鼠源抗AFB1噬菌体单链抗体库的构建与鉴定

研究以AFB1-BSA免疫的小鼠脾脏细胞为试验材料,利用RT-PCR技术,克隆了抗AFB1抗体重链和轻链可变区基因VH和VL,利用连接肽(Gly4Ser)3将VH和VL链接成单链抗体基因scFv,通过噬菌体展示载体pCANTAB-5E携带将其电转化E.coli TG1,经氨苄青霉素平板筛选,构建了库容为2.13×109 cfu/μg DNA的噬菌体单链抗体库,抗体库的克隆阳性率达到100%,且多样性良好,为高活性抗AFB1单链抗体的筛选提供了材料基础。     

多药耐药性肿瘤细胞相关噬菌体抗体库的构建

利用噬菌体抗体库技术 ,构建了多药耐药性肿瘤细胞相关噬菌体抗体库。以多药耐药性肿瘤细胞 KB- A1为免疫原免疫 Bal B/c小鼠 ,从其脾脏提取总 RNA,并分离出 m RNA。RT- PCR扩增出 VH 和 VL 基因 ,经重叠延伸反应组装成单链抗体 ( Sc Fv)。将其克隆入噬菌粒载体p CANTAB 5 E中 ,电转化 E. coli TG1 ,成功构建了库容为 0 .8× 1 0 6的噬菌体抗体库 ,为获得多药耐药性相关分子及肿瘤细胞特异的噬菌体抗体奠定了基础     

噬菌体展示外源多肽的免疫原性研究

利用噬菌体展示技术将丝状噬菌体fd基因8克隆入pKK223-3质粒中，将白色念珠菌特异表位基因插入到修饰后的质粒载体中，并制备了杂合噬菌体，利用纯化的该表位抗原PA免疫小鼠，结果表明该噬菌体-表位抗原PA在有无佐剂存在的情况下均产生抗体，用ELISA方法采用双波长（450/630nm)检测抗体，其在有无佐剂存在的情况下光密度值无明显差别，免疫后小鼠脾淋巴细胞在有无佐剂的情况下均产生明显增殖，未见明显的毒副作用，具有良好的安全性，该方法产生的抗体与传统的合成多肽相比具有方法简便、低价高效等特点，它将成为生产高效低价疫苗的有效途径，该噬菌体-表位抗原PA为真菌疫苗的研制打下基础。     

小鼠噬菌体抗体库的构建和N-肽结合单链抗体的筛选

成功构建一库容量为１．７＊１０＾８的小鼠噬菌体抗体库，并从中淘选出对Ｎ－肽有结合适性的单链抗体。首先从未免疫小鼠的脾脏中提取总ＲＮＡ，分离出ｍＲＮＡ，经逆转录合成其总ｃＤＮＡ。随后通过ＰＣＲ分别扩增出抗体重链和轻链可变区ＶＨ、ＶＬ的基因片断，再经重组ＰＣＲ将两片断由一编码（Ｇｌｙ４Ｓｅｒ）３十五肽的接头连在一起，并克隆到噬菌质粒ｐＣＡＮＴＡＢ５Ｅ中，电击转化大肠杆菌ＴＧ１。经辅助噬菌体Ｍ１３ＫＯ７     

从噬菌体抗体库中淘选人绒毛膜促性腺激素的单链抗体

由鼠源噬菌体抗体库淘选到展示有人绒毛膜促性腺激素单链抗体的噬菌体克隆.用ELISA方法进行检测,从结合力最强的克隆中分离单链抗体基因,插入经改造的高效表达载体pET-21a(+)中,转化大肠杆菌,诱导表达.从培养基中可检测到可溶性抗人绒毛膜促性腺激素的单链抗体.     

小鼠凝血因子Ⅸ基因组DNA的结构分析和ES细胞基因打靶研究

为了利用ＥＳ细胞基因打靶技术建立人血友病Ｂ的转基因小鼠模型，用小鼠ＦＩＸ基因ｃＤＮＡ为探针，筛选１２９Ｓｖ小鼠的基因组ＤＮＡ噬菌体文库，从２＊１０＾５个噬斑中得到５个独立的阳性噬菌体克隆。     

EL-4荷瘤鼠单链噬菌体抗体库的构建

目的:构建一个EL-4荷瘤鼠的单链噬菌体抗体库,为筛选高特异性和高亲和力的单链抗体做准备.方法:于C57小鼠腋区接种EL-4细胞,待肿瘤长大后,从脾细胞中提取总RNA,RT-PCR技术扩增小鼠抗体重、轻链可变区基因(VH、VL),用Linker奖VH和VL基因连成单链抗体可变区片段(scFv).双酶切后(Not Ⅰ、Sfi Ⅰ)与预备好的pCANTAB5E噬粒载体连接,转化入感受态TG1,构建EL-4荷瘤鼠的单链噬茵体抗体库.随机抽取转化后的20个克隆,用以检测外源DNA的转入情况.结果:PCR扩增出的VH约有340bp和VL约有320bp,scFv的长度约有750bp.转化后的TG1约有1.67×107个茵落,随机挑取20个克隆,双酶切显示五分之一的茵落转化了外源DNA片段,有效库容为3.34×106.结论:成功构建了EL-4荷瘤鼠的单链噬茵体抗体库.     

噬菌体筛选技术筛选与联萘酚(BINOL)相结合的抗体

利用噬菌体展示技术筛选只结合1种对应体的BINOL抗体,建立人类单链抗体(scFv)噬菌体文库——Griffin.1文库,以固相化抗原淘筛抗体库,ELISA鉴定噬菌体抗体.从经过4轮富集的次级抗体库中挑选到噬菌体克隆,用该克隆制备的单链抗体阳性克隆,经ELISA证实具有良好的抗原特异性.从人类scFv噬菌体文库中获得抗BINOL的特异性抗体.     

129小鼠CgA基因cDNA 5''''端部分片段及CgA基因的克隆

通过RT-PCR方法,从129小鼠脑组织中克隆到CgA基因cDNA 5'端部分片段,序列分析结果表明,所克隆到的片段序列与文献中完全一致.以此片段为探针,从129小鼠基因组库中筛选获得阳性噬菌体1-1,克隆了CgA基因,并与文献中的小鼠CgA基因限制性内切酶图谱进行了比较分析.     

抗克伦特罗噬菌体单链抗体库的构建、筛选及鉴定

利用噬菌体展示技术构建克伦特罗(CBL)单链抗体(scFv)库,从中筛选CBL特异性噬菌体scFv,从而成功扩增出抗CBL的VL,VH基因片段并采用重叠延伸PCR拼接为全长的scFv基因片段,抗体库库容约为1.6×104,经4轮吸附—洗脱—扩增的富集,采用酶联免疫吸附测定(ELISA)法筛选到6个具有CBL结合活性的噬菌体scFv,为进一步大量表达CBL单链抗体奠定了基础.     

抗牙周病厌氧菌（Bg）单链抗体基因的构建与表达

从牙周病厌氧菌（Ｂａｃｔｅｒｏｉｄｅｓｇｉｎｇｉｖａｌｉｓ）单克隆抗体的杂交瘤细胞株出发，通过基因工程方法──ＰＣＲ技术，调出其抗体的重链和轻链基因可变区序列，体外重组后得到单链抗体ＳｃＦｖ（ｓｉｎｇｌｅｃｈａｉｎｆｒａｇｍｅｎｔｖａｒｉａｂｌｅ）片段，克隆到了ｐＣＡＮＴＡＢ５载体上，在ＥｓｃｈｅｒｉｃｈｉａｃｏｌｉＴＧｌ中得到了成功表达。     

抗α毒素单链抗体基因表达载体的构建

将 2种抗A型产气荚膜梭菌α毒素单链抗体 (ScFv)基因ScFv - 2E3和ScFV - 1A8分别克隆至表达质粒pUC119,pET - 2 0b ,pET - 2 8a和pHOG2 1中 ,构建了重组质粒PUC -2E3和pUC - 1A8,pET2 0b - 2E3和pET2 0b - 1A8,pET2 8a - 2E3和pET2 8a - 1A8以及pHOG - 2E3和pHOG - 1A8,然后分别转化至大肠杆菌中 ,提取质粒 ,并进行酶切鉴定和核苷酸序列分析 ,结果表明构建的重组质粒中均含目的ScFv基因片段 ,说明已成功构建了 8个含ScFv基因的表达质粒     

两种抗α毒素单链抗体基因的序列分析

应用RT－PCR技术,从两株分泌具有中和活性的抗A型产气荚膜酸菌α毒素单克隆抗体（McAb)的杂交瘤细胞株2E3和1A8中,分别扩增出抗体VH和VL基因,用Linker(Gly4Ser）3基因,将VH和VL基因连接成ScFv基因2E3－ScFv和1A8－ScFv,并将其克隆至pGEM-T载体中,经核苷酸序列分析证实,VH和VL基因以及Linker基因拼接正确,2E3－ScFv基因全长为729bp,经计算机分析,VH和VL基因均为新发现的基因序列,符合功能性重排的鼠抗体可燮区基因特征,2E3－ScFv的VH和VL基因分别属于鼠免疫球蛋白重链Ⅱ（B）和轻链kⅢ家簇;而1A8－ScFv的VH和VL基因分别属于鼠免疫球蛋白重链Ⅱ（A）和轻链кⅥ家簇。     

鼠源单抗3G1单链抗体基因的构建及序列分析

利用Rr—PCR方法，从分泌抗汉滩病毒mAb的杂交瘤细胞系3Gl中扩增出抗体VH和VL基因，通过PCR方法获得1inker—VL基因，进而利用加端PCR技术，在扩增出的单抗体VH和1inker—VL基因两端加上限制性酶切位点，分别克隆入pUCl8中并测序．3Gl VH基因长360bp，编码120个氨基酸；VL基因长324bp，编码108个氨基酸．在pUCl8载体中将VH和1inker—VL基因连接成ScFv基因．     

抗CPAα毒素单链抗体基因的克隆和表达及其生物学特性研究

应用RT—PCR技术 ,从分泌具有中和活性的抗A型产气荚膜梭菌 (CPA)α毒素单克隆抗体的杂交瘤细胞中 ,扩增出单链抗体 (ScFv)基因 ,并将其定向克隆于表达载体 pHOG2 1中 ,构建重组表达载体 pHOG 2E3,转化至大肠杆菌XL1 Blue中 ,筛选出表达菌株XL1 Blue(pHOG 2E3)。SDS PAGE分析结果表明 ,在 2 0℃用IPTG诱导培养时 ,表达的ScFv蛋白占菌体总蛋白的 2 5 %,ScFv蛋白主要以包涵体的形式存在 ,但在胞周质和培养上清中也能检测到ScFv蛋白 ,其中在胞周质中表达的ScFv蛋白占菌体可溶性蛋白的 4 %。生物学试验结果表明 ,ScFv基因表达产物不仅能够中和α毒素的磷酯酶C活性 ,而且对攻击致死剂量α毒素的小鼠产生良好的被动保护作用     

Expression of a VHC kappa chimaeric protein in mouse myeloma cells

The heavy (H) and light (L) chains of antibodies consist of variable (V) and constant (C) regions. The V regions of the heavy and light chains form the antibody combining site. To determine whether a V region could be functional when joined to a polypeptide other than its own C region, we constructed a chimaeric gene encoding the V region of a mouse heavy chain and the C region of a mouse kappa light chain ( VHC kappa). The heavy-chain gene is derived from an A/J mouse hybridoma cell line 36-65 whose antibody product (gamma 1, kappa) is specific for the hapten azophenylarsonate. We report here that, when introduced into a mouse myeloma cell line, the chimaeric gene is expressed and a protein of the expected molecular weight is secreted into the medium. As light chains tend to dimerize we expected that the VHC kappa protein might associate with light chain from the cell line 36-65 to form an antibody-binding molecule. Affinity binding experiments and Ka determination indicate that this is the case. Dimers of this type offer a novel and interesting alternative to existing antibody-binding molecules.     

Amplification of immunoglobulin lambda constant genes in populations of wild mice

The lambda immunoglobulin light chain (Ig lambda) locus of BALB/c inbred mice consists of two variable region gene segments (V lambda)1-3, and four constant region gene segments (C lambda)1,2,4,5. Each C lambda gene segment is associated with a unique joining segment (J lambda)2,4-7, and they are organized in two paired units, J3C3-J1C1 and J2C2-J4C4 (refs 4, 8). Using cDNA probes specific for C lambda 1 and C lambda 2 (ref. 9) we have analysed the genomic organization of the C lambda gene segments in wild-derived and inbred strains of mice. Although Southern blots of the genomic DNA of inbred mice show a constant pattern of hybridization, wild-derived mice show a high degree of variation in the number, size and intensity of hybridizing fragments. We have now found that, per haploid genome, mice of a Mus musculus musculus stock isolated from Sladeckovce, Czechoslovakia (CzII) have at least 12 C lambda segments, and mice of a Mus musculus domesticus stock 'Centreville Lights' from Centreville, Maryland (CL) have at least 8 C lambda segments. There appears to have been relatively recent amplifications of the C lambda gene segments in wild mice.     

Expression of functional interleukin-2 receptors in human light chain/Tac transgenic mice

The growth of mature T lymphocytes is regulated by interaction between interleukin-2 (IL-2) and its receptor. Three distinct binding sites for IL-2, namely low- (Kd 10 nM), intermediate- (Kd 100 pM) and high- (Kd 10 pM) affinity sites, have been found on human and primate T lymphocytes. Chemical crosslinking of labelled IL-2 to human T cells shows that two polypeptide chains, p55 (L chain) and p75 (H chain), bind IL-2 with low and intermediate affinities respectively. The high-affinity binding was shown to arise from ternary complex formation of IL-2, L and H chains. Construction of mutants of the L-chain complementary DNA indicated that the L chain is not directly involved in growth signal transduction. Nevertheless, expression of the IL-2 receptor L chain is tightly regulated by antigen or mitogen stimulation. To investigate the L chain function, we have produced transgenic mice using human L-chain cDNA of the IL-2 receptor under the control of a constitutive promoter. Studies on the L-chain transgenic mice showed that functionally active IL-2 receptors with high affinity were expressed on unstimulated spleen and thymus cells. The results indicate that the H chain of the IL-2 receptor is constitutively expressed in T cells.     

Efficacy of anti-CD20 chimeric Fab’ fragment on proliferation of B lymphoma cells

The variable domain of heavy chain (V_H) and light chain (V_L) genes of anti-CD20 monoclonal antibody HI⁴⁷ were cloned from anti-CD20 ScFv expression vector pCANBTEcd20 by PCR and ligated into vector pYZF to construct chimeric anti-CD20 Fab’ fragment expression vector pYZFcd20. Chimeric anti-CD20 Fab’ fragment was expressed inE. coli 16C9 and purified by protein G affinity chromatography. Competitive inhibition assay showed that anti-CD20 Fab’ fragment inhibited binding of HI⁴⁷ to CD20 on the surface of Daudi cells. Results from MTT assay indicated that chimeric anti-CD20 Fab’ fragment inhibited the proliferation of Daudi cells, IC₅₀ = 69 μg/mL. Affinity of chimeric anti-CD20 Fab’ fragment was determined, K_a was about 8.9×10⁸ (mol/L)⁻¹.