茶尺蠖小RNA病毒5'端非编码区的克隆和测序及与哺乳动物小RNA病毒的比较分析

用Trizol从纯化的茶尺蠖Ectropis oblique小RNA病毒（EoPV）中提取病毒基因组RNA,逆转录后加poly(dT),然后进行两步PCR扩增基因组5′端。克隆测序后,对其5′端非编码区的核苷酸序列进行分析,发现具有哺乳动物小RNA病毒的5′端非编码区的一些特征：A/T含量丰富、起始密码子上游AUG和小顺反子多。利用mfold预测了EoPV 5′端非编码区的二级结构,存在4个茎环结构,有哺乳动物内部核糖体进入位点（IRES）的保守区域,即含保守基序GNRA的茎环A和A/C丰富的环B及多聚嘧啶区域。据此推测EoPV基因组翻译采用IRES起始机制。     

茶尺蠖小RNA病毒5′端非编码区的克隆和测序及与哺乳动物小RNA病毒的比较分析

用Trizol从纯化的茶尺蠖Ectropis oblique 小RNA病毒(EoPV)中提取病毒基因组RNA,逆转录后加poly(dT),然后进行两步PCR扩增基因组5′端.克隆测序后,对其5′端非编码区的核苷酸序列进行分析,发现具有哺乳动物小RNA病毒的5′端非编码区的一些特征:A/T含量丰富、起始密码子上游AUG和小顺反子多.利用mfold预测了EoPV 5′端非编码区的二级结构,存在4个茎环结构,有哺乳动物内部核糖体进入位点(IRES)的保守区域,即含保守基序GNRA的茎环A和A/C丰富的环B及多聚嘧啶区域.据此推测EoPV基因组翻译采用IRES起始机制.     

从茶尺蠖中分离到一株微小RNA病毒

从核型多角体病毒(NPV)感染致死的茶尺蠖幼虫尸体中分离到一株微小RNA病毒(EoPV)透射电镜观察纯化的病毒粒子为无囊膜、无表面特征、直径约26nm的球状颗粒。16％的SDS-PAGE显示它有两个分子量为31．5kDa和28．8kDa的衣壳蛋白，后者的含量是前者的2、5倍。3′端克隆序列分析表明EoPV基因组3′有poly(A)尾，编码RNA聚合酶(RdRp)，含有微小RNA病毒RNA聚合酶的八个保守基序，同源性分析表明它与榕透翅毒蛾微小RNA病毒(PnPV)亲缘关系最近。这些特点表明该病毒为一株新的微小RNA病毒。     

表达绿色荧光蛋白的重组鸡传染性支气管炎病毒的构建

为探讨鸡传染性支气管炎病毒(IBV)作为载体表达外源基因的可行性,本研究根据IBV H120疫苗株的全基因组序列设计引物,采用RT-PCR方法分10个片段对其基因组进行扩增,并克隆至pMD19-T载体中;同时构建IBV基因组5a基因编码区被增强型绿色荧光蛋白(EGFP)基因替换的重组质粒。采用体外拼接策略,将BsaI酶切处理的10个基因片段顺序连接,构建5a基因编码区被EGFP基因替换的基因组全长cDNA,其5’端具有完整的T7 RNA聚合酶启动子核心序列,3’端具有polyA尾巴结构。然后通过T7 RNA聚合酶体外转录系统合成病毒基因组RNA,脂质体转染BHK-21细胞进行病毒拯救。结果表明成功的从基因组全长cDNA拯救出重组病毒H120-5a/EGFP株,其在鸡胚中能有效的复制和传代,并表达绿色荧光蛋白;5a基因的缺失并不影响病毒对鸡胚的致病性。本研究为进一步开展IBV的分子致病机理、载体疫苗等研究奠定了基础。     

从茶尺蠖中分离到一株微小RNA病毒

从核型多角体病毒(PV)感染致死的茶尺蠖幼虫尸体中分离到一株微小RNA病毒(oPV)透射电镜观察纯化的病毒粒子为无囊膜、无表面特征、直径约26nm的球状颗粒.16%的SDS-PAGE显示它有两个分子量为31.5 kDa和28.8 kDa的衣壳蛋白,后者的含量是前者的2.5倍.3′端克隆序列分析表明EoPV基因组3′有poly(A)尾,编码RNA聚合酶(dRp)含有微小RNA病毒RNA聚合酶的八个保守基序,同源性分析表明它与榕透翅毒蛾微小RNA病毒(nPV)亲缘关系最近.这些特点表明该病毒为一株新的微小RNA病毒.     

禽流感病毒H5N1全基因组克隆与分析

为明确广东地区分离的一株禽流感病毒H5N1的遗传背景,建立流感病毒反向遗传的平台。对该株禽流感病毒进行了空斑纯化与组织细胞培养,检测其在MDCK细胞中的增殖特性;利用H5N1病毒通用引物,通过RT-PCR对该病毒全基因组的8条片段进行全长克隆及测序分析;将H5N1的8条全长基因组片段分别插入反向遗传通用载体中,构建禽流感病毒H5N1的感染性克隆。结果表明,该H5N1毒株在MDCK细胞中可不依赖胰酶进行有效增殖与复制,可使MDCK细胞出现典型细胞病变,具有高致病性禽流感病毒的细胞增殖特征。RT-PCR克隆得到该H5N1毒株的PB2、PB1、PA、HA、NP、NA、M和NS八条全长片段,经测序分析确认该毒株的基因序列,其内部编码序列出现多处突变,其中HA连接肽为多个连续碱性氨基酸,表明该毒株可不依赖胰酶进行有效复制,与细胞培养结果一致,未出现抗药性的遗传突变。PCR与测序证明,插入H5N1八个全长基因组片段的载体序列完全正确,表明成功构建了该毒株的感染性克隆。为明确病毒遗传信息,建立流感病毒反向遗传的平台,为进一步研究禽流感病毒相关疫苗提供了研究基础。     

犬瘟热病毒小熊猫株反向遗传系统的构建及其鉴定

以驯化致弱的犬瘟热病毒小熊猫株(Canine distemper virus,CDV)为模板,构建犬痘热病毒感染性cDNA克隆.对其全基因组序列测定后,用RT-PCR的方法获得组成全长基因的7个片段,通过酶切、拼接将7段CDVcDNA序列插入到真核表达载体pCI的MCS上,构建犬瘟热病毒小熊猫株的全长cDNA质粒(pCI-CDV-LP),同时分别克隆CDV小熊猫株N、P、L蛋白ORF构建三个辅助质粒.酶切鉴定和序列测定表明,pCI载体中插入的核酶及CDV cDNA序列正确无误,使用转染试剂Lipofectamine TM 2000将全长质粒和三个辅助质粒共转染中国仓鼠肾细胞(BSR),经RT-PCR、间接免疫荧光和病毒感染VERO-SLAM细胞试验鉴定,成功拯救出CDV小熊猫株,显示CDV小熊猫株反向遗传系统构建完成,为犬瘟热病毒致病机理及免疫研究奠定基础.     

一株A型口蹄疫流行毒株全序列的测定及其全长感染性克隆的构建

【目的】测定一株A型口蹄疫流行毒株的全基因组序列,并构建其全长感染性克隆。【方法】参照已公布的A型口蹄疫病毒序列设计引物,将分离的口蹄疫病毒株A/Sea-97/CHA/2014全基因组分为4个重叠的片段进行RT-PCR扩增,并对其进行序列测定与分析。利用酶切连接法将4个基因片段依次克隆至p Blue Script SKhdv载体中,构建该流行毒株的全长c DNA克隆p QAHN。pQAHN经NotⅠ线性化后转染表达T7 RNA聚合酶的BSR/T7细胞,拯救病毒。【结果】口蹄疫病毒全基因组序列测定结果表明该毒株基因组全长8 171 bp[不包括poly(C)区段和poly(A)尾巴],开放阅读框为6 996 bp,编码2 332个氨基酸,5′和3′非编码区分别为1 091 bp和95 bp。VP1系统发生树分析表明该毒株与A/GDMM/CHA/2013毒株亲缘关系最近,相似性为99.1%。线化全长质粒转染BSR/T7细胞68 h后可观察到典型的细胞病变。拯救病毒的间接免疫荧光、RT-PCR和序列测定结果表明成功拯救出了具有感染性的FMDV。拯救病毒与亲本病毒的噬斑表型及生长曲线试验表明二者具有相似的生长表型和增殖能力。【结论】该研究为我国口蹄疫病原生态分布、分子流行病学调查以及A型FMD新型疫苗的研究提供了有益的材料。     

负链不分节段RNA病毒反向遗传系统中通用型真核表达载体的构建

本研究选择pVAX1作为供体载体,通过分子克隆方法,分别用人工合成的锤头型核酶、丁型肝炎核酶序列(用于获得转录后病毒基因组RNA的精确末端)和含有9个常用限制性酶切位点的linker序列(用于病毒基因组插入的多克隆酶切位点)取代真核表达载体pVAX1的多克隆酶切位点,将其改造成负链不分节段RNA病毒反向遗传系统的通用型表达载体。通过酶切鉴定和序列测定表明,pVAX1载体中插入的核酶及linker序列正确无误,并将CTN株狂犬病病毒全长基因组cDNA插入pVAX-R中,通过与辅助质粒的共转染成功拯救CTN株狂犬病毒,证明通用型真核表达载体构建成功,为快速建立负链不分节段RNA病毒反向遗传系统奠定基础。     

流行性乙型脑炎病毒(JEV)全长感染性克隆的制备及恢复病毒的获得

根据JEV病毒减毒株SA14—14—2基因组序列,设计覆盖全长的4对重叠引物,以提取的活疫苗病毒RNA为模板,RT—PCR扩增出4个片段,并克隆到质粒载体中,进一步构建两个半端分子克隆,然后将全长cDNA序列克隆到一个新改造的低拷贝质粒载体pBR—kpn中,构建我国流行性乙型脑炎病毒(JEV)基因组全长cDNA克隆。经过体外转录后得到的转录子转染BHK-21细胞,重新获得JEV的恢复病毒,通过生物学特性、分子生物学水平、蛋白水平等几个方面对恢复病毒进行鉴定。结果获得了稳定的全长cDNA克隆,转录子转染BHK-21细胞后,第4天开始出现细胞病变(CPE),第6～7天时CPE为 ,经过Vero细胞进一步放大培养后,间接免疫荧光实验和RT—PCR实验均为阳性。证实了构建的JEV的全长cDNA克隆有感染性,为进一步的研究奠定了基础。     

Human adenine phosphoribosyltransferase. Complete amino acid sequence of the erythrocyte enzyme

We defined the amino acid sequence of adenine phosphoribosyltransferase isolated from human erythrocytes. Peptide fragments formed by cleavage at arginine, lysine, glutamic acid, and methionine were purified by high pressure liquid chromatography and sequenced by manual Edman degradation. The complete primary structure of human adenine phosphoribosyltransferase was established by sequence analysis of 19 peptide fragments. Presumed homology between the human and rodent enzymes was used to order fragments that had inadequate overlapping sequences. The enzyme has 179 residues with a calculated subunit molecular weight of 19,481. Mass spectrometry indicated that the NH2-terminal residue is acetylated. Human adenine phosphoribosyltransferase has sequence homology with xanthine-guanine phosphoribosyltransferase from Escherichia coli in 110-amino acid region encompassing the NH2-terminal section of the enzyme.     

The delineation of a decapeptide gonadotropin-releasing sequence in the carboxyl-terminal extension of the human gonadotropin-releasing hormone precursor

The human gonadotropin-releasing hormone (GnRH) precursor consists of the GnRH sequence followed by a 59-amino acid carboxyl-terminal extension. A 56-amino acid peptide within this extension has been shown to stimulate gonadotropin release, and this activity has been localized to its amino-terminal region. A series of seven overlapping peptide fragments corresponding to the first 24 amino acids of the carboxyl-extension of the GnRH precursor were synthesized and tested for their ability to stimulate luteinizing hormone and follicle-stimulating hormone release from cultured human anterior pituitary cells. All active peptide fragments were found to incorporate the decapeptide sequence Asn-Leu-Ile-Asp-Ser-Phe-Gln-Glu-Ile-Val which is regarded as a minimal structural requirement delineated for gonadotropin-releasing activity. A further flanking sequence extending this active region from its carboxyl terminus was found to enhance gonadotropin-releasing activity although the flanking sequence itself was inactive. The gonadotropin release stimulated by the active peptides wa shown to be a dose- dependent, specific, and calcium-dependent phenomenon which occurred independently of the GnRH receptor on the pituitary gonadotrophs as a GnRH antagonist did not inhibit activity.     

Isolation of ovocleidin-116 from chicken eggshells, correction of its amino acid sequence and identification of disulfide bonds and glycosylated Asn.

Fractionation of the soluble chicken eggshell matrix by chromatographic methods yielded 13 endogenous proteolytic fragments of the eggshell-specific proteoglycan core protein ovocleidin-116. The N-terminal amino acid sequences of these fragments in general confirmed the recently cDNA-deduced sequence of ovocleidin-116, with one exception. One fragment yielded a completely new sequence and was instrumental in detecting a frame shift error in the nucleotide sequence. The correction yielded a new sequence which was 38 amino acids shorter than before and contained a 57-amino acid long novel C-terminal sequence. The predicted sequence of ovocleidin-116 contained two consensus N-glycosylation sites, only one of which (Asn62) was found to be fully modified. A disulfide bond was identified between Cys31 and 42 implying that Cys329 and 421 form a second disulfide bond. Finally, the yield of fragments indicated that ovocleidin-116 is a major component of the chicken eggshell matrix.     

The cloning and nucleotide sequence of the serine protease gene (aspA) of Aeromonas salmonicida ssp. salmonicida

The gene for Aeromonas salmonicida serine protease has been cloned into phagemid pTZ18R in two restriction fragments, 2.0-kb PstI and 2.3-kb KpnI, of genomic DNA. The nucleotide sequences of the two fragments have been determined, in both directions, after subcloning, by double-stranded sequencing of nested deletions. An open reading frame of 1863 bp translated into a sequence of 621 amino acids, a 24-amino acid signal peptide and a 597-amino acid mature enzyme of molecular mass 64,173 Da. The consensus sequence, NGTS, of a serine protease substrate primary binding site was identified and a putative ribosome-binding site GGAG occurred 6 bp upstream of the ATG initiation codon.     

Nucleotide sequence of the DNA complementary to avian (chicken) preproparathyroid hormone mRNA and the deduced sequence of the hormone precursor

The nucleotide sequence of avian (chicken) prepro-PTH (prepro-PTH) mRNA was determined from a 2.3-kilobase fragment of complementary chicken parathyroid DNA cloned in E. coli MM 924. Northern blot analysis of chicken parathyroid mRNA, using both bovine and chicken cDNA probes, showed that the mRNA (2.3 kilobases) for chicken hormone precursor was approximately 3 times the size of mRNA for mammalian prepro-PTH. Cleavage of the cloned DNA with restriction endonuclease Pstl resulted in three fragments, each of which was subjected to sequence determination. The hormone sequence deduced from the DNA showed that chicken prepro-PTH mRNA encoded a 119-amino acid precursor which included a 25-amino acid signal sequence, a six-residue prohormone peptide, and an 88-amino acid hormone. The hormonal peptide was four residues longer than all known mammalian homologs and included gene deletions and insertions. There was significant homology of sequence in the biologically active 1-34 region with mammalian hormones, but much less in the middle and carboxyl-terminal regions. This is the first nonmammalian PTH sequence to be determined and should prove useful in studying evolution of the gene as well as structure-function relationships of the hormone.     

Structure of full-length cDNA coding for sulfatide activator, a Co-beta-glucosidase and two other homologous proteins: two alternate forms of the sulfatide activator

Full-length cDNA clones have been isolated for an mRNA which codes for four different but homologous proteins--a sulfatide activator protein, a co-beta-clucosidase, and two other proteins of similar structures. The primary structure as deduced from the nucleotide sequence is highly homologous to the precursor of the rat Sertoli cell sulfated glycoprotein 1. The full-length clone was 2,734-bp long, starting from 8 bases above the initiator ATG and terminating with a poly A tail. The nucleotide sequence confirmed an earlier prediction based on the amino acid sequence that a previously published sequence contained errors. On the other hand, the amino acid sequence now closely agrees with the recent revised sequence published by the same group except for several amino acids near the N-terminus. Two alternate forms of the sulfatide activator were detected, differing from each other by the presence or absence of 3-amino acid insertion.     

Human skeletal-muscle aldolase: N-terminal sequence analysis of CNBr- and o-iodosobenzoic acid-cleavage fragments

Fructose-1,6-bisphosphate aldolase was purified from human skeletal-muscle by affinity elution chromatography. Four CNBr-cleavage fragments were purified by gel filtration, and their N-terminal amino acid sequences were determined. Cleavage with o-iodosobenzoic acid at the three tryptophan residues also yielded fragments suitable for N-terminal sequence analysis. Thus, the sequence of 272 of the 363 residues was established. These sequence results allow many of the discrepancies between the two published rabbit skeletal-muscle aldolase sequences to be resolved. The human aldolase sequence reported here is 96% identical to a "consensus" rabbit aldolase sequence. A comparison with a partial sequence of Drosophila aldolase (103 residues) shows 80% identity. The determination of the amino acid sequence of human aldolase is important for the interpretation of the crystal structure of this enzyme.     

Identification and characterization of a novel sericin gene expressed in the anterior middle silk gland of the silkworm Bombyx mori

Sericin is a group of proteins expressed in the middle silk gland that covers the surface of fibroin in the cocoon filament of Bombyx mori. Sericin consists of several serine-rich proteins with different molecular masses. Sericin A is one of the proteins and is produced in the anterior portion of the middle silk gland. To identify the gene coding for the protein, we determined the primary structures of its partial peptides, and the gene was searched using the silkworm genomic databases. Three contigs containing the corresponding nucleotide sequences were identified and categorized as one group. The gene structure covering the 5' flanking and the 3' end was determined by PCR fragments from genomic DNA, RT-PCR, and 5' and 3' RACE. The amino acid sequence deduced from the nucleotide sequence mainly consists of two serine-rich regions of 86-amino acid motif and 8-amino acid repeated sequence. The expression of the gene is limited to the anterior and middle parts of the middle silk gland. In addition, because the sericin gene appeared different from the sericin 1 and 2 genes reported earlier, we designated the newly discovered gene as sericin 3.