猪丙型肝炎病毒核心蛋白结合蛋白6同源基因的克隆化研究

利用不同种属动物之间重要基因序列高度同源的理论,应用分子生物学与生物信息学技术和方法。克隆猪丙型肝炎病毒(HCY)核心蛋白结合蛋白6(HCBP6)的同源基因。首先应用酵母双杂交技术,以表达HCV核心蛋白的表达栽体作为曾饵,对于百万级的肝细胞cDNA文库酵母进行配合、筛选,首先获得人HCBP6的全长鳊码基因。然后应用美国国立生物工程中心(NCBI)建立的核苷酸序列数据库(CenBank)的同源基因的检索,搜索与之同源的来源于猪的表达序列标签(EST)。然后根据基因同泺性的原则,确定猪HCBP6的同源基因。获得了与HCC核心蛋白结合蛋白的36个基因片段,其中之一命名为HCBP6。根据基因同源性搜索,获得了来源于猪的EST基因序列片段。最终确立了猪HCBP6的同源基因。利用不同物种之间基因同泺性的原理、NCBI数据库GenBank同源基因的搜索,获得了猪HCBP6同源基因。生物信息学技术在后基因组时代具有重要地位和作用。     

家兔BMP7基因的克隆及其生物信息学分析

在对已知部分编码序列(CDS)进行分析的基础上, 采用RT-PCR分步扩增以及RACE方法, 对家兔BMP7基因3′和5′末端未知序列进行了克隆与生物信息学分析。测序结果综合分析表明, 所获序列共计1 654 bp, 包括家兔BMP7近全长前肽、全长成熟肽CDS及3′非翻译序列(3′UTR), 将已有的序列向5′和3′端分别延伸了395 bp和628 bp。序列对比表明, 克隆的家兔BMP7 CDS部分与人、小鼠的对应序列的同源性分别为91.89%和89.32%, 预测的氨基酸序列同源性分别为96.51%和96.01%。家兔BMP7 3′UTR长446 bp, 与人、小鼠对应序列同源性分别为57.38%和45.57%; 具有2个转录终止信号位点。推测家兔BMP7成熟蛋白有BMPs特有的7个位置固定的半胱氨酸残基和TGF-β家族指纹。家兔BMP7 3′UTR区转录终止信号的可选择性可能与基因转录后调控有关。     

小型猪CYP3A88基因克隆与其他动物的序列比较

目的克隆我国资源小型猪品系巴马香猪肝脏中的CYP3A88基因,并进行生物信息学分析。方法应用RACE（Rapid Amplification of cDNA Ends）技术对其全长进行扩增,测序,利用Internet和GenBank数据库对其序列进行生物信息学分析。结果首次克隆并鉴定了我国资源小型猪品系巴马香猪肝脏中CYP3A88（GenBank登录号：EF625347）的编码区,获得大小为1965bp的全长cDNA,编码区长为1512bp,编码503个氨基酸;比较核苷酸序列,与小型猪CYP3A39相似性高达94%,而与人等其它动物的CYP3A相似性则在86%以下;推导和分析氨基酸序列表明,与小型猪CYP3A其它成员（CYP3A39、CYP3A29、CYP3A22）进行对比,其相似性分别为92%,89%,80%,而将小型猪与人的CYP3A分别比对,小型猪CYP3A88与人CYP3A4相似性最高,为77%;对其二级结构预测,它可能含12个α螺旋,4个β折叠;经NCBI上的CDD程序分析可知,其39～491氨基酸区域为P4503A亚家族保守结构区域;经聚类分析,小型猪和狗的CYP3A与人有较近的进化关系;通过同源建模法对其在线建模,人CYP3A4晶体结构作为其模建模型,得到了其经典的三维结构。结论在猪CYP3A家族四个基因中,CYP3A88在序列和高级结构上均与人CYP3A4的最为相似。     

人腺苷酸环化酶激活多肽cDNA克隆和序列分析

以人睾丸组织总ＲＮＡ为材料,用ＲＴ－ＰＣＲ方法合成了人腺苷酸环化酶激活多肽（ＡＣＡＰ）编码区（５３０ｂｐ）和全长ｃＤＮＡ（１９３０ｂｐ）片段．并分别将这些ｃＤＮＡ片段克隆入ｐＵＣ１８载体的ＳｍａⅠ限制性内切酶位点．对重组质粒分别采用直接ＤＮＡ双链末端终止法和在核酸外切酶Ⅲ和核酸酶Ｓ１作用下连续缺失ＤＮＡ后,相继克隆,构成一系列连续缺失的缺失体的方法,测定了全部核苷酸顺序．结果表明：ＡＣＡＰ编码区的ｃＤＮＡ顺序与已报道的有１２处碱基的改变,其中１１处碱基顺序的改变不引起编码的氨基酸变化,只有第３８５位的Ｔ→Ａ后,才引起其编码的氨基酸由Ｓｅｒ→Ｔｈｒ,但由于Ｓｅｒ和Ｔｈｒ的理化性质极其相似,这一变化可能并不导致蛋白质的生物活性的变化．这些改变可能是由于种族、群体或个体的差异．     

小鼠Uncv基因克隆及真核表达

目的 克隆小鼠的Uncv基因并在真核细胞表达.方法 采用RT-PCR方法扩增小鼠皮肤组织中Uncv基因编码区,以真核表达质粒pcDNA 3.1-Flag为载体,构建Uncv真核表达质粒,将重组载体转染Hela细胞并用Western blot法检测基因表达.结果 构建Uncv基因真核表达载体pcDNA 3.1-Flag/Unev,重组质粒在Hela细胞中有效表达约95×103的融合蛋白.结论 成功构建真核表达载体pcDNA 3.1-Flag/Uncv,并且在真核细胞中有效表达,为研究Uncv基因生物学功能奠定基础.     

Human mRNA polyadenylate binding protein: evolutionary conservation of a nucleic acid binding motif.

We have isolated a full length cDNA (cDNA) coding for the human poly(A) binding protein. The cDNA derived 73 kd basic translation product has the same Mr, isoelectric point and peptidic map as the poly(A) binding protein. DNA sequence analysis reveals a 70,244 dalton protein. The N terminal part, highly homologous to the yeast poly(A) binding protein, is sufficient for poly(A) binding activity. This domain consists of a four-fold repeated unit of approximately 80 amino acids present in other nucleic acid binding proteins. In the C terminal part there is, as in the yeast protein, a sequence of approximately 150 amino acids, rich in proline, alanine and glutamine which together account for 48% of the residues. A 2,9 kb mRNA corresponding to this cDNA has been detected in several vertebrate cell types and in Drosophila melanogaster at every developmental stage including oogenesis.     

Cloning and expression of isocitrate lyase from human round worm Strongyloides stercoralis

A full length cDNA (1463 bp) encoding isocitrate lyase (EC 4.1.3.1) of Strongyloides stercoralis is described. The nucleotide sequence of this insert identified a cDNA coding for the isocitrate lyase. The conceptually translated amino acid sequence of the open reading frame for S. stercoralis isocitrate lyase encodes a 450 amino acid residue protein with an apparent molecular weight of 50 kDa and a predicted pl of 6.39. The sequence is 69% A/T, reflecting a characteristic A/T codon bias of S. stercoralis. The amino acid sequence of S. stercoralis isocitrate lyase is compared with bifunctional glyoxylate cycle protein of Caenorhabditis elegans and isocitrate lyases from Chlamydomonas reinhardtii and Myxococcus xanthus. The full length cDNA of S. stercoralis was expressed in pRSET vector and bacteriophage T7 promoter based expression system. S. stercoralis lyase recombinant protein, purified via immobilized metal affinity chromatography, showed a molecular mass of 50 kDa on polyacrylamide gels. The role of isocitrate lyase in the glyoxylate cycle and energy metabolism of S. stercoralis is also discussed.     

Energy-linked anion transport. Cloning, sequencing, and characterization of a full length cDNA encoding the rat liver mitochondrial proton/phosphate symporter

A full length cDNA clone encoding the precursor of the rat liver mitochondrial phosphate transporter (H+/Pi symporter) has been isolated from a cDNA library using a bovine heart partial length phosphate transporter clone as a hybridization probe. The entire clone is 1263 base pairs in length with 5'- and 3'-untranslated regions of 16 and 168 base pairs, respectively. The open reading frame encodes for the mature protein (312 amino acids) preceded by a presequence of 44 amino acids enriched in basic residues. The polypeptide sequence predicted from the DNA sequence was confirmed by analyzing the first 17 amino-terminal amino acids of the pure phosphate transporter protein. The rat liver phosphate transporter differs from the bovine heart transporter in 32 amino acids (i.e. approximately 10%). It contains a region from amino acid 139 to 159 which is 37% identical with the beta-subunit of the liver mitochondrial ATP synthase. Amino acid sequence comparisons of the Pi transporter with Pi binding proteins, other H+-linked symporters, and the human glucose transporter did not reveal significant sequence homology. Analysis of genomic DNA from both rat and S. cerevisiae by Southern blots using the rat liver mitochondrial Pi carrier cDNA as a probe revealed remarkably similar restriction patterns, a finding consistent with the presence in lower and higher eukaryotes of homologous Pi carrier proteins. This is the first report of the isolation, sequencing, and characterization of a full length cDNA coding for a protein involved in energy-coupled Pi transport.     

Molecular identification and sequence analysis of Hillarin, a novel protein localized at the axon hillock.

The monoclonal antibody Lan3-15 identifies a novel protein, Hillarin, that is localized to the axon hillock of leech neurons. Using this antibody we have identified a full length cDNA coding for leech Hillarin and determined its sequence. The gene encodes a 1274 residue protein with a predicted molecular mass of 144013 Da. Data base searches revealed that leech Hillarin has potential orthologues in fly and nematode and that these proteins share two novel protein domains. The W180 domain is characterized by five conserved tryptophans whereas the H domains share 21 invariant residues. In contrast to the arrangement in fly and nematode the cassette containing the W180 and H domains is repeated twice in leech Hillarin. This suggests that the leech Hillarin sequence originated from a duplication event of an ancestral protein with single cassette structure.     

The ovalbumin gene family: complete sequence and structure of the Y gene.

The "ovalbumin Y" gene, one of three which constitute the ovalbumin gene family in chicken has been completely sequenced. The exact location of exons can be derived from the comparison with the ovalbumin gene sequence and from the map previously established by electron microscopy analysis. During evolution of the Y gene, selective pressure has operated to retain a sequence coding for an ovalbumin-like protein. The location of splice junctions, the length of protein coding exons and the reading phase are as in the ovalbumin gene. The overall homology between the Y and ovalbumin protein coding sequences is 72.6% (resulting in a 58% homology for the amino acid sequences). A significantly high number of base changes within coding sequences are present in clusters, which appear in several cases to be correlated with the occurrence of direct repeats. The 3' untranslated sequences of the Y and ovalbumin mRNAs have diverged much more, and the Y sequence contains a peculiar U(T) rich region. Corresponding introns of the ovalbumin and Y genes differ extensively both in sequence and in length. They share however characteristic biases in their base distribution.     

前体mRNA剪接蛋白Tra2β1的抗体制备及鉴定

Tra2 β1是Tra2 β前体mRNA剪接调节蛋白家族中的一个组织分布最广、表达量最多的成员 ,它在选择性前体mRNA剪接中有调节功能 ,而在基础性剪接中不是必需的 .为便于对该蛋白功能的进一步研究 ,需要制备能特异地检测Tra2 β1蛋白的抗体 .选择包含Tra2 β1的N端 12个氨基酸的特异编码序列的Tra2 β2全长编码序列 (共 117个核苷酸 ) ,将其克隆至pGEX 3X表达载体形成GST融合基因 ,并以诱导表达和纯化的该融合蛋白为抗原免疫新西兰兔 ,获得了相应的抗体 .Western印迹和免疫细胞化学分析结果显示 ,获得的抗体能特异地检测Tra2 β1蛋白     

完全重瓣型山茶花品种‘红十八学士'CjHDEF基因cDNA的序列分析

我国完全重瓣型山茶花品种‘红十八学士’(Camellia japonica‘Hongshibaxueshi’)CjHDEF基因的cDNA序列(GenBank登录号:HM773024)生物信息学分析表明,该基因属于花发育B类功能基因的AP3基因家族成员,其cDNA全长1013bp中有一个完整的681bp开放阅读框,编码226个氨基酸。该基因编码蛋白质分子量26.09kD,理论等电点9.03;不稳定系数达43.20,表明该蛋白为不稳定蛋白;其编码蛋白属于MIKC型蛋白,该蛋白预测的二级结构和三级结构结果相符,主要以α-螺旋和无规卷曲为主,延伸链所占比重最小;含有6个蛋白激酶C磷酸化位点,表明该基因与‘红十八学士’花器官发育的细胞生长和分化密切相关;有多达11个磷酸化特定位点,说明该基因在花器官发育的细胞信号传递过程中占有重要地位。     

白毛黑眼兔眼部虹膜Trp1、Trp2基因克隆与分析

目的 克隆日本大耳白兔白毛黑眼系(白毛黑眼兔)眼部虹膜Trp1、Trp2基因,获取其完整的外显子序列.进一步推测这两个基因编码的蛋白,并分析其特征.方法 从白毛黑眼兔的黑色虹膜组织中提取RNA,并反转录成cDNA.利用来自小鼠、大鼠和人的同源引物,扩增获得白毛黑眼兔Trp1、Trp2基因外显子片段.然后对已知片段进行3' RACE和5'RACE,从而获得白毛黑眼兔Trp1、Trp2基因外显子完整序列.利用相关软件对获得序列进行翻译和分析.结果 首次获得了白毛黑眼兔Trp1、Trp2基因外显子全序列.该实验兔Trp1基因的编码序列全长1604个碱基,其核苷酸序列与人的相似度为87.9％,与小鼠的相似度为82.7％.TRP1成熟蛋白包含513氨基酸,氨基酸序列与人的相似度为89.8％,与小鼠的相似度为86.6％.该实验兔Trp2基因序列全长1554个碱基,其核苷酸序列与人的相似度为83.2％,与小鼠的相似度为81.9％.TRP2成熟蛋白包含494个氨基酸,其序列与人的一致度为84.2％,与小鼠的一致度为84.4％.结论 本研究获得的TRP1、TRP2的序列与已知的家兔酪氨酸相关蛋白家族成员TYR的序列进行比对,结果显示这三种蛋白之间有较高的相似度,并且TRP1和TRP2蛋白序列表现出了酪氨酸酶家族结构上的保守性和特有的结构特征.     

NRDRiso酶cDNA的序列测定及生物信息学分析

通过鉴定分析人肝组织中辅酶II依赖性视黄醇脱氢酶不同剪接体全长cDNA核苷酸序列与氨基酸序列的结构特征,为今后进一步研究体内维甲酸的代谢情况奠定基础。根据人、小鼠NRDR编码区的一致性序列,设计一对引物,应用RTPCR方法从人肝组织中得到一条377bp的新的cDNA片段。采用RACE法得到了NRDR新亚型cDNA,并以生物信息学软件分析其生物学特征。 测序得知该cDNA长为1003bp,以NADP-dependent retinol dehydrogenase/reductase short isoform（NRDRiso）登录GenBank。其读码框为525bp,拟编码174个氨基酸的蛋白。     

Purification of juvenile hormone esterase and molecular cloning of the cDNA from Manduca sexta.

Juvenile hormone esterase (JHE) is a highly specific enzyme important for regulating the onset of metamorphosis in lepidopteran insects. After affinity chromatography of the hemolymph proteins of Manduca sexta, the pure JHE protein was digested with Lys-C and the resultant peptides were purified by microbore HPLC. Two peptides were selected for sequencing. Based upon these amino acid sequences, degenerate RT-PCR was performed in order to amplify a partial cDNA sequence from mRNA from the fat body of M. sexta. A 1512bp partial cDNA was generated and found to be highly homologous to the JHE from Heliothis virescens. 5' and 3' RACE were performed to obtain the full length cDNA sequence. The cDNA has a total length of 2220bp, with a 1749bp coding region. The deduced protein sequence contains 573 amino acids.     

白桦肌动蛋白(Actin)基因全长cDNA克隆与序列分析

以白桦(Betula platyphylla Suk.)次生木质部为材料,用改良CTAB方法提取总RNA。根据植物肌动蛋白(Actin)基因编码区的保守序列设计引物后进行RT-PCR,并采用RACE技术扩增出Actin基因全长序列。该基因cDNA全长1 785 bp,序列分析表明,该基因编码区1 134 bp,编码377个氨基酸,5′非编码区157 bp,3′非编码区495 bp。所得序列与GenBank中注册的其它植物肌动蛋白核苷酸序列的相似性均在80%以上,氨基酸序列的相似性高达96%以上。此基因已在GenBank注册（EU588981）。根据高等植物肌动蛋白相似性构建了进化树,表明白桦肌动蛋白与蓖麻肌动蛋白之间的亲缘关系最为密切,在进化中分化时间最为接近。     

Expression of human terminal deoxynucleotidyl transferase in Escherichia coli

A cloned DNA fragment related to pT17 containing a partial cDNA sequence of human terminal deoxynucleotidyl transferase was used as a probe to screen for the full length cDNA sequence of the enzyme in a lambda gt11 library constructed from human lymphoblastoid KM-3 cDNA. A recombinant containing a 2068-base pair insert was isolated and recloned into the EcoRI site of the sequencing plasmic pUC-8 as two subclones, pT711 and pT106. DNA sequencing and hybridization studies showed that pT711 contains the pT17 sequence and an additional 172 upstream nucleotides. pT711 represents the coding sequence for the carboxyl half of the terminal transferase protein. pT106, containing a 965-base pair insert, hybridizes to the same mRNA as pT711 on Northern blots and contains an open reading frame that is in phase with the reading frame of the insert in pT711. Amino acid sequencing of the 58-kDa peptide of the calf thymus terminal transferase failed, indicating that the N terminus is blocked. N-Terminal sequencing of a 56-kDa form of the protein produced 24 amino acids corresponding to the translated human cDNA coding sequence starting at residue 398 of the insert in pT106 with 83% homology between bovine and human sequence. The initiation codon is assigned to an ATG sequence at nucleotide 329 of the insert in pT106. Comparison of the translated human terminal transferase sequence with peptides from the calf thymus enzyme showed that the homology between the human and bovine enzyme is better than 90% among 263 amino acids determined. The coding sequences in pT106 and pT711 were recloned into an expression plasmid pUC-19 downstream from the lac promoter and in phase with the coding sequence of the lac Z gene. Lysates of bacteria carrying the reconstructed coding sequence of human terminal transferase contain a fused protein of 60 kDa that reacts with rabbit antibody to terminal transferase on immunoblots and exhibits enzyme activity. Isolation of this fused protein from bacterial lysates with mouse monoclonal antibody to human terminal transferase produces the expected protein of 60 kDa.     

A general strategy for cloning double-stranded RNA: nucleotide sequence of the Simian-11 rotavirus gene 8

Using Simian-11 rotavirus RNA, a strategy has been developed for the production of full length cloned copies of the genes of double-stranded (dsRNA) viruses. Genomic RNA segments were polyadenylated and reverse transcribed to yield a mixture of full length cDNA copies of both possible polarities. The cDNAs were annealed, filled in to complete any partial copies, tailed and inserted into the PstI site of pBR322 using dG/dC tailing. Cloned rotavirus cDNA gene copies were assigned to genomic RNA segments by Northern hybridization. The complete sequence of gene 8 which codes for NCVP3, a non-structural protein of SA11 rotavirus, was determined from a cloned gene copy. It is 1059 bases in length and has an open reading frame which could code for a protein containing 317 amino acids. The apparent 5' and 3' terminal non coding regions are 46 and 59 bases in length, respectively. The sequence ATGTGACCOH at the 3' end of the plus strand is conserved in four of the eleven genes examined. The cloning procedures used should be generally applicable to viruses with segmented dsRNA genomes.     

Drosophila choline acetyltransferase uses a non-AUG initiation codon and full length RNA is inefficiently translated

RNA from a partial cDNA clone containing the entire protein coding sequence of Drosophila melanogaster acetyl-CoA:choline O-acetyltransferase (EC 2.3.1.6; choline acetyltransferase) can be translated into active enzyme. This is unusual since this partial cDNA clone contains no appropriate ATG (AUG) initiation codon. In this study we use in vitro deletion and point mutants to identify GTG as the starting codon for protein translation. We also report the sequence of a full length Drosophila choline acetyltransferase cDNA and demonstrate that RNA produced by this clone is translated into active choline acetyltransferase but at a significantly reduced efficiency when compared to the partial cDNA clone. These results indicate that translational control may be an important regulatory step in production of Drosophila choline acetyltransferase.     

Expression of a phenylcoumaran benzylic ether reductase-like protein in the ovules of <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>

Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to identify differentially expressed proteins in wild-type (DP 5690) and fiberless (SL 1-7-1) cotton ovules. One protein, designated V2 was unique to ovules of the fiber producing DP 5690 line. The protein was purified from 2D-PAGE of 4 d post anthesis DP 5690 ovules and partially sequenced. The short amino acid sequence was nearly identical to the deduced amino acid sequence for cotton phenylcoumaran benzylic ether reductase (PCBER) protein. A consensus sequence was assembled from ESTs encoding cotton PCBER genes, primers were designed, and a full length gene was amplified from plasmid DNA from a 72 h etiolated cotton cotyledon library. The polymerase chain reaction generated a 950 bp product with unique EcoRI (5′) and (3′) KpnI restriction sites for directional insertion into the expression vector pPICZA. Nucleotide sequencing was performed, and the full length coding region was 924 bp encoding a protein of 308 amino acids. The molecular mass and pI measured (2D PAGE) were similar to the theoretical protein.