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。     

利用线性表达框高通量表达人源单克隆抗体

目的:建立不通过克隆步骤高通量表达来源于人单个B细胞的抗体轻、重链基因的方法。方法和结果:PCR扩增3个末端重叠的DNA片段,即1CMV启动子和编码抗体引导区序列的片段;2抗体Ig G1重链恒定区序列和牛生长激素(BGH)poly(A)信号序列,轻链Igκ恒定区序列和BGH poly(A)信号序列,轻链Igλ恒定区序列和BGH poly(A)信号序列;以及3抗体基因可变区序列V_H、V_κ或V_λ。3个片段通过重叠延伸PCR构建全长线性片段即线性表达框,将此来源于人单个B细胞的配对的抗体轻、重链线性表达框共转染293E细胞,72 h收集上清检测到表达的抗体。结论:构建的抗体基因线性表达框是无须克隆,快速高通量表达抗体基因进行筛选分析的策略。     

抗人前列腺干细胞抗原单克隆抗体可变区基因的5＇RACE扩增及序列分析

目的：克隆并分析抗人前列腺干细胞抗原单克隆抗体轻链和重链的可变区基因。方法：从分泌抗人前列腺干细胞抗原单克隆抗体的杂交瘤细胞株中提取总RNA,根据小鼠IgG恒定区序列设计特异性引物,通过5’RACE法扩增其轻链和重链的可变区基因,克隆入pMD18-T载体,测序并分析其可变区序列。结果：3株抗人前列腺干细胞抗原单克隆抗体的重链可变区基因序列全长均为423bp,编码141个氨基酸残基;轻链可变区基因序列全长均为393bp,编码131个氨基酸残基;在GenBank中对氨基酸序列进行比对分析,均符合小鼠IgG可变区基因的特征;根据Kabat法则对3株抗体轻链和重链可变区氨基酸序列进行分析,确定了3个抗原互补决定区、4个框架区和前导肽。结论：通过5＇RACE法得到了3株抗人前列腺干细胞抗原单克隆抗体轻链与重链可变区基因,为进一步研究抗体三维结构、人源化改造奠定了基础。     

抗D－双聚体单抗轻链可变区基因的克隆

以分泌小鼠抗人纤维蛋白降解产物D－双聚体单克隆抗体杂交瘤细胞株的mRNA为模板,用小鼠免疫球蛋白轻链可变区基因的通用引物,通过RT－PCR扩增基因,与载体重组后,经酶切鉴定和核苷酸序列分析,证明克隆含抗D－双聚体单抗的轻链可变区基因,长度为330 bp．     

抗人CD3单抗重,轻链可变区基因的体外扩增,克隆和序列分析

根据免疫球蛋白重链和轻链可变区基因５＇端序列和Ｊ区序列,化学合成适合于体外扩增抗体重、轻链可变区基因的二对引物。从体外培养的ＯＫＴ３杂交瘤细胞中提取总ＲＮＡ,反转录生成ｃＤＮＡ,以ｃＤＮＡ为模板,分别加入合成的重、轻链可变区引物进行ＰＣＲ,扩增出抗体重、轻链可变区基因片段。将扩增产物分别插入ｐＵＣ１９质粒,筛选出阳性克隆,用链终止法进行ＤＮＡ序列测定。所测重链可变区基因全长３５７ｂｐ,编码１１９个     

抗人CD3单抗重、轻链可变区基因的体外扩增、克隆和序列分析

根据免疫球蛋白重链和轻链可变区基因５’端序列和Ｊ区序列,化学合成适合于体外扩增抗体重、轻链可变区基因的二对引物。从体外培养的ＯＫＴ３杂交瘤细胞中提取总ＲＮＡ,反转录生成ｃＤＮＡ,以ｃＤＮＡ为模板,分别加入合成的重、轻链可变区引物进行ＰＣＲ,扩增出抗体重、轻链可变区基因片段。将扩增产物分别插入ｐＵＣ１９质粒,筛选出阳性克隆,用链终止法进行ＤＮＡ序列测定。所测重链可变区基因全长３５７ｂｐ,编码１１９个氨基酸,轻链可变区基因全长３２１ｂｐ,编码１０７个氨基酸。     

β淀粉样肽单克隆抗体可变区基因的5′RACE扩增及序列分析

目的:克隆并分析抗β淀粉样肽单克隆抗体轻链与重链可变区基因。方法:从分泌抗β淀粉样肽单克隆抗体的杂交瘤细胞株A8中提取总RNA,根据恒定区序列设计基因特异性引物,通过5′RACE法扩增抗体的轻链和重链可变区基因,测定并分析可变区基因序列,并克隆入pMD18-T载体。结果:重链可变区基因序列全长450bp,编码150个氨基酸残基;轻链可变区基因序列全长429bp,编码143个氨基酸残基。在GeneBank中对氨基酸序列进行比对分析,二者均符合小鼠IgG可变区基因的特征。根据Kabat法则对A8抗体轻链和重链可变区氨基酸序列基因进行分析并确定了3个抗原互补决定区(CDR)、4个框架区(FR)和信号肽。结论:通过5′RACE法得到了抗β淀粉样肽单克隆抗体轻链与重链可变区基因,为进一步研究抗体三维结构,以及对该抗体进行人源化改造奠定了基础。     

人免疫球蛋白重链可变区基因引物设计方法的改良

针对抗体胚系基因数据库的数据不断更新和完善,为获得人全部免疫球蛋白(Ig)重链可变区基因,改进引物设计方法,自主设计针对可变区基因高度保守的框架区1(FR1)和框架区4(FR4)的引物,提取未经免疫的健康人外周血单个核细胞,通过RT-PCR扩增重链可变区基因.其DNA序列与GenBank数据库和IMGT/V-QUEST软件比对,序列分析符合人免疫球蛋白重链基本框架结构,为胚系基因重排产生的序列.多个克隆的测序结果对比分析显示了良好的多样性.获得的重链序列为研制基因工程抗体及构建噬菌体抗体库奠定了物质基础,也为扩增其他物种Ig可变区基因的引物提供新的设计思路.     

2009年中国流行的甲型流感病毒（H1N1）血凝素特征分析

目的研究甲型流感病毒（H1N1）暴发流行以来中国各地甲型流感病毒血凝素（HA）的特征。方法搜索甲型流感病毒（H1N1）暴发流行以来中国各地报道的血凝素（HA）的氨基酸序列,比较当年不同时期血凝素（HA）的氨基酸序列的变化,并比较2009年报道的血凝素（HA）的氨基酸序列和2008年、2007年报道的血凝素（HA）的氨基酸序列作比较,以分析和前2年血凝素（HA）氨基酸序列相比所发生的变化。结果2009年中国各地甲型流感病毒（H1N1）的血凝素（HA）的氨基酸序列（人源）的同源性为99%-100%,但和2008年以及2007年的同源性非常低,分别为70%-77%和71%-90%。结论2009年暴发流行的甲型流感病毒（H1N1）的血凝素氨基酸序列较往年发生了很大程度的变异,这可能是今年甲型流感病毒（H1N1）暴发流行的主要原因。     

H5N1亚型禽流感病毒血凝素蛋白单链抗体基因的克隆及表达

构建并表达H5N1亚型禽流感病毒血凝素蛋白单链抗体,为禽流感靶向治疗药物的研制制备靶向载体。从分泌血凝素单克隆抗体的杂交瘤细胞株中提取mRNA,采用RT-PCR法扩增出重链和轻链可变区基因,通过SOE-PCR法将重链和轻链通过Linker连接起来构建单链抗体基因,将获得的单链抗体基因装入原核表达载体pET28a(+)中,构建重组质粒并表达,以Western blot鉴定单链抗体的特异性。结果成功构建了单链抗体基因,全长714bp,经原核表达,所构建的单链抗体可与H5亚型禽流感病毒HA蛋白特异结合,为禽流感的靶向治疗奠定了基础。     

抗H5N1病毒嵌合IgA抗体基因的构建及其在CHO细胞中的表达

为了表达具有中和活性的抗禽流感H5N1病毒人-鼠嵌合IgA抗体,采用RT-PCR法克隆具有中和活性的抗禽流感H5N1-HA鼠源单克隆抗体的轻重链可变区基因及相应的信号肽编码序列,分别与人免疫球蛋白IgA2重链恒定区、Kappa恒定区基因拼接,构建表达质粒pEF-IGHA9和pEF-IGK9,共转染二氢叶酸还原酶缺陷型CHO(CHO-dhfr-)细胞,用ELISA检测培养上清中嵌合IgA抗体的表达,对纯化的嵌合抗体进行SDS-PAGE、Western blotting印迹分析。结果成功地在CHO细胞中表达了抗禽流感H5N1病毒人-鼠嵌合IgA抗体,为制备抗H5N1重组分泌型IgA预防性抗体制剂奠定了良好的基础。     

Cloning and characterization of cDNAs coding for heavy and light chains of a monoclonal antibody specific for pre-S2 antigen of hepatitis B virus.

Binding specificity of a monoclonal antibody (mAb) (kappa, gamma 2b) H8 which can react with the pre-S2 peptide of hepatitis B virus (HBV) was determined by Western blot analyses. From the hybridoma cell line secreting mAb H8, poly(A)+ RNA was prepared and used as a template for cDNA synthesis and cloning. Full-length cDNAs coding for the heavy and kappa light chains of the mAb were cloned from the cDNA library and characterized by nucleotide (nt) sequence analyses and N-terminal amino acid sequencing. The sequence analyses revealed that both heavy and light chain-specific cDNAs are functional, and the variable regions of the heavy and light chains are members of mouse heavy chain subgroup III(c) and light chain group I, respectively. Comparison of the nt sequences with mouse immunoglobulin genes listed in the GenBank data base show that the cDNAs have not been previously reported. The cDNAs will be used for the construction of a therapeutic antibody for HBV infection.     

三株交叉反应性的流感病毒HA抗体与H1N1流感病毒的致病机制分析

目的:结合临床甲型流感病例分析流感病毒可能的致病机理。方法:收集87份陕西省2009年甲型H1N1流感重症,危重症及死亡病例的血常规参数,对其淋巴细胞、红细胞和血小板三个指标分析。制备针对甲型H1N1的单克隆抗体,采用抗体亚类鉴定试剂盒分析其抗体轻链和重链的亚型,通过血凝活性实验检测三株抗体的血凝抑制活性,通过ELISA检测三株抗体与人和小鼠的血红蛋白、红细胞、白细胞膜和血小板膜的反应,通过免疫组化分析三株流感病毒抗体与正常小鼠肺组织的结合。结果:流感病毒感染后的死亡病例中淋巴细胞、红细胞和血小板均明显降低。三株抗体与人和小鼠的淋巴细胞、红细胞和血小板均有不同程度的交叉反应;免疫组化结果同时也证实三株HA抗体与小鼠的肺组织有不同的结合力。结论:流感病毒致病的原因可能与流感病毒感染机体后产生的抗体可与血液和组织中的成分结合有关。     

Rapid Generation of Human-Like Neutralizing Monoclonal Antibodies in Urgent Preparedness for Influenza Pandemics and Virulent Infectious Diseases

Background

The outbreaks of emerging infectious diseases caused by pathogens such as SARS coronavirus, H5N1, H1N1, and recently H7N9 influenza viruses, have been associated with significant mortality and morbidity in humans. Neutralizing antibodies from individuals who have recovered from an infection confer therapeutic protection to others infected with the same pathogen. However, survivors may not always be available for providing plasma or for the cloning of monoclonal antibodies (mAbs).

Methodology/Principal Findings

The genome and the immunoglobulin genes in rhesus macaques and humans are highly homologous; therefore, we investigated whether neutralizing mAbs that are highly homologous to those of humans (human-like) could be generated. Using the H5N1 influenza virus as a model, we first immunized rhesus macaques with recombinant adenoviruses carrying a synthetic gene encoding hemagglutinin (HA). Following screening an antibody phage display library derived from the B cells of immunized monkeys, we cloned selected macaque immunoglobulin heavy chain and light chain variable regions into the human IgG constant region, which generated human-macaque chimeric mAbs exhibiting over 97% homology to human antibodies. Selected mAbs demonstrated potent neutralizing activities against three clades (0, 1, 2) of the H5N1 influenza viruses. The in vivo protection experiments demonstrated that the mAbs effectively protected the mice even when administered up to 3 days after infection with H5N1 influenza virus. In particular, mAb 4E6 demonstrated sub-picomolar binding affinity to HA and superior in vivo protection efficacy without the loss of body weight and obvious lung damage. The analysis of the 4E6 escape mutants demonstrated that the 4E6 antibody bound to a conserved epitope region containing two amino acids on the globular head of HA.

Conclusions/Significance

Our study demonstrated the generation of neutralizing mAbs for potential application in humans in urgent preparedness against outbreaks of new influenza infections or other virulent infectious diseases.     

MDCK单克隆细胞库的建立及检定

目的:建立甲型H1N1流感病毒疫苗株高适应MDCK单克隆细胞库,用于培养生产流感病毒疫苗,为细胞代替鸡胚制备流感病毒疫苗提供保证。方法:用有限稀释法将MDCK细胞进行单克隆化,通过血凝和TCID50筛选甲型H1N1流感病毒疫苗株的高适应性单克隆化细胞株,扩大培养建立细胞库并进行检定。结果:共制备了97株单克隆化MDCK细胞,筛选到甲型H1N1流感病毒疫苗株高适应性MDCK单克隆细胞株,扩大培养建立了细胞库,经检定符合《中华人民共和国药典.三部》(2010版)对细胞库的要求。结论:建立了甲型H1N1流感病毒疫苗株高适应性MDCK单克隆细胞库,为细胞培养生产流感病毒疫苗奠定了基础。     

Studies on the influence of different designs of eukaryotic vectors on the expression of recombinant IgA

Production and application of therapeutic monoclonal antibodies are second only to vaccines in the world pharmaceutical market. The most common therapeutic antibodies are monoclonal antibodies (mAbs) of the IgG isotype that are produced in eukaryotic CHO cells. In recent years, there has been a considerable interest in developing treatment medications based on IgA antibodies, which can have a wide range of effector functions on human mucous membranes. To study the expression level of immunoglobulin A (IgA) in mammal cells, we designed a set of bipromoter (CMV and EF1α) vectors. The vectors contain gene fragments that encode the heavy chain variable domain (VH) and the light chain variable domain (VL) of the human monoclonal antibody FI6v3 against the hemagglutinin of influenza virus A. They also contain gene fragments that encode the light chain (kappa type) constant domain and the heavy chain constant domain of the human antibody IgA1. The expression vectors differed in the orientation of the promoters and the presence or absence of introns. Two variants of the full-length light and heavy chains were cloned into a eukaryotic expression vector in head-to-head and head-to-tail orientations. The resulting plasmids were transfected into CHO-DG44 and HEK-293T cells. The antibody expression level for the stable transfection of CHO-DG44 and HEK-293T cell cultures was determined by ELISA. The results of the experiments showed that the expression of FI6v3-IgA1 antibodies significantly increased when eukaryotic cells were transfected with the plasmid pBiPr-ABIgA1FI6-Iht in which the heavy chain of IgA1 contains introns and the promoters are arranged head-to-tail.     

Structural and functional implications of a restricted antibody response to a defined antigenic region on the influenza virus hemagglutinin.

A group of hybridoma antibodies that recognize structurally overlapping epitopes on the influenza virus hemagglutinin have been analyzed for the sequence of their immunoglobulin heavy and light chain variable regions. All VH regions derive from the same gene family, and only two Vk genes, from different families, are involved. The repetitive and restricted use of these variable region genes indicates that considerable structural requirements influence the generation of antibodies specific for this region of the hemagglutinin. The degree of amino acid variability which is permissive for interaction with this region suggests that two thirds of the possible replacement mutations may abolish either antibody function or specificity. Analysis of the somatic mutation which occurred in the individual antibodies indicates that the light chains acquired replacement mutations at the rate predicted for random mutation. The heavy chains, however, accumulated a 3-fold excess of replacement mutations over that predicted for random accumulation, correlating with the dominant role they apparently play in determining fine differences in the specificity of these antibodies. The effect of somatic mutation on the clonal amplification and diversification of these B cell lineages is discussed.     

Cloning and complete nucleotide sequence of mouse immunoglobulin gamma 1 chain gene.

The 6.6 kb DNA fragment coding for the immunoglobulin γ1 chain was cloned from newborn mouse DNA using λgtWES·λB as the EK2 vector. The complete nucleotide sequence (1823 bases) of the γ1 chain gene was determined. The cloned gene contained the entire constant region gene sequence as well as the poly(A) addition site, but not the variable region gene. The results indicate that the variable and constant region genes of immunoglobulin heavy chain are separated in newborn mouse DNA. The constant region genes of other gamma chains (that is, γ2a, γ2b and γ3) are not present in the cloned DNA fragment. The sequence demonstrates that the γ1 chain gene is interrupted by three intervening sequences at the junction of the domains and the hinge region, as previously shown in the γ2b and α chain genes and in the γ1 chain gene cloned from myeloma. The results suggest that the intervening sequence was introduced into the heavy chain gene before divergence of the heavy chain classes, and also support the hypothesis that the splicing mechanism has facilitated the evolution of eucaryotic genes by linking duplicated domains or prototype peptides not directly adjacent to one another. Comparison of the nucleotide sequence of the γ1 chain gene around the boundaries of the coding and intervening sequences with those of other mouse genes revealed extensive divergence, although short prevalent sequences of AG-GTCAG at the 5′ border of the intervening sequence and TCTGCAG-GC at the 3′ border were deduced. A limited homology of nucleotide sequences was found among domains and between the hinge region and the 5′ portion of the CH2 domain. Comparison of 3′ untranslated sequences from the γ1 and γ2b chain genes and the mouse major β-globin gene shows significant homology and a palindrome sequence surrounding the poly(A) addition site.     

H5N1禽流感病毒环介导等温扩增快速检测方法的建立

目的：建立H5N1禽流感病毒环介导等温扩增（LAMP）快速检测方法。方法：从GenBank中获得H5N1禽流感病毒血凝素（HA）基因序列,应用DNAStar软件MegAlign程序分析其序列,利用PrimerExplorerV3软件在序列保守区域设计LAMP引物,即外引物、内引物和环引物,同时以所克隆的阳性质粒为模板,对试验中的几个重要参数进行优化。结果：LAMP检测方法对H5N1禽流感病毒的灵敏度达到4～6个拷贝,其引物对于H1、H9亚型禽流感病毒和新城疫病毒无非特异性扩增,表现出良好的H5亚型特异性。结论：建立的H5N1禽流感病毒环介导等温扩增快速检测方法灵敏度高、特异性强、重复性好,为快速检测禽流感病毒提供了新方法和新思路。