杆状病毒表达EV71病毒样颗粒的装配、制备纯化与鉴定

将EV71P1和3CD基因片段克隆入同一杆状病毒穿梭质粒Bacmid中,构建出重组杆状病毒表达质粒Bac-mid-P1-3CD;脂质体介导其转染Sf9昆虫细胞获得共表达P1和3CD的重组杆状病毒(AcMNPV-P1-3CD)。用IFA和Western-blot法对表达产物进行鉴定和分析。电镜结果显示P1经3CD切割装配成了大小约为27nm的类球形颗粒(即EV71VLPs)。进一步分析影响杆状病毒表达系统的因素以对表达条件进行优化,结果显示MOI值和时间均可影响目的蛋白的表达,其中时间是主要因素。选择优化后条件利用无血清培养基对贴壁Sf9细胞在多层细胞培养器中进行VLPs的大量表达,密度梯度离心法纯化,SDS-PAGE结果可见三条大小约为39kD、34kD和26kD的VP1、VP0和VP3特异性条带。纯化后EV71VLPs颗粒结构完好,为下一步EV71蛋白结构的基础研究和基因工程疫苗的研究奠定了基础。     

基于啮齿类衣壳蛋白的新基因治疗载体

目前,英国Royal Postgraduate Medical School的技术转移附属机构RPMS Technology公司正在请求在离体试验中很有希望的基因治疗载体的研究许可。该载体利用啮齿类病毒多瘤的主要衣壳蛋白(VP1)来转移DNA。 据研究人员声称,VP1假衣壳能在重组杆状病毒中产生并易于纯化。现正在测试用空的假衣壳包装转     

杆状病毒DNA聚合酶基因的研究概况*

杆状病毒DNA聚合酶基因属于杆状病毒早期基因,是杆状病毒复制的必需基因。它编码病毒诱导的DNA聚合酶,能与其它复制因子一起与杆状病毒DNA的同源区和非同源区的顺式作用元件相互作用起始DNA复制。此基因作为杆状病毒系统发育分类的依据,较之包涵体蛋白、egt基因有更大的优势。     

不同载体表达的人类细小病毒B19壳蛋白VP2的抗原性比较研究

构建表达质粒pcDNA3 VP2,将其转染CHO细胞建立了稳定表达的细胞系;用间接免疫荧光法和Western印迹证明了表达的VP2蛋白的特异性。对昆虫杆状病毒系统表达的VP2蛋白作初步纯化。分别用由大肠杆菌、CHO细胞和昆虫杆状病毒表达系统表达的VP2蛋白,以间接免疫荧光法和ELISA法检测人群血清中的VP2抗体,结果表明,间接免疫荧光法的敏感性高于ELISA法。     

杆状病毒DNA聚合酶基因的研究概况

杆状病毒DNA聚合酶基因属于杆状病毒早期基因,是杆状病毒复制的必需基因。它编码病毒诱导的DNA聚合酶,能与其它复制因子一起与杆状病毒DNA的同源区和非同源区的顺式作用元件相互作用起始DNA复制。此基因作为杆状病毒系统发育分类的依据,较之包涵体蛋白、egt基因有更大的优势。     

人多瘤病毒BK株VP1的C端阳电荷残基对病毒样颗粒形成的影响

VP1是人多瘤病毒BK株的主要结构蛋白,使用重组杆状病毒表达系统在体外表达 VP1 可以形成病毒样颗粒(VLP)。为了探讨VP1的C末端阳电荷残基R 281, R 285, K 288, R 290, R 292, K 293, R 294,和 K297 对VLP形成和其结合DNA的影响,我们分别改变将阳电荷残基变成丙氨酸,然后表达 VP1 蛋白。结果发现用丙氨酸替代K 288,R 290,R 292,K 293,R 294后仍能形成VLP, 但与野毒株相比,在 VLP分泌以及衣壳蛋白与细胞DNA的结合方面有差异。有趣的是,R 281被丙氨酸取代后仅在细胞中形成少量的 VLP,而 R 285 被丙氨酸取代后不能形成VLP。该研究证实阳电荷氨基酸残基 R 281 和 R 285 是形成 VLP所必须的,K 288、R 290、R 292、K 293、R 294和K 297则影响VLP和DNA的结合。     

人多瘤病毒BK株VP1的C端阳电荷残基对病毒样颗粒形成的影响

VP1是人多瘤病毒BK株的主要结构蛋白,使用重组杆状病毒表达系统在体外表达VP1可以形成病毒样颗粒(VLP).为了探讨VP1的C末端阳电荷残基R-281,R-285,K-288,R-290,R-292,K-293,R-294,和K297对VLP形成和其结合DNA的影响,我们分别改变将阳电荷残基变成丙氨酸,然后表达VP1蛋白.结果发现用丙氨酸替代K-288,R-290,R-292,K-293,R-294后仍能形成VLP,但与野毒株相比,在VLP分泌以及衣壳蛋白与细胞DNA的结合方面有差异.有趣的是,R-281被丙氨酸取代后仅在细胞中形成少量的VLP,而R-285被丙氨酸取代后不能形成VLP.该研究证实阳电荷氨基酸残基R-281和R-285是形成VLP所必须的,K-288、R-290、R-292、K-293、R-294和K-297则影响VLP和DNA的结合.     

霍乱弧菌分型噬菌体VP3基因组序列的测定与分析

测定和分析霍乱弧菌分型噬菌体VP3基因组序列,并为ElTor型霍乱弧菌两类菌株的分型方法原理提供研究基础。鸟枪法构建VP3噬菌体全基因组随机文库;测序拼接成最小重叠群,引物步移法填补缝隙序列,拼接后获得VP3全基因组序列。PCR随机扩增噬菌体DNA片段并酶切鉴定;预测可能存在的开放读码框(ORF);对VP3和相关噬菌体的DNA聚合酶基因作进化树分析,协助判定VP3的分类;对预测的部分启动子区利用报道基因进行活性分析。VP3全基因组为环状双链DNA,长度39504bp;酶切鉴定结果与序列一致。确定了49个ORF,注释了27个ORF的编码产物,其中有20个基因产物与T7样噬菌体同源,包括RNA聚合酶(RNAP)、参与DNA复制的蛋白、衣壳蛋白、尾管及尾丝蛋白、DNA包装蛋白等。DNA聚合酶(DNAP)进化树分析表明VP3与T7样噬菌体有同源性。将预测的10个启动子序列克隆到lacZ融合质粒pRS1274上,经检测均具有启动子活性。测定和分析VP3的基因组序列,基因组结构与进化树分析提示VP3属于T7噬菌体家族。     

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.     

Low pH-dependent endosomal processing of the incoming parvovirus minute virus of mice virion leads to externalization of the VP1 N-terminal sequence (N-VP1), N-VP2 cleavage, and uncoating of the full-length genome

Minute virus of mice (MVM) enters the host cell via receptor-mediated endocytosis. Although endosomal processing is required, its role remains uncertain. In particular, the effect of low endosomal pH on capsid configuration and nuclear delivery of the viral genome is unclear. We have followed the progression and structural transitions of DNA full-virus capsids (FC) and empty capsids (EC) containing the VP1 and VP2 structural proteins and of VP2-only virus-like particles (VLP) during the endosomal trafficking. Three capsid rearrangements were detected in FC: externalization of the VP1 N-terminal sequence (N-VP1), cleavage of the exposed VP2 N-terminal sequence (N-VP2), and uncoating of the full-length genome. All three capsid modifications occurred simultaneously, starting as early as 30 min after internalization, and all of them were blocked by raising the endosomal pH. In particles lacking viral single-stranded DNA (EC and VLP), the N-VP2 was not exposed and thus it was not cleaved. However, the EC did externalize N-VP1 with kinetics similar to those of FC. The bulk of all the incoming particles (FC, EC, and VLP) accumulated in lysosomes without signs of lysosomal membrane destabilization. Inside lysosomes, capsid degradation was not detected, although the uncoated DNA of FC was slowly degraded. Interestingly, at any time postinfection, the amount of structural proteins of the incoming virions accumulating in the nuclear fraction was negligible. These results indicate that during the early endosomal trafficking, the MVM particles are structurally modified by low-pH-dependent mechanisms. Regardless of the structural transitions and protein composition, the majority of the entering viral particles and genomes end in lysosomes, limiting the efficiency of MVM nuclear translocation.     

草鱼呼肠孤病毒VP7蛋白单克隆抗体的制备与应用

草鱼呼肠孤病毒(Grass carp reovirus, GCRV)是导致该病的主要病原, 研究将Ⅰ型草鱼呼肠孤病毒GCRV-873株的外衣壳蛋白VP7基因进行原核表达, 获得高度纯化VP7重组蛋白, 通过免疫BALB/c小鼠, 首次制备筛选得到高效价单克隆抗体。结果显示, GCRV-I vp7基因可在原核表达系统中高效表达, 主要以包涵体形式存在, 大小约为40 kD。免疫小鼠后筛选到了5株IgG类型阳性杂交瘤细胞株, 其中3株亚型为IgG1, 2株亚型为IgG2a。Western Blot实验和直接免疫荧光实验显示, 该抗体可特异识别GCRV-873, 并且ELISA检测原核重组蛋白的效价高达204800, 亲和常数为4.04×109。研究制备的VP7蛋白单克隆抗体, 为GCRV-I病毒诊断技术开发及病毒感染机制的深入研究提供实验基础。     

VP5 of infectious bursal disease virus is not essential for viral replication in cell culture.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, encodes in its bisegmented double-stranded RNA genome four structural virion proteins, VP1, VP2, VP3, and VP4, as well as a nonstructural protein, VP5. Recently, the establishment of an infectious cRNA system for IBDV has been described (E. Mundt and V. N. Vakharia, Proc. Natl. Acad. Sci. USA 93:11131-11136, 1996). Here, we report the isolation of a VP5- IBDV mutant constructed by site-directed mutagenesis of the methionine start codon of VP5, followed by cRNA transfection. The resulting virus mutant was replication competent in cell culture, which indicates that VP5 is not required for productive replication of IBDV. Absence of VP5 expression was verified by lack of reactivity with newly established anti-VP5 monoclonal antibodies and polyclonal sera. VP5- IBDV exhibited a delay in replication in chicken embryo cells compared to the VP5+ parental virus. However, final yields were similar. Our results thus show that VP5 is nonessential for IBDV replication, which makes it a prime candidate for the construction of deleted, marked vaccines.     

In vivo proteins of defective interfering particles of poliovirus

DX particles of poliovirus are deletion mutants that do not induce synthesis of capsid proteins or the precursor of capsid proteins (NCVPla) during infection. However, cells infected with DX particles synthesize two proteins, p68 and p25, that are not detected during growth of standard virus, and a protein of 27 000 (p27) which is comparable in molecular weight to VP3. Peptide maps of these proteins were obtained by partial digestion with Staphylococcus aureus V8 protease and elastase. The peptide map of p68 corresponded approximately 70% with the peptide map of NCVPla, and antiserum against virions reacted with p68. These data suggest that p68 is a large fragment of NCVPla. Digestion of purified structural proteins VP1, VP2, and VP3 yielded distinct peptide maps, but p25 was resistant to both V8 protease and elastase and did not react noticeably with anticapsid antibody. Peptide maps obtained for in vivo viral proteins migrating with a molecular weight of 27 000 were complex, indicating the presence of at least two and possibly three proteins. Cells infected with standard gs and gr viruses produced authentic VP3, but cells infected with defective interfering particles did not. However, one gr variant of standard virus contained a mutation in structural protein VP2.     

Disassembly of simian virus 40 during passage through the endoplasmic reticulum and in the cytoplasm

The nonenveloped polyomavirus simian virus 40 (SV40) is taken up into cells by a caveola-mediated endocytic process that delivers the virus to the endoplasmic reticulum (ER). Within the ER lumen, the capsid undergoes partial disassembly, which exposes its internal capsid proteins VP2 and VP3 to immunostaining with antibodies. We demonstrate here that the SV40 genome does not become accessible to detection while the virus is in the ER. Instead, the genome becomes accessible two distinct detection procedures, one using anti-bromodeoxyuridine antibodies and the other using a 5-ethynyl-2-deoxyuridine-based chemical reaction, only after the emergence of partially disassembled SV40 particles in the cytoplasm. These cytoplasmic particles retain some of the SV40 capsid proteins, VP1, VP2, and VP3, in addition to the viral genome. Thus, SV40 particles undergo discrete disassembly steps during entry that are separated temporally and topologically. First, a partial disassembly of the particles occurs in the ER, which exposes internal capsid proteins VP2 and VP3. Then, in the cytoplasm, disassembly progresses further to also make the genomic DNA accessible to immune detection.     

Polyoma Virus DNA: Complete Nucleotide Sequence of the Gene Which Codes for Polyoma Virus Capsid Protein VP1 and Overlaps the VP2/VP3 Genes

The nucleotide sequence of part of the late region of the polyoma virus genome was determined. It contains coding information for the major capsid protein VP1 and the C-terminal region of the minor proteins VP2 and VP3. In the sequence with the same polarity as late mRNA's, all coding frames are blocked by termination codons in a region around 48 units on the physical map. This is the region where the N-terminus of VP1 and the C-termini of VP2 and VP3 have been located (T. Hunter and W. Gibson, J. Virol. 28:240-253, 1978; S. G. Siddell and A. E. Smith, J. Virol. 27:427-431, 1978; Smith et al., Cell 9:481-487, 1976). There are two long uninterrupted coding frames in the late region of polyoma virus DNA. One lies at the 5' end of the sequence and contains potential coding sequences for VP2 and VP3. The other contains 383 consecutive sense codons starting with the ATG at nucleotide position 1,218, extends from 47.5 to 25.8 units counterclockwise on the physical map, and is located where the VP1 gene has been mapped. The VP1 gene overlaps the genes for proteins VP2/VP3 by 32 nucleotides and uses a different coding frame. From the DNA sequence, the amino acid sequence of VP1 was predicted. The proposed VP1 sequence is in good agreement with other data, namely, with the partial N-terminal amino acid sequence and the total amino acid composition. The VP1 coding frame terminates with a TAA codon at 25.8 map units. This is followed by an AATAAA sequence, which may act as a processing signal for the viral late mRNA's. When both nucleotide and amino acid sequences are compared with their counterparts in the related simian virus 40, extensive homologies are found over the entire region of the two viral genomes. Maximum homology appears to occur in those regions which code for the C-termini of the VP1 proteins. The overlap region of VP1 with VP2/VP3 of polyoma virus is shorter by 90 nucleotides than is that of simian virus 40 and shows very limited homology with the simian virus 40 sequence. This leads to the suggestion that the overlap segments of both viruses have been freed from stringency imposed on drifting during evolution and that proteins VP2 and VP3 of polyoma virus may have been truncated by the appearance of a termination codon within the sequence.     

Mutation affecting late gene expression in polyoma virus maps in the late region.

A mutation in polyoma virus strain 3049 which results in the overproduction of capsid proteins has been mapped to the late region of the genome between the HindIII site at 45.0 map units and the BamHI site at 58.6 map units. This region contains the coding sequence for VP3 and a portion of VP2, but does not include the late promoters or the coding sequence for the late leaders. The possible role of VP2 or VP3 in the regulation of genetic expression in polyoma virus is discussed.     

Genomic and morphological features of a banchine polydnavirus: comparison with bracoviruses and ichnoviruses

Many ichneumonid and braconid endoparasitoids inject a polydnavirus (PDV) into their caterpillar hosts during oviposition. The viral entities carried by wasps of these families are referred to as "ichnoviruses" (IVs) and "bracoviruses" (BVs), respectively. All IV genomes characterized to date are found in wasps of the subfamily Campopleginae; consequently, little is known about PDVs found in wasps of the subfamily Banchinae, the only other ichneumonid taxon thus far shown to carry these viruses. Here we report on the genome sequence and virion morphology of a PDV carried by the banchine parasitoid Glypta fumiferanae. With an aggregate genome size of approximately 290 kb and 105 genome segments, this virus displays a degree of genome segmentation far greater than that reported for BVs or IVs. The size range of its genome segments is also lower than those in the latter two groups. As reported for other PDVs, the predicted open reading frames of this virus cluster into gene families, including the protein tyrosine phosphatase (PTP) and viral ankyrin (ank) families, but phylogenetic analysis indicates that ank genes of the G. fumiferanae virus are not embedded within the IV lineage, while its PTPs and those of BVs form distinct clusters. The banchine PDV genome also encodes a novel family of NTPase-like proteins displaying a pox-D5 domain. The unique genomic features of the first banchine virus examined, along with the morphological singularities of its virions (IV-like nucleocapsids, but enveloped in groups like some of the BVs), suggest that they could have an origin distinct from those of IVs and BVs.