PCR介导的cDNA文库矩阵排列筛选方法

描述了一种快速、有效的ｃＤＮＡ文库筛选策略。其主要过程是将ｃＤＮＡ文库重组子进行矩阵排列,进而用特异引物进行ＰＣＲ逐级筛选以分离目的基因．     

黑麦1R染色体的微切微克隆研究

显微分离出黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）１Ｒ染色体,用ＣｏｈｅｓｉｖｅａｄａｐｔｅｒｓｓｉｎｇｌｅｐｒｉｍｅｒＰＣＲ（ＣＡＳＰＰＣＲ）方法进行体外扩增,以ＤＩＧ１１ｄＵＴＰ标记扩增产物为探针,进行Ｓｏｕｔｈｅｒｎ分子杂交,结果表明扩增产物来自黑麦１Ｒ染色体。用１／１０体积的连接物转化Ｅ．ｃｏｌｉＤＨ５α,获得１００００多个重组菌落。经酶切分析,克隆子的插入片段为２５０～５００ｂｐ,为进一步筛选１Ｒ染色体的分子标记打下了基础     

植物发育基因克隆技术的新进展

过去２０年,人们已经分离到了相当数量的与发育相关的基因。这主要借助于蛋白质产物的分离纯化,然后根据其氨基酸结构推算其相应的核苷酸序列,并据此合成寡核苷酸探针,最终从ｃＤＮＡ文库或基因组文库中筛选出目的基因。而未知其编码产物的发育基因的分离克隆则是非常困难的工作,以前广泛应用的主要方法有ＤＮＡ标签法（ＤＮＡｔａｇｇｉｎｇ）［１,２,３］,作图克隆法（ｍａｐ－ｂａｓｅｄｃｌｏｎｉｎｇ）［４］,差别筛选法（ｄｉｆｅｒｅｎｔｉａｌｓｃｒｅｅｎｉｎｇ）［５］和扣除杂交法（ｓｕｂｔｒａｃｔｉｖｅｈｙｂｒｉｄｉｚａｔｉｏｎ）［６,７,８］。这些方法虽然都取得了一定的成功,但由于各自的缺陷性,而限制了它们更加广泛的应用。近年来在ＰＣＲ技术的基础上,人们已经建立了若干种分离克隆植物发育基因的新方法。１９９２年,ＬｉａｎｇＰ和ＰａｒｄｅｅＡ［９］首次提出并运用了ｍＲＮＡ差别显示技术（ｍＲＮＡｄｉｆ－ｆｅｒｅｎｔｉａｌｄｉｓｐｌａｙｒｅｖｅｒｓｅｔｒａｎｓｃｒｉｐｔｉｏｎ－ＰＣＲ,ＤＤＲＴ－ＰＣＲ）来进行基因的分离。实践表明,ｍＲＮＡ差别显示技术在分离、鉴定差别表达的新基因方面与以前的各种技术相比有其独特的优越性,但同时它也存在着重复性较差等缺点。因此,最近研究工作者又在其基础上,发展了诸如代表性差式分析（ｒｅｐｒｅｓｅｎｔａｔｉｏｎａｌｄｉｆｅｒｅｎｃｅａｎａｌｙｓｉｓ,ＲＤＡ）［１０,１１］和抑制性扣除（或减去）杂交（ｓｕｐｐｒｅｓｓｉｏｎｓｕｂｔｒａｃｔｉｖｅｈｙｂｒｉｄｉｚａｔｉｏｎ,ＳＳＨ）［１２］等一些更新的方法。下面就此作一简要概述。     

DNA分子标记在柑桔中的应用

ＤＮＡ分子标记是最为理想的遗传标记 ,依其多态性检出所用的分子生物学技术 ,大致可分为Ｓｏｕｔｈｅｍ杂交技术为核心的分子标记和ＰＣＲ技术为核心的分子标记。前者的代表性技术有ＲＥＬＰ(ｒｅｓｔｒｉｃｔｉｏｎｆｒａｇｍｅｎｔｌｅｎｇｔｈｐｏｌｙｍｏｒｐｈｉｓｍ)和ＤＮＡ指纹技术 (ＤＮＡｆｉｎｇｅｒｐｒｉｎｔｉｎｇｔｅｃｈｎｉｑｕｅｓ)。后者的代表性技术有ＲＡＰＤ(ｒａｎｄｏｍａｍｐｌｉｆｉｅｄｐｏｌｙｍｏｒｐｈｉｃＤＮＡ)、ＳＣＡＲ(ｓｅｑｕｅｎｃｅｃｈａｒａｃ ｔｅｒｉｓｔｉｃａｍｐｌｉｆｉｅｄｒｅｇｉｏｎ)…     

疾病基因克隆的策略及主要方法

目　　录一、定位克隆 ( ｐｏｓｉｔｉｏｎａｌｃｌｏｎｉｎｇ)策略　 (一 )家系连锁分析法　 (二 )等位基因共占法　 (三 )人群相关分析法　 (四 )ｃＤＮＡ筛选二、消减杂交 (ｓｕｂｔｒａｃｔｉｖｅｈｙｂｒｉｄｉｚａｔｉｏｎ)策略　 (一 )消减杂交法和抑制性消减杂交法　 (二 )差示反转录ＰＣＲ法和差异消减显示法　 (三 )代表性差异分析法和Ｓ1核酸酶介导的缺失基因探针富集法　 (四 )基因组错配扫描法　 (五 )比较基因组杂交法　 (六 )ＤＮＡ微陈列杂交系统三、两类策略的联系基因组全序列测定可望提前完成 ,而以功能鉴定为核心的功能…     

猪肥胖基因cDNA的克隆与分析

肥胖基因（ａｂ）是近年刚被克隆的新基因,该基因产物Ｌｅｐｔｉｎ是反映体内脂肪含量和调节体重的重信号因子,首次报道猪ｏｂ基因全长序列,并对不同种物ｏｂ基因的同源性进行了比较,以λＵｎｉ－ＺＡＰ＾ＴＭ为载体构建了猪脂肪ｃＤＮＡ文库,根据已知的人和鼠ｏｂ基因序列设计ＰＣＲ引物,利用ＰＣＲ法筛选猪脂肪ｃＤＮＡ文库,并用ＲＴ－ＰＣＲ从脂肪ＲＮＡ中扩增的３６６ｂｐ猪ｏｂ基因片段作探针,获得了全长３２７７ｂｐ的     

侵染检测技术的原理和应用

侵染检测 (Ｉｎｖａｄｅｒａｓｓａｙ)技术是无需进行聚合酶链式反应 (ＰＣＲ )的一种ＤＮＡ(ＲＮＡ)的检测与定量分析技术[1] ,具有无污染、特异性与敏感度高、操作简便等优点 ,可以广泛运用于基因突变、单核苷酸多态性(ｓｉｎｇｌｅｎｕｃｌｅｏｔｉｄｅｐｏｌｙｍｏｒｐｈｉｓｍ ,ＳＮＰ)检测、核酸定量分析以及基于病人基因型的指导临床个体化用药中[2 ] 。它是美国ＴｈｉｒｄＷａｖｅＴｅｃｈｎｏｌｏｇｙ公司的专利技术[3] 。以下对其基本原理与应用前景作一简要介绍。1 .信号探针的切割1 .1　反应体系组成　　侵染检测反应体系中 ,包…     

番茄OVATE基因调控果实梨形发育

ＰｒｏｃＮａｔｌＡｃａｄＳｃｉＵＳＡ ,2 0 0 2 ,99:1 330 2～ 1 330 6早期研究表明番茄果实的梨形性状由位于第二染色体的一个隐性基因ｏｖａｔｅ所控制 ,ｏｖａｔｅ基因位于分子标记ＴＧ645附近 ,以此标记为探针筛选番茄ＢＡＣ文库将该基因定位于一个长为 1 0 5ｋｂ的ＤＮＡ片段内 ,进一步的分子标记定位表明该基因在含有 8个ＯＲＦ的 55ｋｂＤＮＡ内。通过ＰＣＲ扩增了圆形番茄这一区域ＤＮＡ并进行测序 ,结果表明突变体的ＯＲＦ6中有三处发生了突变。ＯＲＦ6由两个外显子和 1个内含子组成 ,有一个点突变和一个 2ｂｐ的插入或缺失突变…     

原位杂交和原位PCR技术在鱼类基因定位中的应用

染色体原位杂交（ ｉｎ ｓｉｔｕ ｈｙｂｒｉｄｉｚａｔｉｏｎ, ＩＳＨ）是基因物理定位的主要方法之一,它根据核酸分子碱基互补配对的原理,将标记的外源核酸探针与染色体上变性处理后的单链ＤＮＡ互补配对,再经检测而将靶顺序在染色体上的位置显示出来。 原位ＰＣＲ技术是将原位杂交与ＰＣＲ技术有机的结合,在原位扩增目的ＤＮＡ片段,并在原位检测其扩增产物,因而兼有ＰＣＲ及原位杂交技术二者的优越性：既可以同时获得组织及细胞的形态结构信息与分子信息,进行定性、定位分析,又具有比原位杂交更好的敏感性与专一性。 染色体原位…     

昆虫核酸分子系统学研究进展

本文从研究对象、方法、类群、内容等方面综述了近十年来昆虫核酸分子系统学研究进展概况。文中首先介绍了ＲＦＬＰＡ、探针杂交及ＤＮＡ指纹、ＰＣＲ与ＲＡＰＤ－ＰＣＲ、顺序分析方法及应用情况,列举了在双翅目、膜翅目、同翅目、直翅目等目昆虫中的研究进展,并从居群遗传结构、分类学研究、系统发育和分子进化４个方面总结了昆虫核酸分子系统学的研究内容和主要成果,最后指出ＲＡＰＤ－ＰＣＲ与ＲＦＬＰ联合用于测序是近期昆虫分子系统学上最有应用价值的方法。     

New anti angiogenesis developments through electro-immunization: optimization by in vivo optical imaging of intradermal electro gene transfer

Direct application of high voltage electric pulses of milliseconds duration to the skin of a mouse enhances in vivo intradermal delivery of injected therapeutic molecules such as DNA. The efficacy of gene transfer and expression is dependent on electrical parameters. DNA electrotransfer in tissues increases the associated DNA expression vaccine potency. This protocol is called "electro-immunization". In the present study, we report a new strategy for optimizing electro-immunization. In vivo fluorescence imaging was used to detect the expression of a fluorescent protein (DsRed) and therefore allowed rapid optimization of the protocol. In vivo electrogenetransfer in the skin was well tolerated and DsRed expression was followed for over 2 weeks. Expression was voltage dependent under our conditions. Parameters were selected giving the highest level of expression. Under these optimized conditions, electrotransfer of a plasmid encoding VEGF was evaluated for its immune response as a gene therapy of interest involved in anti-angiogenic strategies. Anti VEGF 165 antibodies in sera of mice were evaluated by ELISA and compared to those obtained after conventional immunization. Comparable titres of antibodies were obtained in both groups. An IgG2a predominance was found in mice immunized with the plasmid whereas a IgG1 predominance was observed in mice immunized classically. Skin electro-immunization is therefore shown as a good route for DNA immunization for anti-angiogenesis concern.     

New anti angiogenesis developments through electro-immunization: Optimization by in vivo optical imaging of intradermal electrogenetransfer

Direct application of high voltage electric pulses of milliseconds duration to the skin of a mouse enhances in vivo intradermal delivery of injected therapeutic molecules such as DNA. The efficacy of gene transfer and expression is dependent on electrical parameters. DNA electrotransfer in tissues increases the associated DNA expression vaccine potency. This protocol is called “electro-immunization”. In the present study, we report a new strategy for optimizing electro-immunization. In vivo fluorescence imaging was used to detect the expression of a fluorescent protein (DsRed) and therefore allowed rapid optimization of the protocol. In vivo electrogenetransfer in the skin was well tolerated and DsRed expression was followed for over 2 weeks. Expression was voltage dependent under our conditions. Parameters were selected giving the highest level of expression. Under these optimized conditions, electrotransfer of a plasmid encoding VEGF was evaluated for its immune response as a gene therapy of interest involved in anti-angiogenic strategies. Anti VEGF 165 antibodies in sera of mice were evaluated by ELISA and compared to those obtained after conventional immunization. Comparable titres of antibodies were obtained in both groups. An IgG2a predominance was found in mice immunized with the plasmid whereas a IgG1 predominance was observed in mice immunized classically. Skin electro-immunization is therefore shown as a good route for DNA immunization for anti-angiogenesis concern.     

东方田鼠感染血清免疫筛选日本血吸虫成虫cDNA文库

东方田鼠 (Microtusfortis ,Mf)对日本血吸虫 (Schistosomajaponicum ,Sj)感染具有抗性 .为探讨Mf感染Sj后是否产生针对虫体某些特异抗原分子的免疫应答 ,用Mf感染血清对Sj成虫cDNA文库进行免疫筛选 .经初筛和复筛 ,共筛选出 12个阳性克隆 .这些阳性克隆经辅助噬菌体自动剪切后PCR扩增显示 ,插入的SjcDNA片段大小在 3 0 0bp至 1 8kb之间 ,其中 1 8kb片段 5个 ,1kb片段 1个 ,3 0 0bp片段6个 .经DNA测序分析 ,鉴定出 3个未曾报道过的Sj新基因 ,分别命名为Sj Mf1、Sj Mf2和Sj胞质氨基肽酶 ,并在GenBank登记注册 .结果说明 ,Mf感染血清可识别Sj的特异性抗原分子 ,这些抗原分子的免疫保护作用值得进一步研究 .     

Protective immunity against Taenia crassiceps murine cysticercosis induced by DNA vaccination with a Taenia saginata tegument antigen

This study investigated the protective capacity of the recombinant Taenia saginata Tso18 antigen administered as a DNA vaccine in the Taenia crassiceps murine model of cysticercosis. This Tso18 DNA sequence, isolated from a T. saginata oncosphere cDNA library, has homologies with Taenia solium and Echinococcus sp. It was cloned in the pcDNA3.1 plasmid and injected once intramuscularly into mice. Compared to saline-vaccinated control mice, immunization reduced the parasite burden by 57.3-81.4%, while lower levels of non-specific protection were induced in control mice injected with the plasmid pcDNA3.1 (18.8-33.1%) or a plasmid with irrelevant construct, pcDNA3.1/3D15 (33.4-38.8%). Importantly, significant levels of protection were observed between the pcDNA3.1/Tso18 plasmid and pcDNA3.1/3D15 plasmid immunized mice. Mice immunized with pTso18 synthesized low levels of, primarily IgG1 sub-class, antibodies. These antibodies were shown to recognize a 66 kDa antigen fraction of T. crassiceps and T. solium. Splenocytes enriched in both CD4+CD8- and CD4-CD8+ T cells from these vaccinated mice proliferated in vitro when exposed to antigens from both T. solium and T. crassiceps cestodes. Immunolocalization studies revealed the Tso18 antigen in oncospheres of T. saginata and T. solium, in the adult tapeworm and in the tegument of T. solium cysticerci. The protective capacity of this antigen and its extensive distribution in different stages, species and genera of cestodes points to the potential of Tso18 antigen for the possible design of a vaccine against cestodes.     

A monoclonal anti-type II collagen antibody with cross-reactive anti-Ig activity specific for the F(ab')2 fragment

An IgG2a hybridoma antibody (BC-10) was obtained by a myeloma fusion with lymphocytes from B10.RIII mice immunized against native bovine type II collagen. This anti-collagen monoclonal exhibited extensive cross-reactivity with several type II collagen species. BC-10 was found to have self-associating properties, but not the specificity of a typical IgG rheumatoid factor, inasmuch as this mAb bound to F(ab')2 fragments of itself and of normal mouse IgG. Self binding was inhibited by the association of BC-10 with type II collagen, and inhibition assays indicated that antibodies with the capacity to inhibit BC-10 binding to collagen were present in the sera from B10.RIII arthritic mice, but not from DBA/1 LacJ arthritic mice. Joint inflammation and histopathologic features consistent with arthritis were observed in mice injected with the BC-10 hybridoma.     

Nude mice injected with DNA released by antigen stimulated human tlymphocytes produce specific antibodies expressing human characteristics

Nude mice were injected with DNA purified from the nucleoprotein complex released by T lymphocytes previously exposed in vitro to inactivated herpes or poliovirus. After five days the serum of these mice was tested for its virus neutralizing activity. Results show that injected nude mice synthesize antiherpetic or antipolio antibodies depending on the antigen used to sensitize the T lymphocytes in vitro. These antibodies were not found in the serum of uninjected control mice or mice injected with inactivated herpes or polio viruses. Mice injected with DNA release by human T cells produced antibodies carrying human allotypes since they could be neutralized by anti-allotype sera. Moreover their antiviral activity was inhibited by anti-human IgM or IgG. However, the mice which were injected with DNA released by antigen stimulated murine T lymphocytes produced antiviral antibodies which were not neutralized by anti-human allotype sera.     

Effect of sperm DNA vaccine on fertility of female mice

Our laboratory has identified a sperm-specific dodecamer peptide sequence, designated as YLP(12), vaccination with which causes a long-term reversible immunocontraceptive effect in female mice. In the present study, the effects of YLP(12) DNA vaccine were examined. YLP(12) 36 bp cDNA was cloned into pVAX1 vector to prepare the DNA vaccine. Two additional vaccine constructs were made by in frame cloning of one and two CpG repeats in the YLP(12)-cDNA vaccine. Five groups of female mice were immunized intradermally by using gene gun with YLP(12)-cDNA, YLP(12)-cDNA-CpG, YLP(12)-cDNA-CpG-CpG, YLP(12)-cDNA mixed with exogenous synthetic CpG oligodeoxynucleotide (ODN), or vector DNA alone, respectively. Vaccination with all three constructs and the YLP(12) vaccine mixed with exogenous ODN raised antibody response both in the sera as well as locally in the vaginal tract. There was no antibody response in the mice injected with the vector alone. In sera, the highest titers were obtained for the IgG class for all constructs and formulation followed by IgA class. In vaginal washings the highest titers were obtained for the IgA class followed by IgG class. Within the IgG class, the titers for the IgG2a subclass were significantly greater than the IgG1 subclass. Immunization with all constructs and formulation caused a significant (P < 0.05 to <0.001) reduction (20-43%) in fertility of female mice. The highest reductions were seen in mice immunized with YLP(12)-cDNA-CpG-CpG (two repeats) (43% reduction) and with the YLP(12) vaccine administered with exogenous CpG ODN (42% reduction). T lymphocytes obtained from DNA-vaccinated mice showed clearly distinguished comparative RT-PCR analysis of cytokine mRNA expression for Th1 and Th2 immune responses compared to T lymphocytes obtained from control animals injected with vector DNA. Expression of both Th1 cytokines (IL-2 and IFN-gamma) and Th2 cytokines (IL-4 and IL-10) was enhanced after DNA vaccination as compared to controls, with a bias towards Th1 response. The immunocontraceptive effects were long-lasting observed up to 1.3 years of the observation period and increased with time. These novel findings indicate that the intradermal immunization with a sperm-specific DNA vaccine causes a long-term circulating and local immune response resulting in immunocontraceptive effects in female mice.     

Comparison between autoantibodies arising during Trypanosoma cruzi infection in mice and natural autoantibodies

The autoantibodies induced in (C57BL/6 x BALB/c)F1 mice during Trypanosoma cruzi (CL strain) infection were analyzed and compared with natural autoantibodies present in healthy mice. Mice were killed at intervals after infection and their sera were tested by enzyme immunoassay against a panel of self- and non-self-Ag: actin, myoglobin, myosin, tubulin, DNA, and TNP-OVA. The level of IgM and IgG autoantibodies against all Ag started to increase from day 15 until 6 wk after the parasite infection. The high level of all autoantibodies persisted 3 mo postinfection, and 1 yr later, half of the mice still had elevated levels of IgM and IgG autoantibodies, particularly antitubulin IgG antibodies. IgM and IgG were isolated from pools of normal and infected mouse sera and their binding capacity to all Ag was compared. The titers of infected mouse sera were increased and the slopes of both IgM and IgG binding curves of autoantibodies to actin, myosin, and tubulin were greater than those of control mouse sera, indicating higher affinities. The average dissociation constant of the IgG2a autoantibody to mouse tubulin was 5 times lower than that of natural antitubulin IgG2a antibodies. Furthermore, absorption of the IgG from infected mouse sera onto a tubulin immunoadsorbent removed half the reactivity with tubulin and also with myosin, actin and parasite extracts. The eluted antibodies bound the same Ag. When IgG were further analyzed by Western blot on proteolytic fragments of tubulin, we found that antibodies from both groups bound to the same broad spectrum of polypeptide bands. However, additional fragments were recognized by antibodies from infected mice. All these results indicate that the autoantibodies naturally present in mice are significantly affected after infection with T. cruzi, in quantity as well as in specificity and affinity.     

超抗原SEA增强小鼠对HBV DNA 疫苗的免疫反应

观察超抗原SEA(D227A)的真核表达载体(pmSEA),对HBVDNA疫苗诱导Balbc小鼠(H2d)免疫应答的调节作用。肌内注射空载体pcDNA3、HBVDNA疫苗加pmSEA佐剂(pHBVS2S+pmSEA)或不加佐剂(pHBVS2S);ELISA法测定血清抗HBs;ELISPOT检测分泌IFNγ的脾淋巴细胞;4h51Cr释放法检测小鼠脾细胞CTL活性。HBVDNA佐剂组免疫小鼠抗HBsAg抗体滴度明显高于不加佐剂组,其IgG1IgG2a的比例不同于多肽免疫组,二者分别为0.282与10。HBVDNA佐剂组均能增强IgG1和IgG2a的产生,是不加佐剂组的1.36、1.73倍。佐剂组小鼠脾淋巴细胞IFNγ的分泌量是不加佐剂组2～3倍。CTL细胞杀伤活性(E:T=100)佐剂组与不加佐剂组分别为:69.77%±7.5%、42.81%±7.7%,差异显著(P<0.05)。HBVDNA疫苗具有较强的免疫原性,能够诱导机体产生特异性的抗体及CTL反应;pmSEA佐剂能够提高小鼠对DNA疫苗的免疫应答,有望成为DNA疫苗的免疫佐剂。