Evidence for the prion hypothesis: induction of the yeast [PSI+] factor by in vitro- converted Sup35 protein

Starting with purified, bacterially produced protein, we have created a [PSI(+)]-inducing agent based on an altered (prion) conformation of the yeast Sup35 protein. After converting Sup35p to its prion conformation in vitro, we introduced it into the cytoplasm of living yeast using a liposome transformation protocol. Introduction of substoichiometric quantities of converted Sup35p greatly increased the rate of appearance of the well-characterized epigenetic factor [PSI+], which results from self-propagating aggregates of cellular Sup35p. Thus, as predicted by the prion hypothesis, proteins can act as infectious agents by causing self-propagating conformational changes.     

Point mutational inactivation of the retinoblastoma antioncogene

The retinoblastoma (Rb) antioncogene encodes a nuclear phosphoprotein, p105-Rb, that forms protein complexes with the adenovirus E1A and SV40 large T oncoproteins. A novel, aberrant Rb protein detected in J82 bladder carcinoma cells was not able to form a complex with E1A and was less stable than p105-Rb. By means of a rapid method for the detection of mutations in Rb mRNA, this defective Rb protein was observed to result from a single point mutation within a splice acceptor sequence in J82 genomic DNA. This mutation eliminates a single exon and 35 amino acids from its encoded protein product.     

Cytomegalovirus recruitment of cellular kinases to dissolve the nuclear lamina

The passage of large-sized herpesviral capsids through the nuclear lamina and the inner nuclear membrane to leave the nucleus requires a dissolution of the nuclear lamina. Here, we report on the functions of M50/p35, a beta-herpesviral protein of murine cytomegalovirus. M50/p35 inserts into the inner nuclear membrane and is aggregated by a second viral protein, M53/p38, to form the capsid docking site. M50/p35 recruits the cellular protein kinase C for phosphorylation and dissolution of the nuclear lamina, suggesting that herpesviruses target a critical element of nuclear architecture.     

甘蔗花叶病毒外壳蛋白基因的克隆及系列表达载体的构建

采用RT-PCR法,从表现玉米矮花叶病症状的玉米叶片中克隆了外壳蛋白(Coat protein)基因。测序和同源性比较结果表明:所克隆的CP基因来自甘蔗花叶病毒(Sugarcane mosaic virus,SCMV)MDB株系,编码219个氨基酸。以CP基因为目的基因,构建了用于基因枪转化的p35SCPfBar,p35SCPrBar,pUbiCPfBar,pU-biCPrBar 4个表达载体和用于农杆菌转化的pCAMBIACPf,pCMABIACPr 2个表达载体。     

Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain

A cytoplasmic protein that greatly enhances the guanosine triphosphatase (GTPase) activity of N-ras protein but does not affect the activity of oncogenic ras mutants has been recently described. This protein (GAP) is shown here to be ubiquitous in higher eukaryotes and to interact with H-ras as well as with N-ras proteins. To identify the region of ras p21 with which GAP interacts, 21 H-ras mutant proteins were purified and tested for their ability to undergo stimulation of GTPase activity by GAP. Mutations in nonessential regions of H-ras p21 as well as mutations in its carboxyl-terminal domain (residues 165-185) and purine binding region (residues 117 and 119) did not decrease the ability of the protein to respond to GAP. In addition, an antibody against the carboxyl-terminal domain did not block GAP activity, supporting the conclusion that GAP does not interact with this region. Transforming mutations at positions 12, 59, and 61 (the phosphoryl binding region) abolished GTPase stimulation by GAP. Point mutations in the putative effector region of ras p21 (amino acids 35, 36, and 38) were also insensitive to GAP. However, a point mutation at position 39, shown previously not to impair effector function, did not alter GAP-p21 interaction. These results indicate that GAP interaction may be essential for ras p21 biological activity and that it may be a ras effector protein.     

H9亚型禽流感病毒血凝素蛋白在原核系统中的表达

应用带有His6尾的pET原核表达系统对禽流感病毒血凝素蛋白进行表达,将禽流感A/Turkey/Wisconsin/66(HgN2)的HA基因克隆至原核表达载体pET-30a中,经酶切、PCR扩增和测序分析确证其插入,并且阅读框正确,获得重组质粒pET30a-H9.将此重组质粒转化到宿主菌BL21中,用诱导剂异丙基硫代...     

锯缘青蟹呼肠孤病毒p40蛋白的体外表达与单克隆抗体制备

锯缘青蟹呼肠孤病毒(Scylla serrata reovirus,SsRV)第9节段(S9)编码的p40蛋白可能为病毒的甲基转移酶(methyltransferase)。为了便于确认p40在病毒复制过程中扮演的角色,将S9片段的开放阅读框(ORF)克隆到原核表达载体pET28a(+),实现了在宿主表达菌E.coli BL21(DE3)中的高效表达,进而纯化了重组表达蛋白p40(rp40),并制备了抗SsRV p40蛋白的2株单克隆抗体(4B7、3E5)。经Western blot分析表明,2株单克隆抗体均可特异地识别rp40蛋白,以及识别SsRV病毒蛋白中分子量为46 ku和40 ku 2条蛋白条带。实验结果不但确认SsRV p40为SsRV的结构蛋白,也提示p40和p46蛋白可能具有相同的抗原表位。为该蛋白的后续功能研究奠定了基础。     

A 32-kD GTP-binding protein associated with the CD4-p56lck and CD8-p56lck T cell receptor complexes

The guanosine triphosphate (GTP)-binding proteins include signal-transducing heterotrimeric G proteins (for example, Gs, Gi), smaller GTP-binding proteins that function in protein sorting, and the oncogenic protein p21ras. The T cell receptor complexes CD4-p56lck and CD8-p56lck were found to include a 32- to 33-kilodalton phosphoprotein (p32) that was recognized by an antiserum to a consensus GTP-binding region in G proteins. Immunoprecipitated CD4 and CD8 complexes bound GTP and hydrolyzed it to guanosine diphosphate (GDP). The p32 protein was covalently linked to [alpha-32P]GTP by ultraviolet photoaffinity labeling. These results demonstrate an interaction between T cell receptor complexes and an intracellular GTP-binding protein.     

Nucleotide sequence of the Rasheed rat sarcoma virus oncogene: new mutations

The nucleotide sequence of the oncogene of the Rasheed strain of rat sarcoma virus was determined. The oncogene (Ra-v-ras) encodes a 29,000-dalton (p29) transforming protein. This protein is distinct from the immunologically related 21,000-dalton protein (p21) of the Harvey murine sarcoma virus in its amino terminus and in having additional mutations in its carboxyl terminus. Although the functional significance of these changes is unknown, they appear to occur only in rat sarcoma virus.     

大麦双受精过程研究初报

通过光学显微观察,大麦双受精过程如下:授粉后35分钟,花粉管进入胚囊;45分钟释放出两个精子;50分钟,一精子先与卵细胞融合,另一精子移向中央细胞的二极核;55分钟,精子附卵核上,另一精子附于一极核上,同时还观察到一精子附于两极核上的现象;60分钟,精子仍贴附在卵核上,另一精子与二极核之一融合;90分钟,精子开始与卵核融合,初生胚乳核形成,其核中的三个核仁未融合;120分钟,精子与卵核融合,初生胚乳核分裂,但未见核仁融合。     

非洲猪瘟病毒p30蛋白单克隆抗体制备及线性抗原表位定位

【目的】制备非洲猪瘟病毒（African swine fever virus,ASFV）p30蛋白的单克隆抗体（monoclonal antibodies,MAbs）并初步分析其所识别的线性抗原表位,为ASFV及其抗体检测方法的建立及p30蛋白结构和功能的研究奠定基础。【方法】将原核表达并纯化的p30重组蛋白作为免疫原,免疫6—8周龄BALB/c雌鼠,每两周免疫1次,共免疫3次,首次免疫是抗原与等体积的弗氏完全佐剂乳化后免疫,第二次和第三次免疫与等体积的弗氏不完全佐剂乳化,3次免疫后1 w断尾采血,间接酶联免疫吸附试验（ELISA）检测血清抗体效价,选择血清效价最高的小鼠进行加强免疫,3 d后取小鼠脾淋巴细胞与SP2/0骨髓瘤细胞按照4﹕1的比例使用PEG进行常规细胞融合。利用重组p30蛋白作为包被抗原,间接ELISA筛选阳性杂交瘤细胞,有限稀释法进行克隆纯化,直至筛出能够稳定分泌抗体的MAbs。将ASFV接种于猪肺泡巨噬细胞,以筛选的MAbs为一抗、兔抗鼠HRP-IgG为二抗,进行间接免疫荧光试验（IFA）。将感染和未感染ASFV的细胞沉淀处理后进行 SDS-PAGE并转印至硝酸纤维素膜,分别以IFA鉴定为阳性的MAbs上清为一抗、兔抗鼠HRP-IgG为二抗,进行Western blotting分析,筛选获得p30 MAbs。根据已知序列设计引物扩增p30ab与p30bc两段截短基因,其中p30ab代表由第86—153位氨基酸残基的截短体,p30bc代表由第120—187位氨基酸残基的截短体,原核表达部分重叠的截短p30蛋白,最终获得重组蛋白GST-p30ab与重组蛋白GST-p30bc。分别以GST-p30ab和GST-p30bc融合蛋白为包被抗原,以5株MAbs为一抗,以兔抗鼠HRP-IgG为二抗, 通过间接ELISA方法初步定位p30蛋白的抗原表位。【结果】以纯化的重组蛋白为包被抗原,经间接ELISA试验筛选出25株可分泌抗重组 p30蛋白的杂交瘤细胞株。IFA结果显示,5株MAbs（8F4、1D3、1H2、6C3和8E11）与ASFV感染的猪肺泡巨噬细胞IFA 试验呈阳性;Western blotting结果显示,5株MAbs均能够与ASFV感染的细胞呈阳性反应,与未感染病毒的细胞呈阴性反应。试验构建的p30截短体重组蛋白GST-p30ab以可溶和包涵体两种形式表达,而GST-p30bc仅以包涵体形式表达,以两组截短体融合蛋白为包被抗原,通过间接ELISA检测出MAbs 8F4、1H2和6C3与两个重组蛋白均能有效结合,证明MAbs 8F4、1H2和6C3抗原识别区域为两组截短蛋白重叠区域,即第120—153位氨基酸;MAbs 8E11与1D3则只能与GST-p30ab蛋白结合, 证明MAbs 8E11与1D3抗原识别区域为两个重组蛋白的非重叠区域,即第86—119位氨基酸。【结论】本研究可溶性地表达了p30蛋白的第86—153位氨基酸截短体重组蛋白,制备了5株p30 MAbs,定位到2个p30蛋白抗原表位。结合ELISA和IFA,可建立十分可靠的ASFV及其抗体的检测手段。     

大肠杆菌不耐热肠毒素B亚单位基因的原核表达

大肠杆菌(Escherichia coli)不耐热肠毒素B亚单位(LTB)不仅是重要的黏膜免疫原,而且作为佐剂在黏膜免疫中起着重要的作用.试验采用PCR方法扩增了肠毒素性大肠杆菌的LTB基因,并将其克隆到pGEX-KG原核表达栽体上,构建了重组质粒pKG-LTB,经酶切和测序鉴定后,转化到大肠杆菌BL21(DE3)进行IPTG诱导表达,经SDS-PAGE和Western-blot分析表明,克隆的LTB基因与谷胱甘肽转移酶(GST)基因获得了高效融合表达,表达的融合蛋白GST-LTB分子量约为35kDa,并具有免疫反应活性.这为进一步研究LTB蛋白黏膜免疫原性和黏膜佐剂的作用奠定了基础.     

鸡p15INK4B基因的克隆表达及纯化

为研究鸡p15INK4B蛋白的功能及为制备此蛋白的单克隆抗体提供材料,应用引物搭桥法合成p15INK4B基因,应用基因工程的手段构建p15INK4B基因的原核表达载体,在E.coliRosetta(DE3)菌进行融合表达,并对表达产物进行亲和层析纯化。结果成功地构建了p15INK4B基因原核表达载体,经亲和层析制备了较高纯度的目的表达产物。     

Biochemical basis of oxidative protein folding in the endoplasmic reticulum

The endoplasmic reticulum (ER) supports disulfide bond formation by a poorly understood mechanism requiring protein disulfide isomerase (PDI) and ERO1. In yeast, Ero1p-mediated oxidative folding was shown to depend on cellular flavin adenine dinucleotide (FAD) levels but not on ubiquinone or heme, and Ero1p was shown to be a FAD-binding protein. We reconstituted efficient oxidative folding in vitro using FAD, PDI, and Ero1p. Disulfide formation proceeded by direct delivery of oxidizing equivalents from Ero1p to folding substrates via PDI. This kinetic shuttling of oxidizing equivalents could allow the ER to support rapid disulfide formation while maintaining the ability to reduce and rearrange incorrect disulfide bonds.     

PCV-2感染对PK-15细胞IL mRNA转录水平的影响

【目的】观察猪圆环病毒2型（PCV-2）感染对猪肾传代细胞（PK-15细胞）炎性细胞因子白细胞介素（IL） mRNA转录水平的影响,探讨宿主与病毒之间的作用关系及细胞炎性反应机制。【方法】以未感染PCV-2的PK-15细胞为对照组,运用相对定量PCR技术,测定和分析PCV2感染PK15细胞后,PCV-2 DNA相对含量的变化,以及炎性细胞因子IL-6、IL-8、IL-12p35、IL-12p40、IL-13、IL-17、IL-18 mRNA转录水平在1,6,12,24,48,和72 h的变化。【结果】PCV-2感染后,PK-15细胞的IL-6、IL-13、IL-17、IL-18 的mRNA转录水平在12 h显著增加,24 h后mRNA转录水平下降;IL-8的mRNA转录水平在48 h时最高,为对照组的2.5倍,但72 h时恢复至与对照组水平相当;随着感染时间的延长,IL-12p35、IL-12p40 的mRNA转录水平显著下降。【结论】PCV-2感染后可引起PK-15细胞中IL-6、IL-8、IL-13、IL-17、IL-18等细胞因子mRNA转录水平增加,而IL-12 mRNA转录水平下降,提示PCV-2感染后引起的PK-15细胞炎性反应与其分泌的炎性细胞因子的改变有关。     

Isolation and characterization of a novel protein (X-ORF product) from SIV and HIV-2

A protein designated p14 was purified from a simian immunodeficiency virus (SIVMne) and was shown by amino acid sequence analysis to be nearly identical to the predicted translational product of a unique open reading frame (X-ORF) in the nucleotide sequences of SIVmac and human immunodeficiency virus type 2 (HIV-2). Thus the X-ORF is proven to be a new retroviral gene. The p14 is present in SIVMne in molar amounts equivalent to those of the gag proteins. This is the first example of a retrovirus that contains a substantial quantity of a viral protein that is not a product of the gag, pro, pol, or env genes. SIV p14 and its homolog in HIV-2 may function as nucleic acid binding proteins since purified p14 binds to single-stranded nucleic acids in vitro. Antisera to the purified protein detected p14 in SIVMne, SIVmac, and a homologous protein (16 kilodaltons) in HIV-2 but did not react with HIV-1. Diagnostic procedures based on this novel protein will distinguish between HIV-1 and HIV-2.     

水稻启动子OsN1p的克隆与功能分析

将OsN1基因上游881 bp启动子(OsN1p)序列取代pBI121中gus基因上游的35S启动子,构建植物表达载体pBIN1p,经农杆菌介导转化水稻品种‘日本晴’,获得转基因植株。GUS组织化学染色结果表明,由该启动子驱动的gus基因能在愈伤组织中较低水平地表达;稻瘟病菌接种转基因植株24 h后,GUS活性为未接种前的4.2倍;5 mmol.L-1水杨酸(SA)和0.5 mmol.L-1茉莉酸甲酯(MeJA)分别喷施转基因植株叶面6 h后,GUS活性分别为处理前的5.9和2.4倍。表明,OsN1p启动子具有启动活性,同时明显具有受稻瘟病菌、SA和MeJA诱导表达的特性。     

Transverse gradient electrophoresis: protein homogeneity test and subfractionation technique

Electrophoresis of protein (including enzyme) was conducted in a gel medium across which, transverse to the direction of protein migration, a continuous (p)H gradient extends. Any splitting of a resultant trace for the change of protein mobility with (p)H suggests both protein heterogeneity and the (p)H conditions under which further purification and subfractionation may best be pursued.     

A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants

The role of guanine nucleotides in ras p21 function was determined by using the ability of p21 protein to induce maturation of Xenopus oocytes as a quantitative assay for biological activity. Two oncogenic mutant human N-ras p21 proteins, Asp12 and Val12, actively induced maturation, whereas normal Gly12 p21 was relatively inactive in this assay. Both mutant proteins were found to be associated with guanosine triphosphate (GTP) in vivo. In contrast, Gly12 p21 was predominantly guanosine diphosphate (GDP)-bound because of a dramatic stimulation of Gly12 p21-associated guanosine triphosphatase (GTPase) activity. A cytoplasmic protein was shown to be responsible for this increase in activity. This protein stimulated GTP hydrolysis by purified Gly12 p21 more than 200-fold in vitro, but had no effect on Asp12 or Val12 mutants. A similar factor could be detected in extracts from mammalian cells. It thus appears that, in Xenopus oocytes, this protein maintains normal p21 in a biologically inactive, GDP-bound state through its effect on GTPase activity. Furthermore, it appears that the major effect of position 12 mutations is to prevent this protein from stimulating p21 GTPase activity, thereby allowing these mutants to remain in the active GTP-bound state.     

Control of the interferon-induced 68-kilodalton protein kinase by the HIV-1 tat gene product

The tat-responsive region (TAR) of the human immunodeficiency virus-1 (HIV-1) exhibits a trans-inhibitory effect on translation in vitro by activating the interferon-induced 68-kilodalton protein kinase (p68 kinase). Productive infection by HIV-1 was shown to result in a significant decrease in the amount of cellular p68 kinase. The steady-state amount of p68 kinase was also reduced in interferon-treated HeLa cell lines stably expressing tat, as compared to the amount of the kinase in interferon-treated control HeLa cells. Thus, the potential translational inhibitory effects of the TAR RNA region mediated by activation of p68 kinase may be downregulated by tat during productive HIV-1 infection.