ,RIA表明表达产物具有结合其特异性抗原CEA的能力 ,以HRP标记的生物素作为抗体进行蛋白质印迹在 41kD处可见表达条带。说明表达物不仅能与生物素结合且能特异性地与CEA结合。     

Polymerase chain reaction (PCR) detection of B cell clonality in Sjögren's syndrome patients: a diagnostic tool of clonal expansion

Sjögren's syndrome (SS) is an autoimmune disease characterized by clonal B cell attack of the exocrine glands and dysregulated expression of B cell‐activating factor (BAFF). Based upon the current data of increased rates of lymphoid malignancy, as non‐Hodgkin's lymphoma (NHL) is associated with SS, we propose the detection of clonal rearrangements of immunoglobulin heavy chain (IgH) gene in those patients as a predictor of malignant clonal expansion. To test our proposal, we examined the IgH clonal rearrangements in SS patients (60) and healthy control subjects (42) having chronic non‐specific sialadenitis, to determine the presence of clonal B cells in minor labial salivary glands (MSG) of SS patients. Clonal B cell expansion was assessed by two polymerase chain reaction (PCR) assays: (i) semi‐nested PCR, against sequences encoding framework regions FR3, FR2 and FR1c of the variable chain IgH gene in B cells present in the MSG infiltrate; and (ii) the PCR–enzyme‐linked immunosorbent assay (ELISA) technique, against the major and minor breakpoint regions of the Bcl‐2 oncogene coupled with a variable segment of the IgH to assess the Bcl‐2/JH translocation. When FR3, FR2 and FR1c primers were employed, we detected B cell monoclonality in 87% of the SS patients and 19% of the control subjects. The association between inflammation severity of the MSG pattern and the presence of B cell clonality was found to be statistically significant (P < 0·01). We concluded that the presence of B cell clonality in MSG can be used as a index of an altered microenvironment favouring the development of lymphoma in SS patients.     

Polymerase chain reaction screening of immunoglobulin heavy chain and T cell receptor γ gene rearrangements: A practical approach to molecular DNA analysis of non-Hodgkin's lymphoma in a surgical pathology laboratory

Characterization of the clonality of non-Hodgkin's lymphoma (NHL) by the rearranged segments of immunoglobulin heavy chain (Ig(H)) or T cell receptor (TCR) genes is not only useful in the confirmation of the diagnosis but also for the future assessment of how a secondary lymphoma, such as a recurrence or another primary lymphoma, occurs. As a practical approach to obtaining and registering this information in a surgical pathology laboratory, FR3 and FR1 regions of Ig(H) gene and TCRgamma gene were concurrently amplified by polymerase chain reaction (PCR) using each pair of consensus primers and the same PCR protocol. Examined samples consisted of 134 primary NHL (phenotypically, 108 B cell and 26 T cell NHL), 19 reactive lymphadenopathies, as well as five secondary lymphomas whose primary lesions were included in this study. Among the primary NHL, the combined PCR analysis disclosed the clonality in 103 of 134 NHL (77%), by FR3 PCR in 77 B cell and two T cell NHL, by FR1 PCR in 59 B cell and one T cell NHL, and by TCRgamma PCR in 11 B cell and 17 of 26 T cell NHL, but in none of the reactive lymphadenopathies. Among the secondary lymphomas, the same pattern of PCR analysis was obtained in two cases (the durations between first and second lymphomas; 6 and 10 months), which suggested recurrence. In contrast, different results were obtained in three cases (17-37 months), which indicated another primary or emergence of the subclones. The results of Southern blot analysis were concordant with the PCR results of the first and the secondary lymphomas. Although the combined PCR analysis cannot replace Southern blot hybridization because of its lower detection rate, it can select those cases suitable for further Southern blot analysis thus reducing the number of unnecessary examinations by nearly 75%. This approach may also be useful in the comparative evaluation of primary and secondary lymphomas.     

抗BAFF单克隆抗体轻链和重链可变区基因的克隆及鉴定

目的:在本实验室成功制备小鼠抗人BAFF单克隆抗体的基础上,通过分子克隆方法获得该抗体可变区基因序列。方法:从1株小鼠抗人BAFF单克隆抗体杂交瘤细胞FMMUB4中提取总RNA,以此为模版反转录成cDNA,用针对小鼠单克隆抗体重链和轻链可变区基因序列的特异性引物分别进行PCR反应,PCR产物连接入载体,经筛选阳性克隆、酶切鉴定后送测序,测序结果进行生物信息学分析。结果:成功克隆了抗BAFF单克隆抗体FMMUB4重链和轻链可变区基因,测序结果证实序列正确。结论:获得了抗BAFF单克隆抗体FMMUB4重链和轻链可变区基因序列,为下一步构建相关基因工程抗体打下良好基础。     

Immunoglobulin heavy chain rearrangements in primary brain lymphomas. A study using PCR to amplify CDR-III

Primary brain lymphomas (PBLs) have only rarely been analysed for immunoglobulin heavy chain (IgH) rearrangements. In this study, DNA was extracted from paraffin blocks in 23 cases of PBL and examined for IgH rearrangements using the polymerase chain reaction (PCR) to amplify the complementarity-determining region III (CDR-III) of rearranged IgH genes. Fifteen of the cases were phenotyped on paraffin-embedded tissue using a pan-B and pan-T antibody (L26 and UCHL-1, respectively). The remaining eight cases were not phenotyped for lack of tissue. For comparison, we used DNA extracted from paraffin blocks of normal brain, lymph nodes with lymphoid hyperplasia, and non-lymphoid malignancies. PCR products were examined by polyacrylamide gel electrophoresis. Among the ten B-cell PBL; four had a pattern indicative of IgH rearrangement, one had a germline pattern, and five had no detectable PCR products. Among the five T-cell PBLs, one had a germline pattern and four had no detectable products. Among the eight untyped PBLs, two had IgH rearrangement, four had a germline pattern, and two gave no detectable products. DNA from non-lymphoid tissues gave a consistent germline pattern, while DNA from polyclonal lymphoid populations (lymph node) had a pattern of polyclonal IgH rearrangement. In a dilution study, a clonal rearrangement could be detected as long as the clone's DNA constituted at least 10 per cent of the total DNA. PCR to amplify CDR-III can be successfully applied to DNA extracted from paraffin blocks, and it detected a clonal rearrangement in 50 per cent of cases that gave a detectable pattern. This allows clonality analysis of tissue unsuitable for conventional Southern blot analysis. Furthermore, B-cell PBLs have IgH rearrangements similar to those of extracranial B-cell neoplasms.     

在昆虫细胞中表达HSV糖蛋白单域抗体的重组杆状病毒的构建

设计一对含EcoRⅠ—起始码和终止码—SalⅠ的鼠抗体V_H引物,用PCR法对重组质粒p1A12V_H中插入基因1A12V_H进行突变,突变的PCR产物的序列测定表明,在1A12V_H基因两端成功地插入了EcoRⅠ—起始码和终止码—SalⅠ。将突变后1A12V_H依次克隆入pSL301质粒和pAcEEUL8质粒中,使ⅠA12V_H基因两端均接上BamHⅠ接头,用BanHⅠ酶切,将1A12V_H基因克隆入杆状病毒表达载体pAcCL29质粒的多角体基因中的唯一BamHⅠ切点处,经计算机分析,用BamHI;EcoRV/EcoRⅠ;SalⅠ三组酶切,筛选出克隆的1A12V_H基因方向与多角体启动子基因方向一致的重组杆状病毒转基因质粒pAc1A12V_H。CsCL—EB梯度平衡离心法纯化pAc1A12V_H,转化AcNPV基因组DNA,然后转染的sf9细胞,空斑筛选和纯化1A12V_H—AcNPV重组杆状病毒,并用PCR法作进一步鉴定。