浓核病毒感染早期家蚕血液、中肠蛋白表达差异比较分析

为了探明在浓核病毒镇江株(BmDNV-ZJ)侵染早期,家蚕部分组织蛋白所产生的免疫抵抗性变化机制,本实验采用差异蛋白质组学技术研究分析了BmDNV-ZJ感染早期,家蚕的中肠、血液组织中特异性表达的差异蛋白.实验结果表明:在浓核病毒侵染初期,感受性家蚕的中肠组织受病毒感染而得到特异性表达的蛋白可能为丝氨酸蛋白酶抑制剂和巯基抗氧化酶蛋白,前者具有调控蛋白酶活性和细胞凋亡的功能,后者具有抗氧化的作用.血液组织受病毒感染诱导而产生的蛋白可能是丝裂原活化蛋白激酶和类抗氧化酶蛋白,前者具有调控细胞凋亡的功能,后者具有抗氧化、消除自由基作用.由于试验中所得的差异蛋白点很少,这表明在BmDNV-ZJ感染早期,蚕体对浓核病毒的感染而产生的反应很小.蚕体可通过被侵染的中肠组织(浓核病毒感染的靶部位)以及血液(免疫组织)共同产生一些抗氧化或调控细胞凋亡的酶蛋白等来抗击浓核病毒的侵染.     

家蚕浓核病毒DNV-3（中国株）的VD2基因组序列分析

浓核病毒BmDNV-3(中国株)基因组中含有两种不同的单链线形DNA分子(VD1,VD2)。该病毒的VD2被分离、纯化、克隆到pUC119载体上,并完成了VD2全基因组序列的测定。序列分析显示:VD2全基因组长为6022个核苷酸,末端拥有524个核苷酸反向重复序列(ITRs)。VD2基因组正链含有2个大的开放阅读框,负链含有1个小的开放阅读框。计算机分析推测该基因组正链上开放阅读框(ORF1)及负链上开放阅读框(ORF3)主要编码病毒的非结构蛋白,而正链上开放阅读框(ORF2)主要编码病毒的结构蛋白。比较BmDNV-3 VD2和BmDNV-2(Yamanashiisolate)VD2基因组全序列,两者同源性达97.7%,并且有132个碱基的替代1、1个碱基的删除和2个碱基插入。研究结果显示BmDNV-3 VD2和BmDNV-2 VD2有很近的亲缘关系,但也发生一定的变异,这为更好地理解浓核病毒种类的多样性,也为研究家蚕浓核病毒进化提供了有益的线索。     

家蚕浓核病毒(镇江)株主要结构蛋白基因的克隆及表达

家蚕浓核病毒(Bombyx mori densovirus,BmDNV)是一种昆虫细小病毒.与其它昆虫细小病毒感染昆虫体内多种组织不同,家蚕浓核病毒只感染家蚕中肠上皮组织的圆筒型细胞,感染该病毒细胞的细胞核可以被孚尔根和甲基绿浓染,在病毒感染的早期中肠上皮组织细胞数量增加,形成褶皱,最后感染细胞脱落到肠腔中[1-3].自从20世纪70年代末日本学者证实家蚕浓核病是由于家蚕浓核病毒感染引起的以来[4],已经分离得到了多个病毒株系[5-8].根据它们在血清学、理化特性、品种感受性和病理特征等方面的差异,分为BmDNV-1(伊那株)和BmDNV-2(以山梨株为代表)[8-11].     

家蚕浓核病毒 Bm DNV-3(中国株)VD1基因组结构与转录分析

为了进一步认识家蚕浓核病毒BmDNV-3(中国株)VD1基因组的结构和功能,VD1被分离、纯化、克隆到pUC119载体上,完成了基因组全序列的测定。序列分析显示VD1基因组全长为6543个核苷酸,末端拥有224个核苷酸反向重复序列(ITRs)。VD1基因组正链含有3个大的开放阅读框(ORF1-3),负链含有1个大的开放阅读框(ORF4)。比较BmDNV-3的VD1和BmDNV-2(Yamanashiisolate)的VD1基因组全序列,两者同源性为98.4%,并且有107个碱基的替代和1个碱基插入,氨基酸突变集中在VD1ORF3和VD1ORF4。Northern杂交结果显示VD1的左边正链上有1.1kb和1.5kb两个转录本,右边的负链上有一个3.3kb转录本。3′和5′-RACE结果显示1.1kb转录本开始于nt290,结束于nt1437;1.5kb转录本开始于nt1423,结束于nt2931;3.3kb转录本开始于nt6287,结束于nt2922。正链上1.5kb转录本和负链上3.3kb转录本拥有10个核苷酸的3′端的共同序列。研究结果显示该病毒基因转录与已报道的其它浓核病毒存在较大的差异性。     

两个对浓核病毒(镇江株)具感性差异的家蚕品种的血液和围食膜蛋白的比较

家蚕品种间对浓核病毒(镇江株)具有不同的感染性,为了进一步探明家蚕对浓核病毒(镇江株)感性差异的分子机制,本文运用蛋白质电泳和质谱技术比较分析了两个对家蚕浓核病毒(镇江株)感性和抗性的近等基因系家蚕品种JS和NIL的血液和围食膜组织蛋白.结果表明:这两个家蚕品种的血液和围食膜组织蛋白组成差异很小.在血液组织蛋白中发现5个差异蛋白点,其中家蚕品种JS血液中有两个差异蛋白,可能分别为酪蛋白激酶和新型组织蛋白;在NIL血液组织中发现3个差异蛋白点,其中两个可能分别为线粒体延伸因子和类丝氨酸蛋白酶,另外1个的含量极显著高于JS,为血淋巴蛋白.在围食膜蛋白中共发现4个差异蛋白点,其中JS围食膜中有1个差异蛋白可能为新型组织蛋白;其余3个蛋白点在含量上相互间存在显著差异.本研究根据组织差异蛋白的功能初步推测家蚕对浓核病毒(镇江株)感性差异与血液蛋白差异无显著相关,与围食膜蛋白差异可能有关.     

家蚕对浓核病毒中国株(BmDNV-3)抗性及感性品系中肠的差异蛋白质分析

【目的】通过比较家蚕Bombyx mori抗性及感性品系的中肠蛋白质表达谱,获得家蚕对家蚕浓核病毒中国株（BmDNV-3）抗性相关的蛋白。【方法】利用双向电泳(2-DE)对感性品种华八35和抗性品种秋丰接种病毒后48 h的蛋白质表达谱进行比较分析,并对其中的差异蛋白进行MALDI-TOF-TOF质谱分析,通过NCBInr和MSDB数据库进行蛋白点的鉴定和功能分析。【结果】获得重复性较好的差异蛋白点16个,其中质谱鉴定出5种蛋白,它们分别是糖基转移酶（glycosyltransferase）、糖基转移酶-S（GlcAT-S）、21.5 kD小热休克蛋白（21.5 kD small heat shock protein）、V-ATP酶（vacuolar ATP synthase）和精氨酸激酶（arginine kinase）。这5种蛋白在抗性品系秋丰中的表达量均高于感性品系华八35。【结论】糖基转移酶和糖基转移酶-S仅在抗性品系中存在,提示它们可能是与抗性有关的蛋白。此外,增强的应激反应和能量代谢也可能与家蚕对BmDNV-3的抗性产生相关。     

伊蚊浓核病毒三维结构的比较分析

细小病毒是目前已知的结构最小的病毒, 其宿主范围很广. 利用冷冻电镜和三维重构技术获得了伊蚊浓核病毒1.2 nm分辨率下的三维结构, 并利用计算机生物信息处理技术和氨基酸序列比对技术比较了该病毒和其他细小病毒的结构和序列的异同. 尽管均属于浓核病毒亚科, 但是无论从衣壳结构还是其蛋白质的氨基酸序列上, 伊蚊浓核病毒和其他昆虫的细小病毒都有很大差异. 相比之下, 伊蚊浓核病毒和人类B19细小病毒的衣壳蛋白相似性较大, 两者的结构蛋白和非结构蛋白一致性也高于伊蚊浓核病毒与其他昆虫的细小病毒的一致性. 此结果表明: 伊蚊浓核病毒和人类B19细小病毒有着密切关系, 前者可能来源于B19病毒的较近期变种.     

家蚕浓核病毒中国株非结构蛋白1(NS1)的表达

利用PCR技术扩增出BmDNV-3 NS1基因,将目的基因与原核表达载体pET-30a进行连接,转化BL21 star菌并在该菌中表达,经Western blot鉴定表达的产物为BmDNV-3 NS1蛋白,纯化NS1蛋白并制备兔多克隆抗体.同时BmDNV-3 NS1基因亚克隆到杆状病毒转移载体pFastBae-HTb-eGFP中,转化BmDH10BAC感受态细胞,提取的重组Bacmid通过脂质体包埋转染家蚕BmN细胞,再以收获的重组病毒感染家蚕幼虫.家蚕BmN细胞和幼虫感染重组病毒2d后均观察到绿色荧光,经SDS-PAGE分析真核表达的产物与预测的NS1-eGFP融合蛋白大小不一致,说明NS1-eGFP融合蛋白被昆虫内源性的蛋白酶降解.降解的产物用NS1蛋白抗体进行Western blot鉴定为BmDNV-3 NS1蛋白.     

家蚕二分浓核病毒ns1基因序列的改造、表达和鉴定

探讨翻译起始区(TIR)部分密码子发生同义突变后,对家蚕二分浓核病毒(BmBDV)ns1基因表达的影响,以及对BmBDV NS1蛋白毒性进行鉴定,设计特异性上游引物,对BmBDV ns1基因中第3、4、9和10个密码子进行同义突变,利用原核表达系统对野生型和改造后的ns1序列进行表达,通过SDS-PAGE电泳对这两种序列的表达产量进行分析。利用Protein Iso~(TM)GST Resin从超声破碎的菌液上清中纯化融合有GST的NS1蛋白,进而对纯化的靶蛋白在细胞水平和家蚕体内进行毒性分析。结果表明:TIR突变后的BmBDV ns1序列,其与野生型序列的表达产量之间没有明显差异;BmBDV NS1蛋白具有抑制细胞增殖和诱导家蚕致死的生化活性。     

利用优化改造的家蚕杆状病毒表达系统提高NS1表达产量

为优化家蚕杆状病毒表达系统,提高外源基因的表达产量。文中通过同源重组技术,用串联的氯霉素基因(Cm)表达盒和绿色荧光蛋白基因(egfp)表达盒将其替换,从而获得Chitinase和Cystein Protease两个基因缺失的家蚕杆状病毒载体。通过转座,将多角体启动子控制的家蚕二分浓核病毒(Bm BDV)ns1基因表达盒,定点插入到改造后的该分子载体中。将重组载体转染Bm N细胞,获得能表达家蚕二分浓核病毒(Bm BDV)NS1的缺失型重组病毒;另外,将多角体启动子控制的ns1基因转座到野生型Bm-bacmid中,获得能表达Bm BDV NS1的野生型重组病毒。将这两种病毒分别皮下注射家蚕,对感染后的家蚕血液中NS1表达水平进行比较,发现缺失Chitinase和Cystein Protease重组病毒感染的家蚕血液中,NS1的表达量是对照组的3倍,从而建立了一种高效表达可溶性NS1蛋白的方法,为靶蛋白的结构与功能研究奠定基础。     

Comparative modeling of the conformational stability of chymotrypsin inhibitor 2 protein mutants using amino acid sequence autocorrelation (AASA) and amino acid 3D autocorrelation (AA3DA) vectors and ensembles of Bayesian-regularized genetic neural networks

Predicting protein stability changes upon point mutation is important for understanding protein structure and designing new proteins. Autocorrelation vector formalism was extended to amino acid sequences and 3D conformations for encoding protein structural information with modeling purpose. Protein autocorrelation vectors were weighted by 48 amino acid/residue properties selected from the AAindex database. Ensembles of Bayesian-regularized genetic neural networks (BRGNNs) trained with amino acid sequence autocorrelation (AASA) vectors and amino acid 3D autocorrelation (AA3DA) vectors yielded predictive models of the change of unfolding Gibbs free energy change (ΔΔG) of chymotrypsin Inhibitor 2 protein mutants. The ensemble predictor described about 58 and 72% of the data variances in test sets for AASA and AA3DA models, respectively. Optimum sequence and 3D-based ensembles exhibit high effects on relevant structural (volume, solvent-accessible surface area), physico-chemical (hydrophilicity/hydrophobicity-related) and thermodynamic (hydration parameters) properties.     

Amino acid sequence predicts folding rate for middle-size two-state proteins

The significant correlation between protein folding rates and the sequence-predicted secondary structure suggests that folding rates are largely determined by the amino acid sequence. Here, we present a method for predicting the folding rates of proteins from sequences using the intrinsic properties of amino acids, which does not require any information on secondary structure prediction and structural topology. The contribution of residue to the folding rate is expressed by the residue's Omega value. For a given residue, its Omega depends on the amino acid properties (amino acid rigidity and dislike of amino acid for secondary structures). Our investigation achieves 82% correlation with folding rates determined experimentally for simple, two-state proteins studied until the present, suggesting that the amino acid sequence of a protein is an important determinant of the protein-folding rate and mechanism.     

Structure of Bombyx mori densovirus 1, a silkworm pathogen

Bombyx mori densovirus 1 (BmDNV-1), a major pathogen of silkworms, causes significant losses to the silk industry. The structure of the recombinant BmDNV-1 virus-like particle has been determined at 3.1-? resolution using X-ray crystallography. It is the first near-atomic-resolution structure of a virus-like particle within the genus Iteravirus. The particles consist of 60 copies of the 55-kDa VP3 coat protein. The capsid protein has a β-barrel "jelly roll" fold similar to that found in many diverse icosahedral viruses, including archaeal, bacterial, plant, and animal viruses, as well as other parvoviruses. Most of the surface loops have little structural resemblance to other known parvovirus capsid proteins. In contrast to vertebrate parvoviruses, the N-terminal β-strand of BmDNV-1 VP3 is positioned relative to the neighboring 2-fold related subunit in a "domain-swapped" conformation, similar to findings for other invertebrate parvoviruses, suggesting domain swapping is an evolutionarily conserved structural feature of the Densovirinae.     

Generation of deviation parameters for amino acid singlets, doublets and triplets from three-dimentional structures of proteins and its implications for secondary structure prediction from amino acid sequences

We present a new method, secondary structure prediction by deviation parameter (SSPDP) for predicting the secondary structure of proteins from amino acid sequence. Deviation parameters (DP) for amino acid singlets, doublets and triplets were computed with respect to secondary structural elements of proteins based on the dictionary of secondary structure prediction (DSSP)-generated secondary structure for 408 selected non-homologous proteins. To the amino acid triplets which are not found in the selected dataset, a DP value of zero is assigned with respect to the secondary structural elements of proteins. The total number of parameters generated is 15,432, in the possible parameters of 25,260. Deviation parameter is complete with respect to amino acid singlets, doublets, and partially complete with respect to amino acid triplets. These generated parameters were used to predict secondary structural elements from amino acid sequence. The secondary structure predicted by our method (SSPDP) was compared with that of single sequence (NNPREDICT) and multiple sequence (PHD) methods. The average value of the percentage of prediction accuracy for a helix by SSPDP, NNPREDICT and PHD methods was found to be 57%, 44% and 69% respectively for the proteins in the selected dataset. For b-strand the prediction accuracy is found to be 69%, 21% and 53% respectively by SSPDP, NNPREDICT and PHD methods. This clearly indicates that the secondary structure prediction by our method is as good as PHD method but much better than NNPREDICT method.     

Generation of deviation parameters for amino acid singlets, doublets and triplets from three-dimensional structures of proteins and its implications for secondary structure prediction from amino acid sequences

We present a new method, secondary structure prediction by deviation parameter (SSPDP) for predicting the secondary structure of proteins from amino acid sequence. Deviation parameters (DP) for amino acid singlets, doublets and triplets were computed with respect to secondary structural elements of proteins based on the dictionary of secondary structure prediction (DSSP)-generated secondary structure for 408 selected nonhomologous proteins. To the amino acid triplets which are not found in the selected dataset, a DP value of zero is assigned with respect to the secondary structural elements of proteins. The total number of parameters generated is 15,432, in the possible parameters of 25,260. Deviation parameter is complete with respect to amino acid singlets, doublets, and partially complete with respect to amino acid triplets. These generated parameters were used to predict secondary structural elements from amino acid sequence. The secondary structure predicted by our method (SSPDP) was compared with that of single sequence (NNPREDICT) and multiple sequence (PHD) methods. The average value of the percentage of prediction accuracy for αhelix by SSPDP, NNPREDICT and PHD methods was found to be 57%, 44% and 69% respectively for the proteins in the selected dataset. For Β-strand the prediction accuracy is found to be 69%, 21% and 53% respectively by SSPDP, NNPREDICT and PHD methods. This clearly indicates that the secondary structure prediction by our method is as good as PHD method but much better than NNPREDICT method.     

Reducing the false positive rate in the non-parametric analysis of molecular coevolution

Background  

The strength of selective constraints operating on amino acid sites of proteins has a multifactorial nature. In fact, amino acid sites within proteins coevolve due to their functional and/or structural relationships. Different methods have been developed that attempt to account for the evolutionary dependencies between amino acid sites. Researchers have invested a significant effort to increase the sensitivity of such methods. However, the difficulty in disentangling functional co-dependencies from historical covariation has fuelled the scepticism over their power to detect biologically meaningful results. In addition, the biological parameters connecting linear sequence evolution to structure evolution remain elusive. For these reasons, most of the evolutionary studies aimed at identifying functional dependencies among protein domains have focused on the structural properties of proteins rather than on the information extracted from linear multiple sequence alignments (MSA). Non-parametric methods to detect coevolution have been reported to be especially susceptible to produce false positive results based on the properties of MSAs. However, no formal statistical analysis has been performed to definitively test the differential effects of these properties on the sensitivity of such methods.     

What is the protein design alphabet?

Selecting a protein sequence that corresponds to a specific three-dimensional protein structure is known as the protein design problem. One principal bottleneck in solving this problem is our lack of knowledge of precise atomic interactions. Using a simple model of amino acid interactions, we determine three crucial factors that are important for solving the protein design problem. Among these factors is the protein alphabet-a set of sequence elements that encodes protein structure. Our model predicts that alphabet size is independent of protein length, suggesting the possibility of designing a protein of arbitrary length with the natural protein alphabet. We also find that protein alphabet size is governed by protein structural properties and the energetic properties of the protein alphabet units. We discover that the usage of average types of amino acid in proteins is less than expected if amino acids were chosen randomly with naturally occurring frequencies. We propose three possible scenarios that account for amino acid underusage in proteins. These scenarios suggest the possibility that amino acids themselves might not constitute the alphabet of natural proteins.     

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。