Simulation, construction and characterization of a multi-functional thrombolytic agent with anti-thrombosis activity

Prourokinase (scu-PA), a thrombolytic agent, was inserted between Gly118 and Ile119 with foreign antithrombosis functional motif (Lys-Gly-Asp-Trp-motif) to construct a multi-functional chimeric molecule. The molecular model of a chimera was simulated and predicted. The recombinant chimeric protein was expressed by the baculovirus-insect cell expression system and purified by affinity chromatography. The physico-chemical characteristics of the chimeric molecule were assayed. The thrombolytic activity was determined to be 90000 IU/mg of fibrinolytic special activity by the fibrin-plate method. The anti-thrombosis activities were also assayed with IC₅₀ of 9.6 μM by an inhibition test of ADP-induced platelet aggregation.     

草菇MAPKKK同源基因的克隆及序列分析

根据担子菌丝裂原活化蛋白质激酶激酶激酶(MAPKKK)蛋白的保守序列设计两对简并引物,通过巢式简并PCR方法获得草菇VV-MAPKKK基因中的保守片段,然后通过和草菇基因组信息比对,获得了VV-MAPKKK基因全长序列。VV-MAPKKK基因长度为4434bp,包含4个内含子,编码1405个氨基酸残基,推定的氨基酸序列与新型隐球菌(Cryptococcus neoformans)、巴西芽生菌(Paracoccidioides brasiliensis)和异旋孢腔菌(Cochliobolus heterostrophus)的MAPKKK同源蛋白相似性分别为58%、57%和56%。对VV-MAPKKK蛋白的系统发生学分析的结果表明,VV-MAPKKK与担子菌中的Hog信号传导途径的MAPKKK同源蛋白聚在同一进化支上,这些数据都支持所获得的VV-MAPKKK为Hog-MAPKKK蛋白在草菇中的同源物的推定。     

The Slowing Rate of CpG Depletion in SARS-CoV-2 Genomes Is Consistent with Adaptations to the Human Host

Depletion of CpG dinucleotides in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genomes has been linked to virus evolution, host-switching, virus replication, and innate immune responses. Temporal variations, if any, in the rate of CpG depletion during virus evolution in the host remain poorly understood. Here, we analyzed the CpG content of over 1.4 million full-length SARS-CoV-2 genomes representing over 170 million documented infections during the first 17 months of the pandemic. Our findings suggest that the extent of CpG depletion in SARS-CoV-2 genomes is modest. Interestingly, the rate of CpG depletion is highest during early evolution in humans and it gradually tapers off, almost reaching an equilibrium; this is consistent with adaptations to the human host. Furthermore, within the coding regions, CpG depletion occurs predominantly at codon positions 2-3 and 3-1. Loss of ZAP (Zinc-finger antiviral protein)-binding motifs in SARS-CoV-2 genomes is primarily driven by the loss of the terminal CpG within the motifs. Nonetheless, majority of the CpG depletion in SARS-CoV-2 genomes occurs outside ZAP-binding motifs. SARS-CoV-2 genomes selectively lose CpGs-motifs from a U-rich context; this may help avoid immune recognition by TLR7. SARS-CoV-2 alpha-, beta-, and delta-variants of concern have reduced CpG content compared to sequences from the beginning of the pandemic. In sum, we provide evidence that the rate of CpG depletion in virus genomes is not uniform and it greatly varies over time and during adaptations to the host. This work highlights how temporal variations in selection pressures during virus adaption may impact the rate and the extent of CpG depletion in virus genomes.     

牛源流行性出血病病毒(EHDV)血清10型毒株在我国的分离鉴定

我国存在多种血清型流行性出血热病毒(Epizootic haemorrhagic disease virus,EHDV)的流行,但尚未有关于EHDV-10型毒株的分离报道。为了解云南省EHDV的流行情况,2012～2015年,本研究在云南省设立江城、师宗、芒市三个监控点,定期采集监控动物血液,接种幼仓鼠肾细胞(Baby hamster kidney cell,BHK-21)进行病毒分离;通过PCR检测、血清中和试验、琼脂糖凝胶电泳和电镜观察等方法对分离病毒进行鉴定;对分离毒株的Seg-2/VP2与Seg-3/VP3基因节段进行克隆、测序与序列分析。2013年在云南省师宗县的哨兵牛上分离出一株EHDV毒株(YNSZ-V277-2013),病毒可引起BHK-21细胞出现圆缩、裂解的细胞病变(Cytopathic effect,CPE);电镜下病毒粒子呈球形,无囊膜,表面有大量纤维突,直径在70～80nm之间;病毒基因组dsRNA的琼脂糖凝胶电泳显示分离毒株与其他血清型EHDV一致,呈现"3-3-3"的电泳带型;序列分析显示YNSZ-V277-2013毒株的Seg-2/VP2与Seg-3/VP3序列与日本EHDV-10型毒株(ON-4/N/98)相似度最高,分别为97.5%/98.5%与98.1%/99.8%,证实分离毒株为EHDV-10型;系统发育分析显示YNSZ-V277-2013毒株的Seg-2与日本EHDV-10型毒株(ON-4/N/98)的亲缘关系最近,Seg-3与分离至日本和澳大利亚的EHDV毒株同属Eastern型。本研究首次报道了EHDV-10型毒株在我国的分离以及分离毒株的Seg-2与Seg-3基因序列特征,为进一步开展中国EHDV-10型的流行病学调查与致病性研究提供了基础。     

红纹黄单胞菌a-氨基酸酯水解酶的克隆、序列分析及热稳定性提高

【目的】克隆红纹黄单胞菌α-氨基酸酯水解酶基因全序列,对序列进行生物信息学分析,并提高酶的热稳定性。【方法】利用多聚酶链式反应(PCR)克隆α-氨基酸酯水解酶基因全序列;应用生物信息学软件对获得的基因序列及编码的蛋白序列进行分析;通过同源建模,预测红纹黄单胞菌α-氨基酸酯水解酶的三维结构;通过定点突变替换氨基酸序列中高度柔性的位点,提高该酶的热稳定性。【结果】从红纹黄单胞菌(Xanthomonas rubrillineans)中扩增得到α-氨基酸酯水解酶基因aeh(GenBank登录号JF744990),核苷酸序列长度1 917 bp,编码638个氨基酸。序列比对和同源性分析显示,该酶与白纹黄单胞菌Xanthomonas albilineans str.GPE PC73的肽酶及地毯草黄单胞菌Xanthomonas axono-podis pv. citri str. 306的戊二酰-7-氨基头孢烷酸酰化酶氨基酸序列相似性最高,分别为91%和83%,系统进化分析表明,该酶与白纹黄单胞菌Xanthomonas albilineans str. GPEPC73的肽酶亲缘性最高。基于预测的三维模型,对高度柔性的位点进行饱和突变,从282株突变体中筛选得到3株T50较野生型高5°C以上的突变体。【结论】对红纹黄单胞菌AEH的氨基酸序列分析有助于探索同源蛋白的进化过程。对高度柔性位点进行饱和突变的策略可以用于提高热稳定性。     

MotViz: a tool for sequence motif prediction in parallel to structural visualization and analyses

Linking similar proteins structurally is a challenging task that may help in finding the novel members of a protein family. In this respect, identification of conserved sequence can facilitate understanding and classifying the exact role of proteins. However, the exact role of these conserved elements cannot be elucidated without structural and physiochemical information. In this work, we present a novel desktop application MotViz designed for searching and analyzing the conserved sequence segments within protein structure. With MotViz, the user can extract a complete list of sequence motifs from loaded 3D structures, annotate the motifs structurally and analyze their physiochemical properties. The conservation value calculated for an individual motif can be visualized graphically. To check the efficiency, predicted motifs from the data sets of 9 protein families were analyzed and MotViz algorithm was more efficient in comparison to other online motif prediction tools. Furthermore, a database was also integrated for storing, retrieving and performing the detailed functional annotation studies. In summary, MotViz effectively predicts motifs with high sensitivity and simultaneously visualizes them into 3D strucures. Moreover, MotViz is user-friendly with optimized graphical parameters and better processing speed due to the inclusion of a database at the back end. MotViz is available at http://www.fi-pk.com/motviz.html.     

传染性法氏囊病病毒中国强毒株A节段cDNA基因的克隆和序列分析

以来自哈尔滨传染性法氏囊病病毒（ＩＢＤＶ） 强毒株（Ｈａｒｂｉｎ 毒株,Ｈ） 的基因组ＲＮＡ为模板,用反转录聚合酶链反应（ＲＴ－ ＰＣＲ） 的方法得到了其Ａ 节段的全长ｃＤＮＡ 片段,分５＇端（１ ６５９ｂｐ） 和３＇端（１ ４４４ｂｐ） 上下两段分别克隆到ｐＧＥＭＢ○Ｒ － Ｔ 载体上,测定了其核苷酸顺序,在长为３ １０１ ｂｐ 中含有两个阅读框ＯＲＦＡ１ 和ＯＲＦＡ２ ,分别编码１ ０１２ 个氨基酸的前体蛋白（ＶＰ２ － ４ －３） 和１４５ 个氨基酸的ＶＰ５,ＯＲＦＡ１ 和ＯＲＦＡ２ 有部分的重叠。将核苷酸序列及推测出的氨基酸序列与已报道的ＩＢＤＶ 血清Ⅰ型和Ⅱ型毒株的相应序列进行了比较,结果表明：Ｈ 毒株与其它血清Ⅰ型毒株之间,在核苷酸水平上存在２５ｂｐ － ２６７ｂｐ 的差异;在氨基酸水平上存在１７ ～４０ 个氨基酸的差异。在ＶＰ２ － ４ － ３ 内比较显示,Ｈ 毒株与Ｐ２ 、Ｃｕ－ １ 之间氨基酸的差异最小为１ ．７％ ,Ｈ 毒株与ＵＫ６６１ 之间氨基酸的差异最大为３ ．９ ％ 。变异主要发生在ＶＰ２ 的可变区（２０６ － ３５０ 位氨基酸） ,在Ｈ 毒株所特有的１２ 个氨基酸当中,该区就占５ 个,代表１ ．７６ ％ 的变异。ＶＰ４、ＶＰ３ 和ＶＰ５区各有     

SSRs isolated from apple can identify polymorphism and genetic diversity in pear

Apple simple sequence repeats (SSRs) were intergenerically applied to the characterization of 36 pear accessions, including 19 Japanese pears (Pyrus pyrifolia), 7 Chinese pears (P. bretschneideri, P. ussuriensis), 5 European pears (P. communis), 3 wild relatives (P. calleryana), and 2 hybrids between P. pyrifolia and P. communis. All of the tested SSR primers derived from apple produced discrete amplified fragments in all pear accessions. Nucleotide repeats were detected in the amplified bands by both Southern blot and sequencing analysis, and nucleotide sequences of pear were compared with those of apple. The differences in fragment size among pear or between pear and apple were, in many cases, due to the differences in repeat number. Interestingly, the DNA sequence of flanking regions in apple was highly conserved in pear. Hybrids from P. pyrifolia×P. communis showed one fragment inherited from each parent in all scorable cases, which suggested that each primer pair amplified fragments originating from the same locus. A total of 79 alleles were detected from seven SSR loci in pear, and all pear varieties except for the mutants could be differentiated. In conclusion, SSRs isolated from apple are highly conserved in pear and could be utilized as DNA markers in the latter genus. Received: 17 July 2000 / Accepted: 22 September 2000