大腹圆蛛主壶腹腺cDNA文库构建和丝蛋白基因筛选

首次通过反转录-置换法和使用pUC18质粒成功构建大腹圆蛛（Araneus ventricosus)主壶腹腺（major ampullate gland)cDNA基因文库,并以鸟枪法从中筛选出具有典型重复结构的大腹圆蛛主壶腹丝蛋白cDNA基因AvF1,大小为1744bp,编码区为1572bp,编码氨基酸524个,分子量为42489.55Da,典型的重复结构为（GGP）nGGX。与现有已知的蛛丝蛋白基因中三带金蛛（Argiope trifas-ciata)鞭毛样丝基因（AtfF)有最高的同源性69.3%。大腹圆蛛主壶腹腺cDNA文库的构建和蛛丝蛋白新基因的克隆,为提供大腹圆蛛蛛丝蛋白基因背景和进一步研究蛛丝蛋白奠定了基础。     

大腹园蛛大壶状腺丝蛋白-1基因部分cDNA的克隆和序列分析

大腹园蛛大壶状腺表达拖丝蛋白新基因的克隆, 为进一步研究蛛丝蛋白基因以及人工表达蛛丝蛋白提供参考依据。文章利用“通用方法”即反转录—置换法构建大腹园蛛(Araneus ventricosus)大壶状腺(Major ampullate gland) cDNA文库, 并筛选出具有典型重复结构的大腹园蛛大壶状腺丝蛋白-1部分cDNA序列AvMaSp1 (GenBank登录号: AY177203)。该部分序列大小为1 408 bp, 编码区为1 288 bp, 编码氨基酸429个, 预测分子量为34.07 kDa, 典型的重复结构为 (GA)nAm(GA)N, 与十字园蛛(Araneus diadematus)丝蛋白基因ADF-1 (GenBank登录号: ADU47853)同源关系最近, 一致性为75.0%。     

蛛丝蛋白基因在大肠杆菌中表达研究

利用大腹园蛛基因组文库筛选获得一段693 bp基因片段,经分析该段基因处于鞭毛状丝基因重复区域,且其中包含了一个完整的重复框架。通过基因密码子优化,在其3′和5′端分别融合蛋白质亲和层析标签,克隆于pET30LIC表达载体中,在不同的大肠杆菌中进行表达试验。实验结果显示:经过密码子优化,融合目的蛋白基因在BL21(DE3)中得到了高效表达,产量达到25~30mg/L,纯化产物纯度达90%以上。SDS-PAGE和W estern-b lotting检测目的融合蛋白均与预期蛋白大小一致。     

三种蜘蛛丝蛋白组成分析

本文应用高压液相色谱（ＨＰＬＣ）法分析了岳麓山的大腹园蛛Ａｒａｎｅｕｓ ｖｅｎｔｒｉｃｏｓｕｓ（Ｃ．Ｋｏｃｈ,１８７８）,机敏漏头蛛Ａｇｅｌｅｎａ ｄｉｆｆｉｃｌｉｓ （Ｆｏｘ,１９３７）,白额巨蟹蛛Ｈｅｔｅｒｏｐｏｄａ ｖｅｎａｔｏｒｉａ （Ｌｉｎｎａｅｕｓ,１７５７）的丝蛋白的氨基酸组成,以ＳＤＳ－ＰＡＧＥ法测定了大腹园蛛不同丝腺体的未成丝的可溶性丝蛋白的分子量。实验结果表明蛛丝蛋白中占优势的     

3种中型蜘蛛卵袋形态特征与纤维组成结构

采用扫描电镜和氨基酸自动分析仪对球蛛科(Theridiidae)温室拟肥腹蛛(Parasteatodatepidariorum)、肖蛸蛛科(Tetragnathidae)肩斑银鳞蛛(Leucauge blanda)及狼蛛科(Lycosidae)猴马蛛(Hippasa holmerae)3种中型蜘蛛卵袋的超微结构和氨基酸组成进行了观察。形态观察表明,这3种蜘蛛的卵袋形态各异,温室拟肥腹蛛卵袋一头尖,呈梨状;肩斑银鳞蛛卵袋呈扁平状;猴马蛛卵袋呈椭球形。扫描电镜观察表明,温室拟肥腹蛛卵袋外覆盖层仅仅由一种均一直径的柱状腺丝组成,而另外2种蜘蛛卵袋外覆盖层主要由柱状腺丝与少量其他丝腺纺出的丝纤维组成。氨基酸组成分析表明,温室拟肥腹蛛卵袋外覆盖层的丝纤维的氨基酸组成与具有保守性的其他种类蜘蛛柱状腺丝心蛋白的氨基酸组成差异较大,这表明其可能含有新的丝心蛋白家族成员。本文根据氨基酸组成与扫描电镜的结果分析探讨了不同直径丝纤维的丝腺来源。     

大腹园蛛拖丝蛋白一级结构初步研究

采用部分酸水解的方法对大腹园蛛（Ａｒａｎｅｕｓｖｅｎｔｒｏｃｏｓｕｓ）拖丝纤维蛋白进行水解,通过反相高液相色谱分离到一些重复小肽片段,对其序列分析表明,该蜘蛛拖丝蛋白与肖蛸科的棒络新妇蛛（Ｎｅｐｈｉｌａｃｌａｖｉｐｅｓ）丝蛋白的序列比较,有一个完全相同的肽段ＧＹＧＰＧ,其余所测片段测存在差异,显示不同种属的蜘蛛丝在一级结构上的同异,并探讨了结构和功能的关系。     

大腹园蛛主壶腹腺蛛丝蛋白末端结构域的克隆表达

牵引丝(dragline silk)由主壶腹腺蛛丝蛋白(major ampullate spidroin, MaSp)组成,是蜘蛛丝中强度最好的丝,同时具有极佳的生物相容性和可降解性,因此引起研究者的研究热潮。目前关于大腹园蛛MaSp结构和成丝机理方面的研究甚少,限制了其仿生应用。本文以大腹园蛛牵引丝的组成蛋白质之一MaSp1为研究对象,通过锚定PCR的方法首次获取了大腹园蛛MaSp1 NT的完整编码基因,并对其进行了克隆、表达、纯化,产量可达60 mg/L;同时对该MaSp1的CT进行表达纯化,产量可达80 mg/L。另外,通过CD色谱分析了MaSp1 NT和CT的二级结构,结果表明二者的二级结构均以α-螺旋为主。上述结果为大腹园蛛MaSp1的结构和成丝机理研究奠定了基础。     

大腹园蛛管状腺丝重组蛋白的克隆表达及纺丝

TuSp1蛋白(tubuliform spidroin 1)是管状腺丝(tubuliform silkfiber)的主要组成成分。管状腺丝作为蛛丝卵袋的外层包卵丝,其结构具有很好的耐腐蚀性和良好的力学性能。目前国内外对大腹园蛛TuSp1蛋白的研究很少,仅有一条基因序列的报道。本课题首次构建含大腹园蛛N端非重复结构域、重复单元以及C端非重复结构域的重组管状腺丝蛋白TuSp1 NT-Rp-CT,并经湿法纺丝获得重组蛋白丝纤维。重组蛋白液圆二色谱分析结果显示,pH由7.0降低到5.5的过程中,始终保持稳定的α-螺旋构象;重组蛋白丝纤维的傅里叶变换红外光谱结果显示,丝纤维中主要二级结构为β-折叠及β-转角;经扫描电镜观察发现,冻干的絮状重组蛋白能自组装成丝纤维,且表面光滑纤细;湿纺后的重组蛋白丝纤维直径较粗,但表面较平整均匀,具有类似天然管状腺丝的形态特征,这些为TuSp1蛋白的成丝机理及仿生纺丝研究提供了理论依据。     

大腹圆蛛Fosmid文库的构建及MaSp基因筛选方法的探讨

介绍了构建大腹圆蛛Fosmid基因组文库及牵引丝蛋白(MaSp)基因克隆筛选的全过程.采用改良CTAB法提取大片段基因组DNA,通过自主构建的电洗脱核酸回收装置分离回收30～40kbDNA片段,经补平磷酸化、与pCC2FOS载体连接、体外包装和转染EPI300TM-T1R,首次构建了无偏向性的大腹圆蛛Fosmid文库,其滴度为4.5×105cfu/mL,覆盖基因组倍数为10.以α-32P标记寡核苷酸探针对文库进行初步筛选,获得含MaSp基因的12个阳性克隆.该文库符合Fosmid文库的品质要求,为进一步筛选并研究大腹圆蛛MaSp基因序列奠定了基础.     

大腹园蛛丝腺的氨基酸组成分析

通过解剖大腹园蛛,获得５种丝腺体,并对各丝腺蛋白进行了氨基酸组成分析和蛋白质相对分子质量测定,比较了不同丝腺体蛋白质组成的差异,发现不同丝腺体蛋白质氨基酸组成中Ｇｌｙ和Ｇｌｕ均有较高含量,而Ｓｅｒ的含量并不高,壶状腺中含有较高的Ｐｒｏ,而管状腺中含有较高的Ａｒｇ．同时发现不同丝腺体蛋白质的相对分子质量存在较明显差异,例如集合腺中不同相对分子质量的蛋白质分布较均匀,而在葡萄状腺中大分子质量的蛋白质明显高于小分子质量蛋白质．     

Molecular cloning and expression of the C-terminus of spider flagelliform silk protein from <Emphasis Type="Italic">Araneus ventricosus</Emphasis>

A cDNA coding for the C-terminus of spider flagelliform silk protein (AvFlag) was cloned from Araneus ventricosus. Analysis of the cDNA sequence shows that the C-terminus of AvFlag consists of 167 amino acids of a repetitive region and 87 amino acids of a C-terminal non-repetitive region. The peptide motifs found in spider flagelliform silk proteins, GPGGX and GGX, were conserved in the repetitive region of AvFlag. Phylogenetic analysis further confirmed that AvFlag belongs to the spider flagelliform silk proteins. The AvFlag cDNA was expressed as a 28 kDa polypeptide in baculovirus-infected insect cells. As a new expression approach for spider silk protein, the combination of polyhedrin and AvFlag creates a polyhedrin AvFlag fusion protein (61 kDa) that is produced as recombinant polyhedra; this provides a basis for the source of spider silk proteins for various applications.     

Amplified UvrA protein can ameliorate the ultraviolet sensitivity of an Escherichia coli recA mutant.

When a recA strain of Escherichia coli was transformed with the multicopy plasmid pSF11 carrying the uvrA gene of E. coli, its extreme ultraviolet (UV) sensitivity was decreased. The sensitivity of the lexA1 (Ind(-)) strain to UV was also decreased by pSF11. The recA cells expressing Neurospora crassa UV damage endonuclease (UVDE), encoding UV-endonuclease, show UV resistance. On the other hand, only partial amelioration of UV sensitivity of the recA strain was observed in the presence of the plasmid pNP10 carrying the uvrB gene. Host cell reactivation of UV-irradiated lambda phage in recA cells with pSF11 was as efficient as that in wild-type cells. Using an antibody to detect cyclobutane pyrimidine dimers, we found that UV-irradiated recA cells removed dimers from their DNA more rapidly if they carried pSF11 than if they carried a vacant control plasmid. Using anti-UvrA antibody, we observed that the expression level of UvrA protein was about 20-fold higher in the recA strain with pSF11 than in the recA strain without pSF11. Our results were consistent with the idea that constitutive level of UvrA protein in the recA cells results in constitutive levels of active UvrABC nuclease which is not enough to operate full nucleotide excision repair (NER), thus leading to extreme UV sensitivity.     

登革2型病毒PrM基因的重组甲病毒RNA介导的抗病毒作用的研究

将扩增的登革 2型病毒株PrM基因导入pSFV载体的SP6启动子下游 ,筛选出含该基因正、反向插入的重组质粒DNA。用SpeI酶分别将重组的和辅助的质粒DNA线性化 ,并将其体外转录成 5′末端含帽子结构的RNA。再将这两种RNA共转染BHK细胞。然后将转染的宿主细胞用登革 2型病毒株攻击 ,并分别观察含正、反义PrM基因的重组甲病毒RNA介导的抗病毒效果。通过碱基序列测定 ,筛选出含PrM基因正、反向插入的pSFV PrM重组质粒。并获得了经重组RNA与辅助RNA共转染细胞而产生的重组病毒颗粒。含有反义PrM基因的重组病毒RNA ,在宿主细胞中具有抗登革 2型病毒复制的作用 ,而且强于含正义PrM基因的重组病毒RNA。     

Expression and characterization of chimeric spidroins from flagelliform-aciniform repetitive modules

Spiders can produce up to seven different types of silks or glues with different mechanical properties. Of these, flagelliform (Flag) silk is the most elastic, and aciniform (AcSp1) silk is the toughest. To produce a chimeric spider silk (spidroin) Flag_R-AcSp1_R, we fused one repetitive module of flagelliform silk from Araneus ventricosus and one repetitive module of aciniform silk from Argiope trifasciata. The recombinant protein expressed in E. coli formed silk-like fibers by manual-drawing. CD analysis showed that the secondary structure of Flag_R-AcSp1_R spidroin remained stable during the gradual reduction of pH from 7.0 to 5.5. The spectrum of FTIR indicated that the secondary structure of Flag_R-AcSp1_R changed from α-helix to β-sheet. The conformation change of Flag_R-AcSp1_R was similar to other spidroins in the fiber formation process. SEM analysis revealed that the mean diameter of the fibers was around 1 ~ 2 μm, and the surface was smooth and uniform. The chimeric fibers exhibited superior toughness (~33.1 MJ/m³) and tensile strength (~261.4 MPa). This study provides new insight into design of chimeric spider silks with high mechanical properties.     

Spider flagelliform silk: lessons in protein design, gene structure, and molecular evolution

Spiders spin multiple types of silks that are renowned for their superb mechanical properties. Flagelliform silk, used in the capture spiral of an orb-web, is one of the few silks characterized by both cDNA and genomic DNA data. This fibroin is composed of repeating ensembles of three types of amino acid sequence motifs. The predominant subrepeat, GPGGX, likely forms a beta-turn, and tandem arrays of these turns are thought to create beta-spirals. These spring-like helices may be critical for the exceptional ability of capture silk to stretch and recoil. Each ensemble of motifs was found to correspond to a different exon within the flagelliform gene. The pattern of sequence similarity among exons indicates intragenic concerted evolution. Surprisingly, the introns between the iterated exons are also homogenized with each other. This unusual molecular architecture in the flagelliform silk gene has implications for the evolution and maintenance of spider silk proteins.     

Comparison of the virulence plasmid genomes of two strains of Shigella which lost the ability to bind Congo red

We determined and analyzed the Shigella flexneri serotype 5 (pSF5) and S. dysenteriae serotype 1 (pSD1) virulence plasmid genomes. The total length of pSF5 is 136513 bp, including 165 open reading frames (ORFs). Of these ORFs, 133 were identified and 32 of those had no significant homology to proteins with known functions. The length of pSD1 is 182545 bp, including 224 ORFs, of which we identified 181. The remaining 43 ORFs were not significantly homologous to proteins with known functions. The insertion sequence (IS) elements are 53787 bp in pSF5, and 49616 bp in pSD1, which represents 39.4% and 27.1% of the genome, respectively. There are 22 IS element types in pSF5 and pSD1, among which we report ISEc8 and ISSbo6 for the first time in the Shigella virulence plasmid. Compared to pCP301, there are a large number of deleted genes and gene inversions in both pSF5 and pSD1. The ipa-mxi-spa locus in pSF5 is completely absent, and the genes related to the O-antigen biosynthesis are partially missing. In contrast, the above genes in pSD1 are integral, with the exception of virF. The whole genome analysis of the two plasmids shows that the loss of genes related to gene invasion or regulation also obliterates the ability of pPF5 and pSD1 to bind Congo red (Crb). Whether these genes determine the Crb function requires continued investigation.     

外源丙氨酸提高蜘蛛牵引丝蛋白天然基因在原核系统中的表达

蜘蛛丝蛋白天然基因的体外表达受诸多因素的限制。本研究在获得生长于中国的Nephila clavipes蜘蛛牵引丝蛋白Spidroin2 cDNA（Genbank Accession No. AF441245）的基础上,利用限制性内切酶双酶切反应构建含有Spidroin2 cDNA的重组表达质粒pET-28b(+)-Sp。将该质粒转化至大肠杆菌BL21（DE3）宿主细胞感受态菌中,以不同浓度的IPTG进行诱导,并通过诱导时间、培养温度、加入外源丙氨酸等途径提高Spidroin2 cDNA的表达量,同时利用多克隆抗体对表达产物进行Western blot检测。重组质粒pET-28b(+)-Sp的测序结果表明Spidroin2 cDNA基因以正确的阅读框插入到原核表达载体中;SDS-PAGE结果表明菌体表达蛋白中存在着大小约为31 kDa的目的蛋白带（加入外源丙氨酸条件下）,Western blot检测结果进一步证实,目的基因在大肠杆菌中得到正确表达。本研究证实,蜘蛛牵引丝蛋白Spidroin2 cDNA可在原核细胞内正确表达,外源丙氨酸的加入对于提高天然蜘蛛丝蛋白基因在原核系统的表达作用明显。     

Comparison of the virulence plasmid genomes of two strains of Shigella which lost the ability to bind Congo red

As an enteric pathogen and Gram negative bacte-rium, Shigella possesses high infectivity and leads to serious illness. Since its discovery in 1898 by Shiga, Shigella species have been studied widely. These studies have elucidated the Shigella pathogenicit…