甘薯贮藏蛋白(sporam in)A基因编码区克隆及序列同源性分析

采用 PCR技术 ,从我国广泛栽培甘薯品种南薯 88基因组中扩增和克隆到甘薯贮藏蛋白 A基因编码区段 ,并测定了其全部核苷酸序列 .该编码区长 65 7bp,编码一个长 2 1 9个氨基酸残基的蛋白质 ,其中信号肽长 37个氨基酸残基 ,成熟蛋白质长 1 82个氨基酸残基 ,其分子量为 2 0 k D.将该片段的核苷酸序列与已登录在 Gen Bank中的另外 6个甘薯贮藏蛋白 A基因编码区序列进行比较 ,发现其同源性高达 90 % ,说明甘薯贮藏蛋白 A基因编码区序列具有高度保守性 .虽然 7个基因编码区的核苷酸总变异为 1 0 % ,但在每两个基因之间的比较则表明其核苷酸的变异范围小于 7% .     

蜡样芽胞杆菌M22铜锌超氧化物歧化酶基因的克隆及表达

采用PCR技术 ,从蜡样芽胞杆菌M2 2基因组DNA扩增到长 132 0bp的基因片段 .该片段含编码 179个氨基酸残基的开放阅读框 ,推定蛋白序列与报道的BacillusanthracisCu ,Zn SOD序列有96 %同源性 ,其中N端 16个氨基酸残基推定为信号肽序列 .将Cu ,Zn SOD编码区插入载体pET 2 2b(+ )构建表达载体pET 2 2b sodC ,转化E .coliBL2 1(DE3) ,IPTG诱导融合蛋白表达 .SDS PAGE显示 ,融合蛋白表观分子质量约 2 4kD ,占菌体裂解液中总蛋白的 2 1 3% .将此表达载体转入SOD缺陷型大肠杆菌PN132 ,赋予了该菌株对paraquat的抗性 .NBT光抑制法测定SOD活性显示 ,与PN132无SOD活性相比 ,重组子在IPTG诱导前SOD活性极低 (1 79U/mg) ,诱导后活性高达 6 9 76U/mg .非变性电泳结果显示 ,该酶活性不同程度地受到 5mmol LH2 O2 和 5mmol LKCN的抑制     

苏云金杆菌毒素调控基因plcR的特性与表达

根据蜡状芽胞杆菌plcR基因和papR基因序列设计特异引物,对6个Bt菌株（WB1、WB7、WB9、HD98、8010、8311）及5个Bc菌株（6A1、6A2、6A3、6A4、6S1）进行了PCR检测.结果显示,3个Bt菌株及4个Bc菌株含有plcR-papR基因.克隆了Bt8010、Bc6A2和6A3的plcR、papR基因,核苷酸序列分析表明,三个菌株的plcR、papR基因与NCBI数据库中的Bt、Bc及Ba相应序列都有很高的相似性.Bt8010的plcR基因编码框由846个核苷酸组成,可编码282个氨基酸;papR基因的编码框由144个核苷酸组成,可编码48个氨基酸.推导的氨基酸序列分析表明,Bt8010 的PapR有21个氨基酸的信号肽序列,PlcR没有信号肽序列.与Bc6A2、6A3和Bc 569相比,Bt8010 的PlcR和PapR在氨基酸序列上与Bc 相应序列存在相对较大的差异.将plcR-papR基因连接到表达载体pHT304中,并转化至大肠杆菌JM109中成功进行了表达,为研究Bt plcR基因的功能奠定了基础.     

不同花色矮牵牛细胞色素b5蛋白的cDNA克隆及序列分析

以云南不同花色矮牵牛的花瓣为材料,提取总RNA,用Oligo(dT)作为引物反转录合成cDNA第一链。以此为模板,用根据国外报道的矮牵牛细胞色素b5蛋白的cDNA序列设计合成的引物进行PCR扩,均扩增到一条约450bp的片段,分别克隆到pGEM-T载体上。对重组克隆进行序列分析,结果表明所克隆到的矮牛细胞色素b5蛋白的cDNA的编码区均含有447个核苷酸,编码149个氨基酸残基,与国外报道的一致;但其核苷酸及氨基酸的序列与国外报道的有所不同,即与国外的相比,紫红色、蓝紫色矮牵牛中的该cDNA的核苷酸有1个不同,而氨基酸完全相同;粉红色、白色矮牵牛中的3个核苷酸不同,并导致了2个氨基酸的不同。暗示该基因对花色的调控可能与其编码cDNA的一级结构有关。     

鲤鱼生长激素基因的PCR扩增、克隆及其序列比较

从鲤鱼(Cyprinus carpio)脑垂体中分离其总RNA,经反转录成为第一条链cDNA。以第一条链cDNA为模板,以合成的两段29个寡聚核苷酸为引物,再经过PCR扩增,合成了鲤鱼的生长激素基因,并把其克隆到大肠杆菌表达载体(P^{blue-ocripx Ks+/-})上。序列分析和限制酶切图谱表明所克隆的鲤鱼生长激素基因的开放读框含有630bp,编码210个氨基酸,其中含有22个氨基酸的信号肽和188个氨基酸的成熟GH序列。我们所克隆的鲤鱼GH基因与Koren发表的鲤鱼GHcDNA的序列在编码区完全一致,但是与chao发表的鲤鱼GHcDNA比较,在编码区核酸序列和氨基酸序列的同源性分别为95．6％和96．7％。     

烟草花叶病毒蚕豆株系基因组全序列分析及结构特征

根据已报道的TMV-U1株系核苷酸序列合成引物 ,利用RT-PCR技术获得了覆盖整个烟草花叶病毒蚕豆株系 (TMV-B)基因组的cDNA重组克隆 .结合末端测序技术 ,完成了TMV-B全基因组序列测定 .TMV-B全基因组共有 6 395个核苷酸组成 ,包括 4个开读框 (ORF) ,分别编码 1 2 6ku(含 1 1 1 6个氨基酸 )、1 83ku(含 1 6 1 6个氨基酸 )、30ku(含 2 6 8个氨基酸 )和 1 7.5ku蛋白 (含 1 5 9个氨基酸 ) .TMV-B与TMV-U1相比全基因组同源率达 99.4% ,两病毒基因组 5′ ,3′非编码区和CP基因完全相同 .TMV-B与TMV-U1之间在 1 2 6ku蛋白中有 6个氨基酸差异 ,5 4ku蛋白中有 2个差异 ,30ku蛋白中有 3个差异 .对导致TMV-B侵染蚕豆的可能致病机理进行了分析 .     

猪MHC-Ⅱ类区DQA新等位基因及新突变位点的发现

根据猪SLA-DQA基因cDNA序列和人HLA-DQA基因组序列设计引物,在猪中成功扩增了一个731bD的片段,该片段包括猪SLA-DQA基因的大部分第2外显子、完整第2内含子和少部分第3外显子,通过克隆和PCR直接测序获得该片段的核苷酸序列。在家系中对第2外显子的核苷酸序列和其编码的α1结构域的氨基酸序列进行了分析,并与GenBank中所有的SLA-DQA第2外显子序列进行比较分析,发现有4个新突变位点和两个新等位基因存在。新等位基因分别是DQA—SLT26和DQA—TC 21—1。DQA—SLT26与单倍型c、d相比有8个核苷酸的差异和4个氨基酸的改变：单倍型c、d在60、65、81、93位的氨基酸分别是Val、Lys、Asp、Lys;而DQA—SLT 26相应的氨基酸是Ala、Glu、Gly、Ile。DQA—TC 21—1与单倍型c、d相比存在1个氨基酸的改变,由单倍型c和d中94位氨基酸His变为Tyr。     

用重叠PCR合成植物甜蛋白brazzein基因

目的:为在泡盛曲霉(Aspergillus awamori)中进行表达,采用重叠PCR合成了植物甜蛋白brazzein基因。方法:根据非洲热带植物Pentadiplandra brazzeana产生的天然甜蛋白brazzein的氨基酸序列及泡盛曲霉糖化酶基因glaA的密码子偏爱性,设计并化学合成了2对3’-端互补的寡聚核苷酸,通过PCR延伸获得2条末端有部分重叠的双链核苷酸片段,再通过重叠PCR扩增,合成了用于泡盛曲霉表达的植物甜蛋白brazzein基因。结果:将brazzein基因克隆到pMD18-T载体,随机挑取6个重组质粒测序,结果1个重组质粒有连续4个碱基缺失,3个重组质粒各有1个碱基缺失,2个重组质粒携带的brazzein基因核苷酸序列完全正确。结论:合成的brazzein基因大小162 bp,编码54个氨基酸,推断的氨基酸序列与Pentadiplandra brazzeana产生的天然brazzein完全一致,表明植物甜蛋白brazzein基因成功合成。     

中国西藏小反刍兽疫病毒China/Tib/Gei/07-30磷蛋白基因的分子特征及其编码蛋白特性分析

对我国西藏小反刍兽疫病毒野生株China/Tib/Gej/07-30进行磷蛋白基因序列测定,并进行分子生物学特征分析。首先应用逆转录聚合酶链式反应扩增出病毒磷蛋白基因片段,对聚合酶链式反应产物进行直接测序,然后对测定的核苷酸和推测的氨基酸序列进行比较分析。小反刍兽疫病毒China/Tib/Gej/07-30磷蛋白基因由1 655个核苷酸组成,编码2个相互交叠的开放阅读框(ORF)。第一个ORF长度为1 530个核苷酸,编码的P蛋白长度为509个氨基酸。第二个ORF长度为534个核苷酸,编码的C蛋白长度为177个氨基酸。第一个ORF通过基因编辑在751位插入1个G核苷酸,转录生成第二个mRNA,长度为897个核苷酸,编码的V蛋白长度为298个氨基酸。小反刍兽疫病毒China/Tib/Gej/07-30的P蛋白与其他分离株氨基酸序列同源性为86.1%～97.3%,C蛋白氨基酸序列相似性为84.3%～94.9%,V蛋白为82.9%～96.3%。China/Tib/Gej/07-30的P蛋白第315～387位氨基酸是一段高度保守的七肽重复序列。     

柞蚕核型多角体病毒泛素类似基因的克隆与序列分析

从感病的柞蚕Antheraea pernyi蛹中分离纯化柞蚕核型多角体病毒 (ApNPV),提取基因组DNA,分别构建ApNPV DNA的HindⅢ和SalⅠ酶切片段文库。对基因文库中1个克隆进行序列分析,得到1个长度为321 bp的序列,其中包含一个编码76个氨基酸的开放阅读框,预测的分子量为8.46 kD,系泛素类似基因。在读码框的上游调控序列中,具有典型的晚期基因启动子序列ataag。氨基酸序列同源性分析结果表明,ApNPV与黄杉毒蛾Orgyia pseudotsugata核型多角体病毒 (OpNPV)的同源性最高 (96.1%),与苜蓿尺蠖Autographa californica核型多角体病毒 (AcNPV) 的同源性为86.8%,与棉褐带卷蛾Adoxophyes orana 颗粒体病毒 (AoGV) 的同源性最低（71.1%）,但与人类、线虫和酵母的泛素同源性分别为77.6%、76.3%和76.3%。一些氨基酸残基在真核生物中保守,在杆状病毒中不保守,个别氨基酸残基是杆状病毒所特有的,这些氨基酸序列的改变对杆状病毒泛素基因的作用有待进一步研究。     

Biochemical analysis of Hyphantria cunea NPV attachment to Spodoptera frugiperda 21 cells

Binding characteristics of Hyphantria cunea nuclear polyhedrosis virus (HcNPV) to Spodoptera frugiperda 21 (Sf21) cells was determined. The cells displayed an affinity of 0.9 × 10¹⁰ M^-1 with about 8900 binding sites per cell. The biochemical nature of HcNPV-binding sites on the cell surface was also partially elucidated. There were 45 to 49% reductions in HcNPV binding following the pretreatment of cells with three proteases, suggesting the involvement of a cellular protein component in virus binding. Tunicamycin, which inhibits N-linked glycosylation and the expression of some membrane proteins on the cell surface, reduced virus binding suggesting a role for glycoprotein(s) in binding. Treatment of cells with wheat germ agglutinin or neuraminidase did not measurably reduce virus binding, indicating that oligosaccharides containing N-acetylglucosamine or sialic acid are not directly involved in HcNPV attachment. The negative effect of methylamine on HcNPV binding seems to be due to the fact that HcNPV entry via an endocytic pathway is blocked by the increased pH of the endosome. Data on energy inhibitors (sodium azide and dinitrophenol) indicates that HcNPV attachment to Sf21 cells may be closely linked to viral entry via receptor-mediated endocytosis. These findings suggest that the binding site moiety has a glycoprotein component, but that direct involvement of oligosacccharides containing N-acetylglucosamine or sialic acid residues in binding is unlikely, and that HcNPV attachment to Sf21 cells might be via receptor-mediated endocytosis. This revised version was published online in August 2006 with corrections to the Cover Date.     

HcNPV半胱氨酸蛋白酶,几丁质酶基因失活分析

将含有美国白蛾核型多角体病毒（Ｈｙｐｈａｎｔｒｉａ ｃｕｍｅａ ｎｕｃｌｅａｒ ｐｏｌｙｈｅｄｒｏｓｉｓ ｖｉｒｕｓ,ＨｃＮＰＶ）半胱氨酸蛋白酶基因（ＣＰ）的自然和几丁质酶基因（ＣｈｉＡ）的片段克隆进ＰＣＲⅡ,构建了转移载体ｐＨｃＣＶｄｅｌ;将含有ＨｃＮＰＶ多角体蛋白全基因（ｐｏｌｈ）序列的片段插入到ｐＨｃＣＶｄｅｌ的ＥｃｏＲＩ位点,得到重组转移载体ｐＨｃＣＶｐｏｌｈ。通过重组转移载体与含有家蚕促     

Identification and characterization of the p35 gene of Bombyx mori nuclear polyhedrosis virus that prevents virus-induced apoptosis.

Nucleotide sequence analysis of the Bombyx mori nuclear polyhedrosis virus (BmNPV) genome revealed the existence of a gene homologous to the p35 gene of Autographa californica NPV (AcNPV), which has been shown to prevent virus-induced apoptosis. The BmNPV p35 gene showed 96.1% nucleotide and 89.6% predicted amino acid sequence identity to the AcNPV p35 gene. A mutant BmNPV (BmP35Z) lacking a functional p35 gene induced apoptosis-like cell degradation in infected BmN cells. However, unlike the p35-deleted AcNPV mutant (vAcAnh), BmP35Z replicated normally and produced polyhedral inclusion bodies. The patterns of protein synthesis and the percentages of viable BmN cells remaining following infection with either wild-type BmNPV or BmP35Z were nearly identical. BmP35Z also replicated in silkworm larvae without showing any apparent apoptotic response in infected hemocytes, fat body, or other tissues. Time to death of larvae infected with BmP35Z was similar to that for wild-type-infected larvae, and significant numbers of polyhedral inclusion bodies were produced. These results indicate that viral factors (or genes) other than p35 or host cell factors play a role in inducing, accelerating, or interfering with apoptotic processes. The evolution of baculovirus genomes is also discussed with reference to comparative analysis of the p35 and p94 gene sequences. The p94 gene is found immediately upstream of p35 in AcNPV; in BmNPV, however, the p94 gene was nearly completely missing, presumably because of large deletions in a BmNPV ancestor virus having a gene similar to the AcNPV p94 gene.     

家蚕NPV SOD基因序列和在大肠杆菌中表达

通过ＰＣＲ 克隆了家蚕核型多角体病毒（ＢｍＮＰＶ） ＳＯＤ基因, 并在大肠杆菌中进行表达, 证明了Ｂｍ ＮＰＶＳＯＤ基因产物确有ＳＯＤ活性, 其活力单位约为５７６ ｕ／ｍＬ 培养液。ＤＮＡ 测序结果表明Ｂｍ ＮＰＶ ＳＯＤ 基因编码１５１ 个氨基酸, 与人的ＳＯＤ１ 基因的核苷酸同源性为５６ ％ , 与ＡｃＮＰＶ 拟为的ＳＯＤ基因同源性为９７ ．２％ 。     

High-efficient expression of porcine IL-2 with recombinant baculovirus infected silkworm,Bombyx mori

Biologically active porcine Interleukin-2(poIL-2) was produced fromin vitro andin vivo baculovirus expression systems, namely theAutographa californica nuclear polyhedrosis virus (AcNPV)-cell culture system and the Hybrid nuclear polyhedrosis virus (HyNPV)— silkworm larva system. The concentration of the recombinant poIL-2(rpoIL-2) in the larvae hemolymph was 1 to 3 mg/mL, which was about 7 to 20 times those of the cell culture systems. The level of this expression efficiency is equal to that with transgenic livestock, secretion products in milk.     

Strong and regulated expression of Escherichia coli beta-galactosidase in insect cells with a baculovirus vector.

The N-terminal region of the gene encoding polyhedrin, the major occlusion protein of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), has been fused to DNA encoding Escherichia coli beta-galactosidase. The fused gene was inserted into the AcNPV DNA genome by cotransfection of insect cells with recombinant plasmid DNA and wild-type AcNPV genomic DNA. Recombinant viruses were selected as blue plaques in the presence of a beta-galactosidase indicator, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Studies of one such virus, L1GP-gal3, indicated that the synthesis of beta-galactosidase is temporally controlled beginning late (20 h) in infection after the release of infectious virus particles from the cell. By 48 h postinfection, a remarkably high level of expression is achieved. On the basis of these results, AcNPV should be a useful vector for the stable propagation and expression of passenger genes in a lepidopteran cell background. A generalized transplacement vector that facilitates the construction and selection of recombinant viruses carrying passenger genes under their own promoter control has also been developed.     

Improving baculovirus transduction of mammalian cells by surface display of a RGD-motif

An RGD-containing peptide, comprising 23 amino acids from the foot-and-mouth disease virus (FMDV) VP1 protein was engineered into the envelope of Autographa californica nuclear polyhedrosis virus surface (AcNPV) using two different display strategies. The RGD-motif is a well-described tripeptide, that by binding to cell surface integrins facilitates virus entry into cells. This epitope was displayed, either by directly modifying the native major envelope protein gp64 of AcNPV, or by incorporating a second, modified version of gp64 onto the virus surface. Transduction efficiencies of four mammalian cell lines were compared by detecting the expression of the reporter gene green fluorescent protein (gfp), delivered by the baculovirus genome. Our results showed that insertion of the RGD-peptide into the envelope protein gp64 leads to enhanced specific uptake of baculoviral particles in mammalian cells, only when a combination of wild-type and mutant gp64 was present on the viral surface. Whenever the RGD-peptide was directly inserted into the native gp64, the overall amount of gp64 envelope protein was diminished, leading to decreased viral uptake.     

Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system

Silkworm is one of the most attractive hosts for large-scale production of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. The bacmid system of Autographa californica nuclear polyhedrosis virus (AcNPV) has already been established and widely used. However, the AcNPV does not have a potential to infect silkworm. We developed the first practical Bombyx mori nuclear polyhedrosis virus bacmid system directly applicable for the protein expression of silkworm. By using this system, the green fluorescence protein was successfully expressed in silkworm larvae and pupae not only by infection of its recombinant virus but also by direct injection of its bacmid DNA. This method provides the rapid protein production in silkworm as long as 10 days, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses.