猪轮状病毒vp4基因的克隆及其在昆虫细胞中的表达

本研究扩增猪轮状病毒中国分离株JL94株VP4蛋白主要抗原编码区基因(1-756 bp),将测序结果与国外分离株进行比较;将该基因片段同载体pMel BacA连接后,与杆状病毒DNA共转染入昆虫细胞Sf9,经蚀斑筛选纯化重组病毒并再感染Sf9细胞获得vp4基因的表达,对表达的VP4蛋白进行Western blot分析和血清中和抗体试验.结果表明JL94株VP4主要抗原编码区基因与国外分离株CRW-8株、Gottfried株该基因片段氨基酸同源性分别为96.43%和67%,说明JL94株与CRW-8株属同一VP4血清型,而与Gottfried株属不同血清型.JL94株VP4主要抗原编码区氨基酸最大变异处位于aa81-aa207.vp4基因在昆虫细胞中表达量占细胞总蛋白的20%,Western Blot证实表达蛋白有良好的生物学活性.所表达的蛋白免疫小鼠产生中和抗体,阻断JL94在MA104细胞上引起的细胞病变.     

猪轮状病毒JL94中国分离株衣壳蛋白基因的分析

根据GenBank发表的猪轮状病毒衣壳蛋白vp4基因、vp6基因和vp7基因保守序列,设计合成3对引物,分别扩增猪轮状病毒中国分离株JL94株的此3段基因并进行序列分析。结果表明,JL94株vp4基因与国外分离株CRW-8株、OSU株、Gottfried株、4F株和4S株的VP4氨基酸同源性分别为97.16％、95％、71.88％、73.45％和70.88％;JL94株vp6与CRW-8株、OSU株、Gottfried株、4F株和4S株的VP6氨基酸同源性分别为96.98％、97.48％、93.20％、97.48％和93％;JL94株vp7基因与CRW-8株、OSU株、Gottfried株、4F株和4S株的VP7氨基酸同源性分别为98％、99.90％、75.40％、84.66％和80％。可见JL94株同CRW-8株、OSU株同源性更高些;vp4和vp7基因在同型间高度保守而不同型间差距较大,vp6基因在同群和非同群间差别不是很明显。同时可以判定JL94属于A群Ⅰ亚群G5P7血清型PRV。研究猪轮状病毒中国分离株JL94株衣壳蛋白基因的特征,为研制高效抗病毒疫苗奠定基础。     

猪轮状病毒vp4基因在大肠杆菌中的表达

以猪轮状病毒JL94株核酸为模板扩增该病毒vp4全基因,对扩增产物进行测序及序列比较;根据VP4的5'端(1bp～750bp)特异片段主要决定其活性的观点,再设计一对引物扩增该主要抗原位点基因,将此主要抗原位点基因同pGEX-6P-1载体连接并转化入E.coli.BL21(DE3)plays,经IPTG诱导表达出蛋白质;对表达的蛋白进行Westernblot分析、纯化和血清中和抗体试验.结果表明JL94株与国外分离株CRW-8株、BEN-307株vp4全基因片段氨基酸同源性分别为96.98%和98.05%,说明JL94株与CRW-8株、BEN-307株属于同一VP4血清型;经IPTG诱导VP4主要抗原位点基因获得了高效表达,表达量占菌体蛋白的26%;Westernblot结果和所表达的融合蛋白免疫小鼠产生的中和抗体能阻断JL94在MA104细胞上引起的细胞病变,说明所表达蛋白有良好的生物学活性.     

猪轮状病毒vp4基因在大肠杆菌中的表达

以猪轮状病毒JL94株核酸为模板扩增该病毒vp4全基因,对扩增产物进行测序及序列比较;根据VP4的5’端(1bp-750bp)特异片段主要决定其活性的观点,再设计一对引物扩增该主要抗原位点基因,将此主要抗原位点基因同pGEX-6P-1载体连接并转化入E．coli．BL2l(DE3)plays,经IPTG诱导表达出蛋白质;对表达的蛋白进行Westernblot分析、纯化和血清中和抗体试验。结果表明：JL94株与国外分离株CRW．8株、BEN．307株vp4全基因片段氨基酸同源性分别为96．98％和98．05％,说明JL94株与CRW．8株、BEN．307株属于同-vP4血清型;经IPTG诱导VP4主要抗原位点基因获得了高效表达,表达量占菌体蛋白的26％;Westernblot结果和所表达的融合蛋白免疫小鼠产生的中和抗体能阻断JL94在MAl04细胞上引起的细胞病变,说明所表达蛋白有良好的生物学活性。     

猪轮状病毒JL94株vp4全基因克隆及原核表达载体构建

根据GenBank已发表的猪轮状病毒vp4基因保守序列,设计一对引物扩增猪轮状病毒地方株JL94株vp4全基因;将扩增产物与载体pMD-18T连接进行序列测定并将测序结果同国外分离株进行序列比较。用限制性内切酶BamHI和SalI将vp4基因从pMD-18T-vp4切下;同样用BamHI和SalI双酶切表达载体pGEX-6P-1;将这2个双酶切产物连接并转化,通过酶切鉴定和PCR鉴定证明完成了vp4原核表达载体的构建。测序结果表明:vp4全基因长2362bp,JL94株同国外分离株BEN307株、Gottfried株vp4全基因片段核苷酸同源性分别为93.44%和69.43%;氨基酸同源性分别为96.06%和71.88%。说明JL94株同BEN-307株属同一VP4血清型,而同Gottfried株属于不同血清型。重组原核表达载体pGEX-6P1-vp4的构建为表达VP4蛋白,研制诊断性抗原和病毒重组活载体疫苗奠定了基础。     

人A组轮状病毒北京地方株VP4血清型特异片段编码基因的序列分析

轮状病毒是引起婴幼儿严重腹泻的重要病原,其第四基因编码主要中和抗原VP4,而VP4可裂解为VP8和VP5两个片段。VP8为抗原型特异性片段。克隆并测定了具有代表性的三个轮状病毒北京株VP4编码基因5′端（VPS＋VPS一部分）887个核苷酸序列并据此推导出其氨基酸序列。结果表明,相同血清型的地方株和标准株之间具有高度同源性（92％～966％）,不同血清型间则变异较大（70．5％～71％）。氨基酸最大变异处位于aa84～172,并对胰酶作用位点在致病性中的可能性进行了讨论。     

A组轮状病毒多个结构蛋白抗原表位的串联表达及其免疫原性的检测

从北京腹泻婴儿粪便提取的轮状病毒(rotavirus,RV)(T114株)的RNA中,克隆到轮状病毒结构蛋白基因vp4,vp6和vp7的全长cDNA,对它们编码的蛋白质序列和可能的抗原表位肽进行了预测,选择了RV主要抗原蛋白VP7、VP6和VP4的4个抗原表位肽,通过人工合成DNA的方式将这些抗原表位肽基因串联融合成一个阅读框RME(rotavirus multipleepitopes,RME)并构建原核表达载体.大肠杆菌表达的RME在ELISA反应中可被RV多克隆抗体识别,纯化的RME蛋白注射免疫小鼠可诱导特异性免疫应答,产生高滴度的同源氨基酸序列特异抗体和人RV抗体,其中针对RME的IgG抗体滴度达到l∶40 000,针对单个抗原表位EV7、EV6和EV4的IgG抗体滴度达l∶10 000～l∶20 000,针对RV Wa株的IgG抗体滴度较低为l∶2 500,但能特异地中和该病毒对MAC145细胞的侵染.上述结果为新型RV基因工程疫苗的研发提供了论据和基础.     

Asia1型口蹄疫病毒分离株结构蛋白基因的克隆与表达

口蹄疫病毒结构蛋白氨基酸的变化是病毒抗原性变异的分子基础,大部分抗原表位位于主要的免疫原蛋白VP1上,部分非线性抗原表位位于VP2和VP3上。本研究首次成功测定了 Asia1 型口蹄疫病毒(YNBS/58)四种结构蛋白基因( p1 区)的核苷酸序列,全长 2199 个碱基,编码 733 个氨基酸,该基因与 Ind63/72、Pka3/54、Israel、China/99、C1/Germany、A22、ZIM7/83/2 毒株的 p1 基因核苷酸序列同源性分别为 88. 4%、86. 0%、89. 3%、68.6%、67.6%、66.8%、50.3%,推导的氨基酸序列同源性分别为 94.1%、93.2%、95.1%、79.9%、77.0%、76.5%、58.1%;将YNBS/58株与 Ind63/72、Pka3/54、Israel株的 vp1、vp2、vp3、vp4 基因和编码蛋白分别进行同源性比较,发现VP1的序列变异最大,VP2、VP3、VP4次之,且VP1的氨基酸变异主要集中在 42-50 位和 137-156 位。实现了YNBS/58株结构蛋白基因在大肠杆菌中的高效表达,其表达的融合蛋白以包涵体形式存在,分子量约为88kDa,占菌体总蛋白的16%左右,并利用镍柱对目的蛋白进行了纯化,纯度达 90%以上,本实验为进一步研究 A sia1型口蹄疫病毒的分子流行病学、p1基因及其编码蛋白的生物学功能奠定了基础。     

Asia1型口蹄疫病毒分离株结构蛋白基因的克隆与表达

口蹄疫病毒结构蛋白氨基酸的变化是病毒抗原性变异的分子基础,大部分抗原表位位于主要的免疫原蛋白VP1上,部分非线性抗原表位位于VP2和VP3上.本研究首次成功测定了Asia1型口蹄疫病毒(YNBS/58)四种结构蛋白基因(p1区)的核苷酸序列,全长2199个碱基,编码733个氨基酸,该基因与Ind63/72、Pka3/54、Israel、China/99、C1/Germany、A22、ZIM7/83/2毒株的p1基因核苷酸序列同源性分别为88.4%、86.0%、89.3%、68.6%、67.6%、66.8%、50.3%,推导的氨基酸序列同源性分别为94.1%、93.2%、95.1%、79.9%、77.0%、76.5%、58.1%;将YNBS/58株与Ind63/72、Pka3/54、Israel株的vp1、vp2、vp3、vp4基因和编码蛋白分别进行同源性比较,发现VP1的序列变异最大,VP2、VP3、VP4次之,且VP1的氨基酸变异主要集中在42-50位和137-156位.实现了YNBS/58株结构蛋白基因在大肠杆菌中的高效表达,其表达的融合蛋白以包涵体形式存在,分子量约为88kDa,占菌体总蛋白的16%左右,并利用镍柱对目的蛋白进行了纯化,纯度达90%以上,本实验为进一步研究A-sia1型口蹄疫病毒的分子流行病学、p1基因及其编码蛋白的生物学功能奠定了基础.     

含禽流感病毒M2e 氨基端抗原表位的重组传染性法氏囊病毒VP2 蛋白的免疫原性鉴定

【目的】构建传染性法氏囊病毒VP2蛋白展示禽流感M2e抗原表位的重组蛋白,研发预防H5或H9亚型禽流感和传染性法氏囊的基因工程疫苗。【方法】根据现有禽流感疫苗株M2e的氨基端12个氨基酸多肽序列(nM2e)序列,结合GenBank中H5和H9亚型禽流感病毒nM2e的比对结果,确定nM2e序列。用融合PCR分别将1拷贝H5或H9的nM2e序列插入IBD B87株VP2基因的PBC区,获得VP2BCnM2e重组基因。将重组基因克隆至杆状病毒表达系统,转染Sf9细胞进行表达。经间接免疫荧光和Western blotting检测Sf9细胞表达重组基因后,扩繁重组病毒,制备疫苗,间隔4周对非免鸡作2次重复免疫,用间接ELISA和鸡胚成纤维细胞中的病毒血清中和试验检测血清中VP2和nM2e的抗体效价。【结果】成功构建含H5或H9 nM2e的VP2BCnM2e重组基因,该重组基因在Sf9细胞中得到表达。经免疫鸡,两重组蛋白均能激发针对VP2和nM2e的抗体,VP2BCnM2eH5组抗体效价高于VP2BCnM2eH9组。【结论】两重组蛋白均具有免疫原性,VP2BCnM2eH5免疫原性更佳。     

Molecular cloning of a GTPase activating protein specific for the Krev-1 protein p21rap1

The rap1/Krev-1 gene encodes a ras-related protein that suppresses transformation by ras oncogenes. We have purified an 88 kd GTPase activating protein (GAP), specific for the rap1/Krev-1 gene product, from bovine brain. Based on partial amino acid sequences obtained from this protein, a 3.3 kb cDNA was isolated from a human brain library. Expression of the cDNA in insect Sf9 cells resulted in high level production of an 85-95 kd rap1GAP that specifically stimulated the GTPase activity of p21rap1. The complete deduced amino acid sequence is not homologous to any known protein sequences, including GAPs specific for p21ras. Northern and Western blotting analysis indicate that rap1GAP is not ubiquitously expressed and appears most abundant in fetal tissues and certain tumor cell lines, particularly the Wilms' kidney tumor, SK-NEP-1, and the melanoma, SK-MEL-3, cell lines.     

Molecular cloning and expression of a G25K cDNA, the human homolog of the yeast cell cycle gene CDC42.

G25K is a low-molecular-mass GTP-binding protein with a broad distribution in mammalian tissues. A cDNA clone was isolated by using oligonucleotides corresponding to the partial amino acid sequence of purified human G25K. The cDNA encodes an 191-amino-acid polypeptide containing GTP-binding consensus sequences and a putative farnesylation site at the C terminus. The sequence exhibits 50 and 70% identities to the mammalian rho and rac proteins, respectively, and an 80% identity to the Saccharomyces cerevisiae CDC42 gene product. Insect Sf9 cells infected with recombinant baculovirus vectors expressing the G25K cDNA produced a 25-kDa protein that bound GTP and was recognized by antibodies specifically reactive to G25K. G25K appears to be the human homolog of the CDC42 gene product, since expression of the G25K cDNA in S. cerevisiae suppressed both cdc42-1 and cdc24-4 temperature-sensitive lethal mutations.     

猪瘟病毒糖蛋白E0基因的克隆及及表达研究

用ＲＴ－ＰＣＲ方法扩增分别获得了中国猪瘟病毒强毒石门株和兔化弱毒株糖蛋白Ｅ０基因ｃＤＮＡ,并克隆到ｐＧＥＭ Ｔ载体中测定其核苷酸序列和推导出其对应氨基酸序列,结果表明这两个毒 间的Ｅ０基因核苷酸序列同源性为９５．３％,氨基酸序列同源性为９４％,有１４个殖基的差异;与几个代表毒株ＡＬＤ株、ＧＰＥ株、Ｂｒｅｓｃｉａ株、Ａｌｆｏｒｔ株和国外测得的兔化弱毒Ｃ株相应序列进行比较,所测石门病毒核苷酸序列与上述     

Characterization of cDNAs encoding p53 of Bombyx mori and Spodoptera frugiperda

Complementary DNAs encoding homologs of the tumor suppressor gene, p53, were characterized from two lepidopteran insects, Bombyx mori (Bm) and Spodoptera frugiperda (Sf). They encoded predicted proteins of 368 (41.2 kDa) (Bm) and 374 (42.5 kDa) (Sf) amino acids. The sequences shared 44% amino acid and 60% nucleotide sequence identity with each other, but exhibited less than 20% amino acid and 46% nucleotide sequence identity to Drosophila melanogaster p53. Despite the sequence diversity, conserved amino acids involved in DNA and zinc binding were present in the lepidopteran sequences. Expression of Sfp53-induced apoptosis in S. frugiperda cells, and antiserum made against recombinant Sfp53 recognized a protein whose abundance increased after treatment with DNA damaging agents.     

猪细小病毒SD-68株vp2基因的克隆及序列分析

对猪细小病毒(PPV)SD-68株印2基因进行的克隆和序列测定表明：SD-68株VP2基因全长1740bD,编码579个氨基酸残基组成的多肽;PPVSD-68株与Kresse株、SY-99株、NADL-2(5075)株、NADL-2(4973)株、US-1株的VP2基因比较,核苷酸的同源性在99％以上,氨基酸的同源性在96％以上。进化树分析表明SD-68株与kresse株的亲缘关系最近;在决定毒株组织嗜性的关键氨基酸位点上(378,383及436),SD-68株与kresse株的差异最小,据此推测SD-68株的组织嗜性与Kresse株相似,即SD-68株属皮炎型PPV;而比较弱毒株NADL-2、SD-68和强毒株kresse VP2的氨基酸差异后发现,215、378和383可能是决定PPV致病性强弱的关键位点。     

Sequence of the T4 recombination gene, uvsX, and its comparison with that of the recA gene of Escherichia coli.

We have determined the nucleotide sequence of the uvsX gene of bacteriophage T4 which is involved in DNA recombination and damage repair, and whose product catalyzes in vitro reactions related to recombination process in analogous manners to E. coli recA gene product. The coding region consisted of 1170 nucleotides directing the synthesis of a polypeptide of 390 amino acids in length with a calculated molecular weight of 43,760. Amino acid composition, the sequence of seven NH2-terminal amino acids and molecular weight of the protein deduced from the nucleotide sequence were consistent with the data from the analysis of the purified uvsX protein. The nucleotide sequence and the deduced amino acid sequence were compared with those of the recA gene. Although a significant homology was not found in the nucleotide sequences, the amino acid sequences included 23% of identical and 15% of conservatively substituted residues.     

苜宿银纹夜蛾核型多角体病毒vp39基因的克隆及其对S_f9细胞形态的影响

最近的研究发现：ＡｃＮＰＶ的ｖｐ３９基因与侵染密切相关［１］．在侵染过程中,ＶＰ３９蛋白与宿主的肌动蛋白结合,使其重排形成缆索（ｃａｂｌｅ）．导致细胞骨架发生变化有利于病毒编码的蛋白酶的水解．最后,子代病毒颗粒大量形成,宿主昆虫体全部液化成为脓水．可...     

Characterization of aquabirnaviruses from flounder Pseudopleuronectes americanus and mummichog Fundulus heteroclitus in the Chesapeake Bay, Virginia, USA

Viruses were isolated in cell culture from tissue homogenates of flounder Pseudopleuronectes americanus and mummichog Fundulus heteroclitus in the Chesapeake Bay, Virginia, USA. Neutralization and immunofluorescence tests with aquabirnavirus (West Buxton strain)-specific polyclonal antisera indicated that both viruses were aquabirnaviruses belonging to Serogroup A, the most common aquabirnavirus serogroup in the United States. This was confirmed by RT-PCR, with primers targeting the VP3 and VP2 gene of aquabirnaviruses. The VP2-specific RT-PCR cDNA amplification product was sequenced and deduced amino-acid sequences were compared with known sequences of the type strains of the 9 serotypes of aquabirnavirus Serogroup A. This demonstrated that the viruses from both flounder and mummichog belong to aquabirnavirus Genogroup 1. The flounder isolate exhibited deduced amino acid sequence similarities of 98.1% with the Jasper strain of serotype A9, and 97.7% with the West Buxton strain of serotype A1. The isolate from mummichog exhibited deduced amino acid sequence similarities of 99.1% with the West Buxton strain of Serotype A1 and 94.8% with the Jasper isolate of Serotype A9. Similarities of deduced amino acid sequences ranged from 79.9 to 86.9%, with representatives of the other 7 serotypes. This is the first report of an aquabirnavirus from mummichog F. heteroclitus and only the fifth report of an aquabirnavirus from a flounder species.     

蚓激酶基因的克隆及在毕赤酵母中的表达

以赤子爱胜蚓(Eisenia fetida)体内的总RNA为模板,通过RT-PCR方法扩增含自身信号肽的蚓激酶基因F238,将其克隆到pUCm-T载体上,并进行测序。GenBank登录号为:DQ202401。测序结果表明基因全长为738bp,共编码245个氨基酸,包括7个氨基酸的信号肽序列和238个氨基酸的成熟肽序列。与粉正蚓(Lumbricus rubellus)F-III-2相比,核苷酸与氨基酸序列的同源性均为99%,仅存在2个碱基的差异,导致2个氨基酸的突变。通过生物信息学方法对蛋白质的理化及结构特性进行分析预测,F238的等电点为4.61,含有11个半胱氨酸,形成3个二硫键。蛋白质分子主要由β折叠组成,具有丝氨酸活性中心,属丝氨酸蛋白酶超家族胰蛋白酶类。以重组质粒pUCm-T-F238为模板,通过PCR方法扩增去信号肽的蚓激酶基因F238-m,构建毕赤酵母(Pichia pastoris)表达载体pPIC9-F238-m,将其线性化后用电穿孔法导入酵母宿主菌GS115中。在MM和MD平板上筛选表型,经甲醇诱导后,SDS-PAGE分析显示表达产物的分子量为28kDa左右,纤维平板法测定活力最高可达100U/mL。