番茄条斑坏死病毒的研究

从上海郊区番茄条斑坏死病果分离的棒状病毒(ToSNV)的外壳蛋白亚基的分子量小于烟草花叶病毒普通株(TMVc)和长叶车前花叶病毒(HRV),ToSNV的外壳蛋白比TMVc少10个氨基酸残基,比HRV少8个氨基酸残基,氨基酸组成中含甲硫氨酸及组氨酸。ToSNV与长叶车前花叶病毒上海分离株(HRVsh)及油菜花叶病毒(YMV_(15))有免疫交叉反应,但与TMVc却无血清学亲缘关系,而且ToSNV外壳蛋白的酶解图谱不同于TMVc。因此推测ToSNV是烟草花叶病毒组中既不同于TMVc又有别于HRVsh和YMV_(15)的一个分离株。     

番茄条斑坏死病毒的研究

从上海郊区番茄条斑坏死病果分离的棒状病毒(ToSNV)的外亮蛋白亚基的分子量小于烟草花叶病毒普通株(TMVc)和长叶车前花叶病毒(HRV),ToSNV 的外壳蛋白比TMVc少10个氨基酸残基,比HRV 少8个氨基酸残基,氨基酸组成中含甲硫氨酸及组氨酸。ToSNV 与长叶车前花叶病毒上海分离株(HRVsh)及油菜花叶病毒(YMV_(15))有免疫交叉反应,但与TMVc 却无血清学亲缘关系,而且ToSNV 外壳蛋白的酶解图谱不同于TMVc。因此推测ToSNV 是烟草花叶病毒组中既不同于TMVc 又有别于HRVsh 和YMV_(15)的一个分离株。     

狂犬病病毒CVS-11核蛋白B细胞线性抗原表位研究

为阐明狂犬病病毒CVS-11核蛋白B细胞线性抗原表位,本研究通过合成肽模拟B细胞线性抗原表位,采用免疫学方法对生物信息学分析获得的狂犬病病毒CVS-11核蛋白潜在B细胞线性抗原表位进行验证。结果显示,狂犬病病毒CVS-11核蛋白355~369、385~400位氨基酸序列合成肽免疫小鼠血清经间接酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA)检测抗多肽抗体效价达到1∶12 800以上;抗多肽抗体在免疫印迹试验(Western blot,WB)中识别变性狂犬病病毒CVS-11核蛋白抗原,在间接荧光抗体试验(Indirect fluorescent antibody,IFA)中识别感染BHK-21细胞的狂犬病病毒CVS-11核蛋白抗原。因此,狂犬病病毒CVS-11核蛋白355~369、385~400位氨基酸序列经证实为B细胞线性抗原表位。     

印度木薯花叶双生病毒外壳蛋白的研究Ⅱ．计算机分析外壳蛋白的一些特性

用NWGCG软件系统分析了印度木薯花叶双生病毒外壳蛋白的一些性质：(1)外壳蛋白是碱性蛋白,其等电点为10．91;(2)在外壳蛋白氨基酸序列中分布有α—helcies,β—Sheets及turns等二级结构;(3)分析了外壳蛋白中可能的表面氨基酸区域,亲水性和抗原决定簇区域;(4)在外壳蛋白的氨基酸序列中存在两个糖基化位点HNT,同时分析了双生病毒外壳蛋白氨基酸遗传密码的利用频率。     

长叶车前花叶病毒(HRV_(sh))外壳蛋白赖氨酸残基的修饰

我们曾报道长叶车前花叶病毒上海分离株(简称HRVsh)的外壳蛋白有二个赖氨酸残基,在PH8.5无变性剂存在的条件下,完整病毒颗粒表面的赖氨酸残基可与三硝基苯磺酸(TNPS)起反应,反应后的TNP-HRVsh病毒颗粒的感染力丧失达90％以上。 本文又进行了甲基乙亚胺甲酯(MEI)对HRVsh赖氨酸残基的修饰反应,修饰后的MEI-HRVsh病毒颗粒的感染力也同样丧失90％以上。 从三硝基苯磺酸修饰的病毒颗粒(TNP-HRVsh)中分离得到的RNA能与天然的HRVsh的外壳蛋白重建病毒颗粒,并具有感染力,说明修饰过程中核酸并不受影响。 进一步用同位素~(35)S,~(32)P双标记病毒,再以TNPS修饰标记的病毒,得到(~(35)S,~(32)P)-HRVsh及TNP-(~(35)S,~(32)P)-HRVsh。将两者分别接种于系统寄主青菜(Brassica chinensis)的一片叶片,一天后在非接种叶片上都可测得~(35)S,~(32)P的放射计数。其中,(~(35)S,~(32)P)-HRVsh的~(35)S/~(32)P比值降低了,而TNP-(~(35)S,~(32)P)-HRVsh的~(35)S/~(32)P比值保持不变。说明HRVsh外壳蛋白赖氨酸残基的修饰并不影响病毒颗粒进入寄主细胞,以及在寄主细胞间的转移。同位素双标记的结果表明,其感染力丧失的原因可能是由于上述修饰作用阻止了病毒在感染中所必须的脱壳过程。     

外壳蛋白羧端序列缺失对烟草花叶病毒侵染性的影响

对野生型烟草花叶病毒(TMV-U1)的外壳蛋白羧端序列进行系列缺失突变,观察到TMV-U1株系的外壳蛋白羧端序列缺失6个氨基酸(保留152个氨基酸),仍能较强系统侵染烟草并高水平表达外壳蛋白,且能在新生叶里复制大量完整的病毒粒子。该研究结果表明：外壳蛋白羧端6个氨基酸序列并非烟草花叶病毒感染和复制所必需,并对利用外壳蛋白羧端缺失型病毒载体表达外源多肽具有一定的启示性。     

HIV-1辅助蛋白Vpr多肽抗体的制备与鉴定

预测Vpr蛋白的B细胞抗原表位,并利用合成的B细胞表位肽制备Vpr特异性抗体。应用生物信息学技术获得Vpr蛋白共享氨基酸序列并预测其潜在B细胞抗原表位,与载体蛋白血蓝蛋白(KLH)偶联合成多肽并免疫家兔,鉴定及纯化获得的多肽特异性抗体。软件预测显示,Vpr蛋白N端的第3～19位(N)和C端的第82～95位(C)氨基酸序列为潜在B细胞抗原表位;ELISA检测抗血清中多肽特异性抗体的效价都达到1:105以上;Western-Blotting结果显示,无论对HIV-1B亚型还是CRF07_BC重组型的Vpr蛋白,其多肽N抗体和C抗体均能特异性识别;免疫沉淀结果显示,Vpr多肽N和C抗体也能特异性结合未变性的野生型Vpr或GFP-Vpr融合蛋白。利用生物信息学技术能成功预测Vpr蛋白B细胞抗原表位,免疫所获得的抗体具有较好的特异性和应用性。     

丙型肝炎病毒核壳蛋白抗原的化学合成及其在血清学诊断中的应用

选取丙型肝炎病毒(HCV)核壳蛋白区两个可能含有优势抗原表位的19和16氨基酸的肽殴(C-19肽和C-16肽),利用固相多肽合成技术进行了化学合成。经用反相高压液相层析(HPLC)及脉冲液相多肽测序技术检定合成肽产品的纯度及序列后,以C-19和C-16肽作为包被抗原组装成检测HCV血清抗体的ELISA诊断试剂。用所组装的合成肽试剂检测中国药品生物制品检定所提供的HCV标准参比血清,并比较利用该试剂和美国Abbott实验室生产的HCV第二代EIA诊断试剂平行检测109份献血员血清的结果,表明C-19肽能够特异、重复、灵敏地检出HCV血清抗体,可以作为我国第一代HCV诊断试剂的合成肽抗原。     

传染性法氏囊病病毒五个抗原表位短肽的鉴定与序列分析

以5株传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)单克隆抗体HNF1、HNF7、B34、2B1和2G8作为筛选分子,对噬菌体展示12肽库进行3轮"吸附-洗脱-扩增"淘洗,从每株单克隆抗体筛选到的噬菌斑中随机挑取12个单克隆蓝色噬菌斑,合计60个,用间接ELISA检测,A值大于1.00;用竞争抑制ELISA分析,单克隆抗体和IBDV抗原均能竞争抑制筛选12肽与固相包被单克隆抗体的反应,抑制率大于40%,表明在该12肽内含有IBDV抗原表位。选取35个单克隆噬菌斑,测定噬菌体gIII部分基因的核苷酸序列,确定了这5个含有不同IBDV抗原表位12肽的核苷酸和氨基酸序列。进一步将其与GenBank中IBDV基因组编码蛋白的氨基酸序列进行比较,发现2B1筛选肽有4个连续氨基酸残基Leu-Ala-Ser-Pro与IBDV基因组A片段编码多聚蛋白的第536-599氨基酸残基一致,推测2B1为线性表位;而HNF1、HNF7、B34和2G8筛选肽均没找到有3个以上连续氨基酸残基与IBDV蛋白序列相同之处,推测可能是构象依赖性表位。     

传染性法氏囊病病毒五个抗原表位短肽的鉴定与序列分析

以5株传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)单克隆抗体HNF1、HNF7、B34、2B1和2G8作为筛选分子,对噬菌体展示12肽库进行3轮"吸附-洗脱-扩增"淘洗,从每株单克隆抗体筛选到的噬菌斑中随机挑取12个单克隆蓝色噬菌斑,合计60个,用间接ELISA检测,A值大于1.00;用竞争抑制ELISA分析,单克隆抗体和IBDV抗原均能竞争抑制筛选12肽与固相包被单克隆抗体的反应,抑制率大于40%,表明在该12肽内含有IBDV抗原表位.选取35个单克隆噬菌斑,测定噬菌体gⅢ部分基因的核苷酸序列,确定了这5个含有不同IBDV抗原表位12肽的核苷酸和氨基酸序列.进一步将其与GenBank中IBDV基因组编码蛋白的氨基酸序列进行比较,发现2B1筛选肽有4个连续氨基酸残基Leu-Ala-Ser-Pro与IBDV基因组A片段编码多聚蛋白的第536-599氨基酸残基一致,推测2B1为线性表位;而HNF1、HNF7、B34和2G8筛选肽均没找到有3个以上连续氨基酸残基与IBDV蛋白序列相同之处,推测可能是构象依赖性表位.     

The primary structure of the coat protein of alfalfa mosaic virus strain VRU. A hypothesis on the occurrence of two conformations in the assembly of the protein shell

The complete primary structure of the coat protein of strain VRU of alfalfa mosaic virus (AMV) is reported. The strain is morphologically different from all other AMV strains as it contains large amounts of unusually long virus particles. This is caused by structural differences in the coat protein chain. The amino acid sequence has mainly been established by the characterization of peptides obtained after cleavage with cyanogen bromide and digestion with trypsin, chymotrypsin, thermolysin or Staphylococcus aureus protease. The major sequencing technique used was the dansyl-Edman procedure. The VRU coat protein consists of 219 amino acid residues corresponding to a molecular weight of 24056. Compared to the coat protein of strain 425 [Van Beynum et al. (1977) Eur. J. Biochem. 72, 63-78], 15 amino acid substitutions were localized. Most of them have a conservative character and may be explained by single-point mutations. A correction is given for the AMV 425 coat protein: Asn-216 was shown to be Asp-216. The prediction of the secondary structure for the two viral coat proteins was not significantly influenced by the various amino acid substitutions except for the region containing residues 65-100. This led us to the hypothesis that the AMV coat protein may occur in two different conformations favouring its incorporation into either a pentagonal or hexagonal quasi-equivalent position in the lattice of the protein shell. The substitutions in the above-mentioned region of the VRU coat protein may have caused a strong preference for the hexagonal lattice conformation. The model is supported by preliminary sequence data of the same coat protein region in AMV 15/64, a strain morphologically intermediate between 425 and VRU.     

The complete amino acid sequence of vitelline coat lysin

The vitelline coat lysin of a top shell, Tegula pfeifferi, is a single polypeptide consisting of 118 amino acid residues and having a relative molecular mass of 13800. The complete amino acid sequence of the vitelline coat lysin was determined by the analyses of five peptides obtained by cyanogen bromide degradation and three fragments obtained by Staphylococcus aureus protease digestion of the protein. The sequence showed the presence of microheterogeneities in the vicinity of the C-terminal half of the molecule and the existence of two homologous domain structures.     

The primary structure of the coat protein of the broad-host-range RNA bacteriophage PRR1.

The complete amino acid sequence of the coat protein of RNA bacteriophage PRR1 is presented. After thermolysin digestion, 26 peptides were isolated, covering the complete coat protein chain. Their alignment was established in part using automated Edman degradation on the intact protein, in part with overlapping peptides obtained by enzymic hydrolysis with trypsin, pepsin, subtilisin and Staphylococcus aureus protease, and by chemical cleavage with cyanogen bromide and N-bromosuccinimide. To obtain the final overlaps, a highly hydrophobic, insoluble tryptic peptide was sequenced for seven steps by the currently used manual dansyl-Edman degradation procedure, which was slightly modified for application on insoluble peptides. PRR1 coat protein contains 131 amino acids, corresponding to a molecular weight of 14534. It is highly hydrophobic, and the residues with ionizable side chains are distributed unevenly: acidic residues are absent in the middle third of the sequence, whereas a clustering of basic residues occurs between positions 44 and 62. PRR1 coat protein was compared with the coat proteins of RNA coliphages MS2 and Q beta, and the minimum mutation distance was calculated for both comparisons. It is highly probable that PRR1. Q beta and MS2 share a common ancestor. The basic region present in the three coat proteins is recognized as an essential structural feature of RNA phage coat proteins.     

Immunochemical analysis of the synthetic peptides incorporated into the protein antigenic determinant of the potato virus X coat]

Three peptides located in the N-terminal region of the potato virus X coat protein were synthesized by hand solid phase method for epitope mapping of this protein. One of these peptides (nanopeptide) interacted with monoclonal antibodies to native virus X. On the basis of these studies it was assumed, that amino acid sequence of the potato virus X coat protein, which included lysine residue in position 19, is located on the virion surface.     

南方菜豆花叶病毒外壳蛋白基因序列的分析

用重组DNA技术及序列分析法测定了南方菜豆花叶病毒RNA基因组3′端1,000个碱基的序列,以及由此序列推导出的整个外壳蛋白的氨基酸顺序,它与巳报导的基本上一致。介绍了用DNA的寡核苷酸水解混合物作为起始引物,以3′端不含PolyA尾巴且不能加上PolyA的病毒RNA作为模板合成互补DNA,及进一步无性繁殖此cDNA的方法。     

马铃薯Y病毒外壳蛋白基因的克隆及序列分析

本文报道应用聚合酶链式反应(PCR)技术,在体外扩增马铃薯 Y 病毒外壳蛋白基因及其克隆和序列分析的结果。病毒 RNA 从马铃薯 Y 病毒感染的烟草叶片中提取,用合成的PCR 3引物及 AMV 逆转录酶合成了单链的 cDNA。利用 PCR 技术,经30个循玎的扩增。得到了一特异的0.8kb 片段。克隆后对此片段进行了限制性内切酶物理图谱分析,并测定了其全序列。实验结果证明,我们克隆到的是完整的马铃薯 Y 病毒的外壳蛋白基因。与国外报道的马铃薯 Y 病毒 N 株相比,其核苷酸序列及推测的氨基酸序列的同源率分别为97.8%和97%。将该基因导入马铃薯以期获得抗 Y 病毒马铃薯的工作正在进行。本文还对 PCR 技术用于扩增植物 RNA 病毒的方法以及用基因工程方法培育抗病毒作物新品种的可行性等进行了讨论。     

香蕉束顶病毒基因克隆和序列分析

对香蕉束顶病毒（ＢＢＴＶ）中国分离株ＤＮＡ组份Ｉ（ＤＮＡ－１）、外壳蛋白（ＣＰ）和运转蛋白（ＭＰ）基因进行了克隆和序列分析。ＢＢＴＶＤＮＡ－１含有１１０３个核苷酸,与南太平洋和亚洲分离株分别有８７％－８８％ ９６．９－９８％的核苷酸序列同源性。由ＤＮＡ－１编码的复制酶含有１８６个在酸残基。与南太平洋和亚洲分离株分别有８４．４％－９５．８％和９７．６％、９８．０％的氨基酸序列同源性。外壳蛋白基因由５     

Structural studies on the coat protein of alfalfa mosaic virus. Isolation and characterization of the tryptic peptides and the alignment of the cyanogen-bromide fragments.

The reduced and carboxymethylated coat protein of alfalfa mosaic virus (AMV 425) was fragmented by means of cyanogen-bromide cleavage. The tryptic peptides from the protein and its four cyanogen-bromide fragments were isolated on a preparative scale by combinations of column and paper separation techniques. The tryptic digest of the carboxymethylated protein contained 24 peptides and two free amino acids. All peptides have been characterized by amino acid analyses and end-group determinations. Together the tryptic peptides account for a total chain length of 228 amino acids. The data are in good agreement with previous reports from this laboratory.     

Viral Coat Protein Peptides with Limited Sequence Homology Bind Similar Domains of Alfalfa Mosaic Virus and Tobacco Streak Virus RNAs

An unusual and distinguishing feature of alfalfa mosaic virus (AMV) and ilarviruses such as tobacco streak virus (TSV) is that the viral coat protein is required to activate the early stages of viral RNA replication, a phenomenon known as genome activation. AMV-TSV coat protein homology is limited; however, they are functionally interchangeable in activating virus replication. For example, TSV coat protein will activate AMV RNA replication and vice versa. Although AMV and TSV coat proteins have little obvious amino acid homology, we recently reported that they share an N-terminal RNA binding consensus sequence (Ansel-McKinney et al., EMBO J. 15:5077–5084, 1996). Here, we biochemically compare the binding of chemically synthesized peptides that include the consensus RNA binding sequence and lysine-rich (AMV) or arginine-rich (TSV) environment to 3′-terminal TSV and AMV RNA fragments. The arginine-rich TSV coat protein peptide binds viral RNA with lower affinity than the lysine-rich AMV coat protein peptides; however, the ribose moieties protected from hydroxyl radical attack by the two different peptides are localized in the same area of the predicted RNA structures. When included in an infectious inoculum, both AMV and TSV 3′-terminal RNA fragments inhibited AMV RNA replication, while variant RNAs unable to bind coat protein did not affect replication significantly. The data suggest that RNA binding and genome activation functions may reside in the consensus RNA binding sequence that is apparently unique to AMV and ilarvirus coat proteins.