人白细胞硫氧还蛋白还原酶的分子克隆和序列分析

目的：克隆人白细胞硫氧还蛋白还原酶（thioredoxin reductase,TR)编码区的cDNA并进行序列分析。方法：采用RT－PCR方法获得TR基因cDNA，选择阳性克隆并测序。结果：通过分子克隆和序列分析，发现TR基因长1554bp,ORF为1494bp，编码498个氨基酸，结论：确定了中国人白细胞TR的cDNA序列，并据此推导出相应的氨基酸序列。     

人胎脑发育早期甲状腺激素受体mRNA的表达

目的 动态观察甲状腺激素受体(TRs)mRNA在人脑发育过程中的表达变化。方法 用RT-PCR半定量法检测3、5月龄人胎各脑区TRs mRNA表达变化。结果 3、5月龄人胎大脑、小脑、海马、丘脑、脑干及脊髓均有TRs mRNA表达，其中3月龄胎脑各脑区TRs mRNA表达以TRα、TRα2为主，TRβ1次之。5月龄胎脑各脑区TRs mRNA表达中以TRβ1，α2为主，TRα1次之。TRs mRNA在大脑、小脑、海马处表达相对丰富。实验中发现的与TRα2始终伴行的特异条带经测序证实，除在371-412位氨基酸处比TRα2少42个氨基酸，余序列两者完全相同。结论 在脑发育过程中，TRs mRNA呈时空性表达，参与调节CNS的分化、成熟。首次验证了人TRα3与TRα2编码区的差异序列。     

中国猪种DQA新等位基因的克隆和分析

目的：克隆和序列分析中国猪种DQA基因cDNA，为猪异种器官移植的免疫识别机理的研究提供基础。方法：采用RT-PCR方法扩增广西巴马猪(GXP)、贵州香猪(GZP)及云南小耳猪(YNP)DQA基因cDNA，克隆入测序载体，然后进行测序及其序列分析。结果：获得具有阅读框架的3个结构和已有序列不同的DQA新等位基因(GXPDQA、GZPDQA和YNPDQA)，基因序列号分别为AY102473，AY102474和AY102475，其长度分别为765、768和768个核苷酸，除末端终止密码外，分别编码254、255和255个氨基酸残基。结论：发现了3个猪DQA新等位基因，同时发现中国猪种DQA基因及其推导的氨基酸与人相应基因的同源性职显高于小鼠。     

组织型纤溶酶原激活物基因的DNA改组

目的：探索利用DNA改组（DNAshuffling)技术，获得更高活性tPA的可能性。方法：以人，恒河猴及大白鼠tPAcDNA为一组基因，进行tPA的DNA改组（DNAfamily shuffling)。以改组后构建的tPA多样性文库转染CHO细胞并进行克隆和筛选。结果：得到了两株有意义的克隆；t9和t17，其中t9克隆表达的tPA活性略高于人tPA，初步的比活性测定结果表明，活性约提高4倍。t17克隆表达的tPA虽然有88个氨基酸的缺失，但仍表现出与人tPA相同的活性，两株克隆经测序证明，为改组后的基因，其序列以人和恒河猴的tPAcDNA序列为主，少数序列来源于大白鼠tPAcDNA。结论：这一探索性结果将为后续几轮的tPADNA改组探明道路，为最终从改组后tPA多样性基因库中筛选到比较理想的重组体打下基础。     

人类睾丸生精细胞凋亡相关基因TSARG3的克隆

目的：克隆人类睾丸生精细胞凋亡相关基因TSARG3。方法：从已获得的小鼠稳睾和正常睾丸对照中表达量有明显差异的表达序列标签片段（BE644537）入手，构建人同源表达序列标签重叠群，应用基因特异性引物和载体特异性引物，在睾丸cDNA文库的DN或进行巢式PCR扩增、测序，对测序结果进行生物信息学分析。结果：从睾丸cDNA文库中分离出人类睾丸凋亡相关基因的5’末端而获得全长cDNA,命名为TSARG3，GenBank登录号为AF419291（保密期为1年），同时应用相同方法克隆了该基因在小鼠中的同源基因，GenBank登录号为AF419292。结论：获得人类睾丸生精细胞凋亡相关基因TSARG3，该基因可能与人类睾丸生精细胞凋亡有关。     

牛IL-18基因的克隆及遗传进化分析

目的：克隆牛白细胞介素18（IL-18）全基因，并对其进行序列分析。方法：从ConA刺激培养的牛外周血淋巴细胞提取总RNA，利用巢式RT-PCR方法扩增出牛IL-18全长cDNA，将其克隆到pMDl8-T载体上，测序后进行序列分析。结果：成功地克隆到了牛IL-18全基因，序列分析表明，实验中所克隆到的牛IL-18序列与GeneBank所登录的牛IL-18核苷酸序列及其推导氨酸序列同源性分别是99．5％和99％。与人、猕猴、野猪、山羊属、马等核苷酸序列及其推导氨基酸序列同源性分别在84．9％-99．5％和74．9％～99％之间，研究结果在国内还未见报道。结论：成功地从牛外周血中克隆到了牛IL-18的基因，其全长为598bp。     

铜绿假单胞菌外毒素A免疫毒素片段的抗原性评估及三级结构模拟

目的 克隆铜绿假单胞菌外毒素A(PEA)基因及分析不同克隆序列中不同性质氨基酸对抗原性及三级结构的贡献.方法 以铜绿假单胞菌基因组DNA为模板,以PEA编码基因特异区段为引物,采用Touchdown PCR克隆PEA编码基因.阳性克隆经菌落PCR鉴定后进行DNA测序及Clustal X(1.83)比对分析;采用生物信息学在线软件BIMAS及SWISS-Model对所得基因编码蛋白进行抗原性评估及三级结构模拟.人为置换关键部位的氨基酸残基,考察不同性质氨基酸对三级结构的影响.结果 从铜绿假单胞菌DNA中获得3个克隆.抗原性分析及三级结构模拟结果显示,3个克隆与公开发表的序列之间存在少数氨基酸的差异;个别区段上氨基酸的改变可引起相应位点抗原性的改变,但仅有个别位点的改变对空间结构产生影响.结论 基于抗原性评估及三级结构分析,获得了不同性质氨基酸对抗原性及三级结构的贡献等信息,为PEA编码基因用于弱化抗原性靶向性毒素的构建奠定了基础.     

从人血液中克隆过氧化氢酶基因

目的 从人血液中克隆过氧化氢酶基因.方法 以新鲜的人血液为材料,提取全血RNA,运用RT-PCR方法扩增过氧化氢酶基因,构建pMD18T/CAT克隆载体,并进行了不同来源过氧化氢酶的氨基酸序列同源分析.结果 从人血液中成功扩增出过氧化氢酶基因,获得了重组载体PMD-18T/CAT,氨基酸序列分析的同源性达80% 以上.结论 从人血液中可以很方便地克隆出过氧化氢酶基因.     

人睾丸前列腺素D合成酶的cDNA克隆和序列分析

目的：对人睾丸Lipocalin型前列腺素D合成酶前列腺素（L－PGDS）cDNA进行克隆和序列分析。方法：用人脑L－PGDS的增特异性引物对人睾丸RNA进行逆转录和PCR扩增,并对扩增产物进行纯化和测序。结果：人睾丸L-PGDS cDNA核苷酸序列与人外周血淋巴细胞的L－PGDS基因区序列的一致为99．5％,与人脑L－PGDS cDNA的一致性为96．0％,相应氨基酸的一致性分别为100％和94．2％,结论：人睾丸－PGDScDNA序列及推导的氨基酸序列的阐明,对进一步探讨L－PGDS蛋白的特性及其在男性生殖中的作用是十分有意义的。     

小鼠MBL-A基因的克隆和序列分析

目的：克隆小鼠甘露聚糖结合凝集素-A(MBLA)基因的全长编码区cDNA。方法：利用RT-PCR方法，从Balb/c小鼠的肝细胞中，分离出MBL-A基因cDNA片段，克隆入pUC-T载体，测序并进行分析。结果：扩增得到的小鼠MBL-A基因cDNA全长720bp，编码240个氨基酸残基，包含了完整的富含半胱氨酸区、胶原区、颈区和糖识别域。分析表明，与Genbank中发表的序列具有99.9％的同源性。结论：获得小鼠MBL-A基因的克隆，为进一步研究MBL-A分子在体内的生物学功能奠定了一定基础。     

Molecular cloning and sequence analysis of the duck enteritis virus UL5 gene

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious, and fatal disease. In the present article, the DEV UL5 gene was cloned and sequenced from a vaccine virus. According to the consensus sequence of herpesvirus UL5 and UL3 gene degenerate oligonucleotide primers were designed and were used in the polymerase chain reaction (PCR) to amplify DNA products with 4577 bp in size. DNA sequence analysis revealed a 2568 bp open reading frame (ORF) encoding a 855 amino acid polypeptide homologous to herpesvirus UL5 proteins. The DEV UL5 gene has a base composition of 769 adenine (29.95%), 556 cytosine (21.65%), 533 guanine (20.76%) and 710 thymine (27.65%). Sequence comparison revealed that the nucleotide sequence of the DEV UL5 gene was highly similar to other alphaherpesviruses. Phylogenetic tree analysis showed that the fifteen herpesviruses viruses analyzed fell into four large groups, and the duck enteritis virus itself branched and was most closely related to meleagrid herpesvirus 1, gallid herpesvirus 2 and gallid herpesvirus 3 subtrees.     

Sequence of the serotype-specific glycoprotein of the human rotavirus Wa strain and comparison with other human rotavirus serotypes

Complementary DNA was synthesized from the double-stranded RNA of the Wa strain of human rotavirus and inserted into the bacterial plasmid pBR322. Clones which contained the gene that codes for the viral glycoprotein (VP7) were identified and the nucleotide sequence was determined. The gene was 1062 base pairs in length with an open reading frame which coded for 326 amino acids. Two potential glycosylation sites were found as well as two hydrophobic regions at the N-terminus of the polypeptide. The untranslated regions at the 5' and 3' ends were 48 base pairs and 33 base pairs long, respectively. Only one nucleotide at position 493 differed from the sequence of the Wa VP7 gene described by Richardson et al. (1984, J. Virol. 51, 860-862). A strong prokaryotic promoter sequence was also found between residues 434 and 462. A comparison of the amino acid sequence of the Wa strain (serotype 1) to the Hu/5 strain of human rotavirus (serotype 2) and SA11, the simian rotavirus (serotype 3), revealed a high degree of homology (79.1% and 83.1%, respectively) between the serotypes, suggesting that rotavirus serotypes are stable. The hydrophilic regions of VP7 of the three serotypes were identified and compared for homology. Four of these regions showed variation between serotypes.     

The adenovirus that causes hemorrhagic disease of black-tailed deer is closely related to bovine adenovirus-3

Summary.  DNA sequence data was obtained from an adenovirus previously shown to be the cause of a distinctive, fatal hemorrhagic disease of black-tailed deer in California. A 256 base fragment of the viral hexon gene was amplified by PCR from purified adenovirus preparations. The amplicon then was cloned and sequenced. Phylogenetic relationships with other mammalian adenoviruses were also determined. Although sequence analysis of this portion of the hexon gene indicates that the black-tailed deer adenovirus is closely related to bovine adenovirus-3, the biologic properties of the two viruses are clearly distinct. Accepted September 2, 1998 Received July 30, 1998     

中国人幽门螺杆菌尿素酶B亚单位的基因克隆及序列分析

目的 克隆人幽门螺杆菌（ｈｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒｉ,Ｈｐ）尿素酶Ｂ基因（ｕｒｅＢ）,并分析其核苷酸序列的特性。方法 用ＰＣＲ技术从临床分离的Ｈｐ菌株基因组中扩增出ｕｒｅＢ基因,将其克隆至ｐＨＰ质粒上进行序列分析。结果 克隆得到的ｕｒｅＢ基因长度为１７１３ｂｐ,其核苷酸序列与ＧｅｎＢａｎｋ公布的序列有６１个碱基存在差异,同源为９６４４％,推定的氨基酸序列同源性为９９．６５％。结论：我们所     

Molecular cloning, expression, and primary sequence of outer membrane protein P2 of Haemophilus influenzae type b.

The structural gene for the porin of Haemophilus influenzae type b, designated outer membrane protein P2, was cloned, and the DNA sequence was determined. An oligonucleotide probe generated by reverse translation of N-terminal amino acid sequence data from the purified protein was used to screen genomic DNA. The probe detected a single EcoRI fragment of approximately 1,700 base pairs which was cloned to lambda gt11 and then into M13 and partially sequenced. The derived amino acid sequence indicated that we had cloned the N-terminal portion of the P2 gene. An overlapping approximately 1,600-base-pair PvuII genomic fragment was cloned into M13, and the sequence of the remainder of the P2 gene was determined. The gene for P2 was then reconstructed under the control of the T7 promoter and expressed in Escherichia coli. The N-terminal sequence of the purified protein corresponds to residues 21 through 34 of the derived amino acid sequence. Thus, the protein is synthesized with a 20-amino-acid leader peptide. The Mr of the processed protein is 37,782, in good agreement with the estimate of 37,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

人脑源性神经营养因子基因扩增及序列测定

目的：从基因组DNA获取编码脑源性神经营养因子基因,并对目的基因进行序列测定。方法：本研究直接从脑组织中提取人的基因组DNA,根据人的脑源性神经营养因子（BDNF）的cDNA序列,设计一对寡核苷酸引物,采用聚合酶链反应（RCR）,体外扩增编码人脑源性神经营养因子基因;扩增产物用自动分析仪进行序列测定。结果：从基因组DNA体扩增出脑源性神经营养因子基因,序列与献报道一致。结论：利用PCR直接从基因组获取目的基因,表明人脑神经营养因子基因为单一外显子,目的基因的扩增成功为进一步研究打下了基础。     

Cloning and Sequencing of an MPB70 Homologue Corresponding to MPB83 from Mycobacterium bovis BCG

MPB70 is secreted in high concentrations by Mycobacterium bovis BCG substrain Tokyo (BCG Tokyo), but little by substrains Pasteur (BCG Pasteur) and M. tuberculosis . The gene encoding a MPB70 homologue secreted by BCG Tokyo was found at the upstream region of the gene encoding MPB70, with approximately 2.3 kilobase pairs (kbp) spacing: the same gene was also found in BCG Pasteur. This gene was cloned and sequenced from BCG Tokyo. The DNA sequence which contained a 663 base pair (bp) open reading frame beginning at position 1 and ending with a TAA codon at position 661 was found. Its theoretical molecular mass was calculated to be 22.068 kDa. This gene was highly homologous to the coding region of mpb70 and the deduced amino acid sequence was very similar to MPB83 reported by Harboe et al . It was speculated that the gene the authors characterized probably corresponded to the mpb 83 gene.     

Comparative sequence analysis of duck and human hepatitis B virus genomes

We have cloned and sequenced an infectious, functionally active genome of a duck hepatitis B virus (DHBV). It is 3,021 base pairs (bp) in length and shows little DNA sequence homology to the genome of human hepatitis B virus (HBV). However, the amino acid sequences of predicted viral gene products are similar between DHBV and HBV, and the genome organization present in DHBV reflects that of HBV. As in the mammalian virus the long minus strand of the DHBV genome encodes three long overlapping reading frames designated as P, S, and C. The fourth open reading frame, termed X, is absent in DHBV. A comparison with a sequence of a second DHBV isolate [Mandart et al, Journal of Virology 49:782-792, 1984] revealed a nucleotide sequence variation of 5.6% and confirmed the presented overall gene organization of DHBV.     

一个含有25bp内含子的阴道毛滴虫Rabl-like新基因

目的 克隆和鉴定一个新的阴道毛滴虫Rabl-like基因（TvRabl-like）及其内含子。方法 我们从一阴道毛滴虫cDNA表达文库中分离出一个cDNA克隆，它与各物种的Rab家族蛋白有较高的同源性，因此我们进一步用BLASTP、RPS-BLAST、ClustalW和MEGA3等分析软件对该cDNA克隆进行了序列分析和进化树分析；用PCR和RT-PCR等技术分别对该基因组和mRNA进行了扩增和测序分析。结果 序列分析结果表明该eDNA克隆长705bp，开放阅读框具603bp，推测肽链含有200个氨基酸。序列比较分析结果提示该cDNA克隆所推测的蛋白质是一个Rabl亚家族的亚型。进化树分析也表明它属于阴道毛滴虫Rabl亚家族。基因组PCR扩增和测序分析表明该基因包含一个25bp的内含子，该内含子具有阴道毛滴虫和其它真核生物较大内含子所具备的典型的5’GT-AG-3’和分支位点基序。RT-PCR产物及其测序分析表明在该基因的转录本中存在着未剪切和剪切后的mRNA，说明确实有内含子的存在。结论 TvRabl-like基因属于阴道毛滴虫Rabl亚家族，该基因含有一个25bp的内含子。该内含子是至今发现的最小的阴道毛滴虫基因内含子之一，很可能也是真核生物中最小的内含子。对诸类最低等真核生物内含子的研究将有助于我们理解真核生物内含子的起源和进化。