苯丙氨酸羟化酶基因G247S、E280G、P362T和A434D突变的体外表达研究和结构分析

目的 探讨苯丙氨酸羟化酶基因4种新错义突变G247S、E280G、P362T和A434D的致病性质以及与临床表型之间的关系.方法 (1)应用体外真核表达体系进行突变体蛋白的瞬时表达;以野生型苯丙氨酸羟化酶(phenylalanine hydroxylase,PAH)的酶活性作为参照,计算突变体的残余酶活性.(2)比对不同种属的PAH酶蛋白序列,分析这些突变位点的氨基酸的保守性.(3)以正常PAH蛋白的晶体结构为基础,应用生物大分子结构模拟软件进行突变位点的三维结构分析,预测氨基酸的替换对酶功能的潜在影响.(4)根据治疗前血苯丙氨酸浓度和饮食苯丙氨酸的耐受量,进行苯丙酮尿症(phenylketonuria,PKU)患儿的临床表型分析.结果 (1)突变体G247S、E280G、P362T和A434D体外表达的残余酶活性分别为野生型的3.1%、0.4%、8.2%和21.7%.(2)Gly 247、Glu 280、Pro362均属于高度保守的氨基酸残基位点,而Ala434则属于比较保守的位点.(3)三维分子结构分析显示G247S和E280G位于酶的活性中心位置,可能分别影响PAH酶与四氢生物蝶呤和铁离子的结合;而突变P362T和A434D则可能分别影响PAH酶二聚体和四聚体化的形成和稳定性.(4)临床表型分析显示携带G247S和E280G突变的患者为经典型PKU,携带P362T的患者既有经典型PKU也有中间型PKU,携带A434D患者均为中间型PKU.结论 (1)E280G、G247S、P362T和A434D突变均为致病性的突变,位于酶活性中心位点的突变影响酶的活性功能更为明显.(2)突变酶分子的结构分析以及对酶功能影响的预测与体外表达的实验结果基本一致.(3)突变基因型、体外表达活性实验以及临床表型之间的关联分析存在着一定的关联性.     

线粒体12S rRNA基因突变与2型糖尿病

目的 观察线粒体12S rRNA 1310、1438及1442位点在中国汉族2型糖尿病患者群体中的突变情况,同时筛查该区域与2型糖尿病发生有关的突变。方法 采用PDR－SSCP及PCR产物直接测序等技术对86例2型糖尿病患者及70名正常对照个体的血细胞线粒体DNA进行突变分析。结果 发现1例患者线粒体DNA 12S rRNA基因1438位点存在G→A的点突变,另1例存在1442位点G→A的点突变,所有对照个体均未发现该两位点的突变。未发现线粒体基因12S rRNA 1310位点C→T点突变。结论 1438位点G→A、1442位点G→A的突变可能与2型糖尿病的发生相关,该两位点突变的具体意义如何尚需进一步研究。1310位点C→T在血细胞中的突变率可能较低,进一步说明2型糖尿病的发生在线粒体遗传上具有一定的异质性。     

HLA-Gα1结构域Met76和Gln79在KIR2DL4特异性识别中的作用研究

目的 探讨HLA-G α1结构域中特异的Met76和Gln79两个位点在HLA-G特异性受体KIR2DL4识别中的作用.方法 采用真核表达载体CD51neg1,克隆表达KIR2DL4胞外区与IgGFc段的可溶性融合蛋白;利用“桥式”PCR和“点突变”方法,将位于HLA-G目的基因α1结构域中编码Met76和Gln79的密码子突变为Ala76,79（HLA-mG）,通过逆转录病毒表达载体分别使野生型HLA-G及其突变体在HLAⅠ分子阴性的K562细胞上表达.流式细胞术分别测定KIR2DL4-IgG Fc融合蛋白与野生型HLA-G及Met76、Gln79→Ala76,79 HLA-G突变体结合的荧光强度,通过比较两者的平均荧光强度分析HLA-G分子Met76、Gln79残基在HLA-G与其受体KIR2DL4识别过程中的作用.结果 Western blot结果显示,本实验成功表达KIR2DL4-IgG Fc融合蛋白.FACS结果表明,野生型HLA-G及Met76、Gln79→Ala76,79 HLA-G突变体在K562细胞上高表达.Met76、Gln79突变为Ala76,79后能显著影响KIR2DL4对HLA-G分子的识别.结论 HLA-Gα1结构域中Met76和Gln79可能是其特异性受体KIR2DL4识别的关键位点.     

线粒体信号肽引导葡萄糖调节蛋白75-增强型绿色荧光蛋白(GRP75-EGFP)融合蛋白定位于线粒体

目的构建葡萄糖调节蛋白75(GRP75)不同结构域、不同形式突变体与线粒体信号肽重组蛋白并鉴定其亚细胞定位。方法通过重叠延伸PCR,将线粒体靶向信号肽序列分别与GRP75不同结构域基因拼接。利用定点突变PCR分别扩增实现62和65位氨基酸突变的磷酸化失活型、磷酸化激活型突变体、482位氨基酸突变的底物结合缺陷型突变体及三位点同时突变重组体。将上述重组基因克隆入p EGFPC1真核表达质粒,酶切和测序验证后分别用脂质体转染He La细胞,Western blot法检测重组蛋白表达水平,激光共聚焦显微镜观察比较重组蛋白亚细胞定位情况。结果成功构建了线粒体信号肽融合或缺失的GRP75结构域、磷酸化失活和激活突变体、底物结合缺陷突变体真核表达质粒。各重组质粒的片段插入、位点突变、信号肽融合情况均符合设计目的,在He La细胞表达后产物的相对分子质量大小均符合预期。EGFP蛋白C端插入信号肽能引导下游融合的GRP75全长蛋白、结构域片段、突变体蛋白表达定位于He La细胞线粒体中,而缺失信号肽的对应重组蛋白则主要分布于胞质和局部核周。结论构建了一系列GRP75重组真核表达质粒,EGFP融合信号肽能引导重组蛋白在线粒体中表达定位。     

人可溶性APRIL突变体的原核表达与纯化

目的 制备人可溶性增殖诱导配体(soluble a proliferation inducing ligand,sAPRIL)的两种突变体,为寻找APRIL 的竞争抑制剂创造条件.方法 采用一步反向PCR法,以人APRIL第186位甘氨酸(186G)和187位谷氨酰胺(187Q)残基为突变位点,构建如下两个突变体DNA,即sAPRIL突变体-1(mutant sAPRIL-1,msAPRIL-1)DNA(186G被赖氨酸残基K置换,187Q缺失)和msAPRIL-2 DNA(186G187Q置换为186K187G);测序后将两突变体DNA分别亚克隆于原核表达载体pQE-80L,继而在大肠杆菌DH5α中表达相应突变体蛋白,经SDS-PAGE和Westernblot鉴定表达产物,经Ni2 -NTA和Sephadex G-75柱层析纯化相应突变体蛋白.结果 一步反向PCR结合DNA测序,得到了与上述设计一致的两个sAPRIL突变体DNA.将两突变体DNA分别亚克隆于pQE-80L后,在大肠杆菌DH5α中成功表达了相应突变体蛋白.经Ni2 -NTA柱层析成功地纯化了两突变体蛋白,经Sephadex G-75柱层析成功获得了两突变体蛋白的三聚体分子.结论 成功制备了sAPRIL的两种突变体蛋白,为寻找基于sAPRIL突变体的抗肿瘤分子奠定了基础.     

碳青霉烯类抗生素诱导耐药铜绿假单胞菌外膜蛋白D2表达与编码基因多点突变

目的 探讨对碳青霉烯类抗生素耐药的铜绿假单胞菌编码外膜蛋白D2(OprD2)基因变异与OprD2表达的关系.方法 由临床分离敏感铜绿假单胞菌,使用不同浓度亚胺培南与美罗培南含药琼脂平板法筛选耐药菌株.应用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、凝胶成像系统分析OprD2.应用聚合酶链反应及测序分析OprD2基因编码区.结果 人工诱导能产生出与临床分离株一样的对碳青霉烯类抗生素耐药的菌株.在SDS-PAGE图谱上亚胺培南与美罗培南耐药株与同源敏感株相比均有OprD2的减少.测序结果显示在多株耐药菌中,OprD2基因编码区3个基因位点同时出现碱基突变:在+84位点产生突变,胞嘧啶突变为胸腺嘧啶(C→T);在+401位点突变(C→A),造成第134位氨基酸苏氨酸突变为天冬氨酸(Thr→Asp);在+959位点突变(A→G),造成第320位氨基酸赖氨酸突变为精氨酸(Lys→Arg).结论 同时出现3个或更多的碱基点突变可能导致铜绿假单胞萧OprD2缺失或表达减少,可能是对碳青霉烯类抗生素产生耐药性的原因.     

人IL-6及其受体拮抗剂的研究进展

人 IL- 6及其受体拮抗剂的研究主要集中于两个方面 :单克隆抗体和突变体。针对人 IL- 6和人 IL- 6 R的活性区域构建的单克隆抗体对于临床治疗多发性骨髓瘤显示出了很好的短期疗效 ,根据与生长激素 (GH)及其受体复合物 GH/ (GHbp) 2的结构对比 ,推测人 IL- 6和 IL- 6 Rα的活性位点 ,结合定点突变技术 ,设计 IL- 6突变体、IL- 6 R突变体和 IL- 6突变体 - IL- 6 Rα融合蛋白 ,它们对天然 h IL- 6的生物活性显示出了明显的拮抗作用     

应用白喉毒素-IL-2融合蛋白研究Q126D突变对IL-2功能的影响

IL 2是一非常重要的由 133个氨基酸组成的TH1细胞因子 ,从N端到C端依次为上、上后再下、下的 4个螺旋结构。位于C端的最后一个螺旋结构在其与相应受体的 β、γ亚基的结合中起重要作用。本文应用位点定向突变技术(Site directedmutagenesis)改造了位于第 4个螺旋结构中的第 12 6个氨基酸 ,使其由谷氨酰胺突变为天冬氨酸 ,并分别与白喉毒素 (DT)及其突变体组成融合蛋白 ,并以DT作指示系统 ,观察了这种改变对IL 2与受体结合及其功能的影响。首先将CRM 197(白喉毒素G5 2E突变株 )克隆入PSM30 8的EcoRⅠ与HindⅢ位点间 ,设计 1对分别…     

人乳头瘤病毒2（HPV2）E2蛋白不同功能区突变对转录抑制作用影响的研究

目的 研究HPV2变异株上E2蛋白不同功能区域的点突变对其转录作用的影响.方法 根据HPV2突变株E2上的突变点设计引物,采用延伸法构建不同的E2突变体,并插入到真核表达质粒pcDNA3.1中.表达不同突变体E2的pcDNA3.1-E2与带有HPV原毒株LCR的CAT报道基因载体进行共转染Hela细胞.通过检测CAT的表达量对不同E2突变体的转录抑制作用进行评估.结果 与全长的原毒株E2相比较,去除N端及C端功能区的E2蛋白均降低了E2蛋白对启动子活性的抑制作用.位于E2蛋白转录激活区（nt 3037）,铰链区（nt 3387）及DNA结合区（nt 3697）的点突变明显影响了其转录抑制功能.结论 HPV2 E2蛋白的转录调节功能与其转录激活区以及DNA结合区密切相关.HPV2突变株上的不同的E2的单个突变点能够明显降低E2蛋白对启动子活性的抑制作用.     

Plk1330/597位丝氨酸磷酸化突变体对胞质分裂的影响

目的:研究Plk的330/597位丝氨酸的模拟磷酸化突变体Plk1S330/597D和不能磷酸化突变体Plk1S330/597A的绿色荧光融合蛋白在HeLa细胞中的定位以及对有丝分裂的影响。方法:将野生型Plk1-GFP质粒、模拟磷酸化型突变体Plk1S330/597D-GFP质粒和不能磷酸化突变体Plk1S330/597A-GFP质粒分别转染HeLa细胞株,免疫荧光法检测突变体融合蛋白Plk1S330/597D-GFP和Plk1S330/597A-GFP的细胞定位的定位,West-ern blot法检测上述突变体融合蛋白的表达。通过有丝分裂期的细胞计数,流式细胞仪检测细胞周期,观察转染突变体后HeLa细胞的有丝分裂进程。结果:模拟磷酸化突变体Plk1S330/597D和不能磷酸化突变体Plk1S330/597A的融合蛋白能正确表达。突变体在细胞有丝分裂过程中均能正确定位于动点和中体,但不能磷酸化突变体Plk1S330/597A融合蛋白对HeLa细胞的有丝分裂有明显的抑制作用,使胞质分裂期的细胞数量较对照组明显增多,产生G2/M期阻滞(P<0.01),并造成染色体分离滞后的现象。结论:Plk1激酶自身的磷酸化状态对其有丝分裂期功能具有重要作用,其330和597位丝氨酸去磷酸化能抑制细胞的有丝分裂,使细胞周期发生G2/M期阻滞。     

Studies with GFP-Vpr fusion proteins: induction of apoptosis but ablation of cell-cycle arrest despite nuclear membrane or nuclear localization

The human immunodeficiency virus type 1 (HIV-1) Vpr protein is known to arrest the cell cycle in G(2)/M and induce apoptosis following arrest. The functions of Vpr relative to its location in the cell remain unresolved. We now demonstrate that the location and function of Vpr are dependent on the makeup of fusion proteins and that the functions of G(2)/M arrest and apoptosis are separable. Using green fluorescence protein mutants (EGFP or EYFP), we found that fusion at either the N- or C-terminus compromised the ability of Vpr to arrest cell cycling, relative to that of His-Vpr or wild-type protein. Additionally, utilizing the ability to specifically identify cells expressing the fusion proteins, we confirm that Vpr can induce apoptosis, but appears to be independent of cell-cycle arrest in G(2)/M. Both N- and C-terminal Vpr/EYFP fusion proteins induced apoptosis but caused minimal G(2)/M arrest. These studies with Vpr fusion proteins indicate that the functions of Vpr leading to G(2)/M arrest and apoptosis are separable and that fusion of Vpr to EGFP or EYFP affected the localization of the protein. Our findings suggest that nuclear membrane localization and nuclear import and export are strongly governed by modification of the N-terminus of Vpr.     

Surface density of the Hendra G protein modulates Hendra F protein-promoted membrane fusion: role for Hendra G protein trafficking and degradation

Hendra virus, like most paramyxoviruses, requires both a fusion (F) and attachment (G) protein for promotion of cell-cell fusion. Recent studies determined that Hendra F is proteolytically processed by the cellular protease cathepsin L after endocytosis. This unique cathepsin L processing results in a small percentage of Hendra F on the cell surface. To determine how the surface densities of the two Hendra glycoproteins affect fusion promotion, we performed experiments that varied the levels of glycoproteins expressed in transfected cells. Using two different fusion assays, we found a marked increase in fusion when expression of the Hendra G protein was increased, with a 1:1 molar ratio of Hendra F:G on the cell surface resulting in optimal membrane fusion. Our results also showed that Hendra G protein levels are modulated by both more rapid protein turnover and slower protein trafficking than is seen for Hendra F.     

RNA干扰MAD2基因对细胞增殖的影响

目的:探讨MAD2对HepG2细胞周期的影响。方法:构建针对MAD2基因的shRNA表达载体,MAD2基因与GFP融合的绿色荧光蛋白真核表达质粒pmad2-EGFP共转染HepG2细胞株,用Western印迹法检测shRNA的抑制效应;用MTT法测定细胞增殖率;用流式细胞仪检测细胞周期。结果:shRNA的重组质粒表达载体能明显抑制MAD2绿色荧光融合蛋白的表达。HepG2细胞转染有效干扰质粒48h后,细胞增殖抑制率达65%。有效干扰质粒引起G0/G1期和S期细胞的明显降低,G2/M期细胞增多。结论:在细胞水平,可通过RNA干扰特异性抑制MAD2基因来抑制细胞的增殖,为研究MAD2基因在细胞增殖中的作用机制奠定基础。     

Activation of GPCRs modulates quantal size in chromaffin cells through G(betagamma) and PKC

Exocytosis proceeds by either full fusion or 'kiss-and-run' between vesicle and plasma membrane. Switching between these two modes permits the cell to regulate the kinetics and amount of secretion. Here we show that ATP receptor activation reduces secretion downstream from cytosolic Ca2+ elevation in rat adrenal chromaffin cells. This reduction is mediated by activation of a pertussis toxin-sensitive G(i/o) protein, leading to activation of G(betagamma) subunits, which promote the 'kiss-and-run' mode by reducing the total open time of the fusion pore during a vesicle fusion event. Furthermore, parallel activation of the muscarinic acetylcholine receptor removes the inhibitory effects of ATP on secretion. This is mediated by a G(q) pathway through protein kinase C activation. The inhibitory effects of ATP and its reversal by protein kinase C activation are also shared by opioids and somatostatin. Thus, a variety of G protein pathways exist to modulate Ca2+-evoked secretion at specific steps in fusion pore formation.     

Evaluation of Glu11 and Gly8 of the H5N1 influenza hemagglutinin fusion peptide in membrane fusion using pseudotype virus and reverse genetics

Summary Influenza viruses gain entry into host cells by binding to cellular receptors and promoting the fusion of the viral envelope with the host cell membrane. The fusion peptide of influenza hemagglutinin (HA) is crucial for fusion. To examine the structural and functional roles of amino acids E11 and G8 of the H5 HA fusion peptide, a series of fusion mutants was generated. We determined the effect of each mutation on fusion activity and infection of rescued recombinant virus by polykaryon formation, cell–cell fusion assay, HA pseudovirus transduction and reverse genetics. Our findings indicate that E11V and E11A mutants dramatically inhibit fusion and that at position 11 a polar residue such as glutamic acid or serine may be desirable for preserving the fusion activity. More interestingly, one mutation (G8E) raised the threshold pH of polykaryon formation. Our results suggest that G8 as well as E11 play an important functional and structural role in membrane fusion and that the polarity of E11 is crucial for fusion activity. Finally, we developed an assay based on a reporter gene plus pseudotyped virus that could sensitively detect fusion activity. Correspondence: Po Tien, Center for Molecular Virology, Chinese Academy of Sciences, Institute of Microbiology, Beijing 100080, P.R. China     

Transfer of antibiotic resistance genes between yeast and mammalian cells under conditions favoring cell fusion

Antibiotic resistance to G418 has been transferred into Chinese hamster cell lines via a plasmid vector. The same plasmid, which also contained the Leu2 gene, has been used to transform Leu2^– yeast (strain MCI6) to leucine prototrophy. Subsequent fusion between transformed yeast and untransformed hamster cells demonstrated that plasmid DNA could be transferred and its genes expressed within the mammalian cell genome. The fusion of transformed hamster cells with untransformed MC16 yeast cells demonstrated that DNA integrated within the mammalian cell genome could be transferred to correct the Leu2 deficiency and also confer G418 resistance on some yeast colonies.     

Identification of epitopes associated with different biological activities on the glycoprotein of vesicular stomatitis virus by use of monoclonal antibodies

Summary Thirteen monoclonal antibodies (MAbs) to the glycoprotein (G) of vesicular stomatitis virus (VSV) serotype Indiana were prepared and examined for their effects on various biological activities of VSV, including in vitro infection, hemagglutination, adsorption to cells, and mediation of cell fusion. Competitive binding assays with these MAbs revealed the presence of at least seven distinct antigenic determinants (epitopes) on the G protein. In some cases, overlappings among epitopes to various degrees were observed as partial inhibition or binding enhancement. The MAbs to all the epitopes but one (epitopes 1–6) reacted with the denatured G protein in a Western immunoblot analysis. Four of the epitopes (epitopes 2, 4, 5, and 7) were involved in neutralization and two (epitopes 1 and 2) in hemagglutination inhibition. None of the MAbs inhibited the adsorption of radiolabeled VSV to BHK-21 cells; the MAbs to epitope 2 slightly enhanced the virus adsorption. All neutralization epitopes except epitope 2 (epitopes 4, 5, and 7) were associated with inhibition of VSV-mediated cell fusion. These results show a direct spatial relationship between the epitopes recognized by the MAbs and functional sites on G protein and further insights into the structure and function of G protein.     

HIV-1 Vpr基因在肿瘤细胞中诱导G2期停滞、细胞致死效应及其核定位功能研究

目的 通过研究ＨＩＶ－１ Ｖｐｒ基因在宫颈癌细胞ＨｅＬａ中诱导细胞周期Ｇ２停滞、细胞致死效应及其核定位功能,探讨将Ｖｐｒ用于肿瘤的治疗的可能性。方法 用脂质体转染的方法,将携带Ｖｐｒ或其突变体ＶｐｒＸ基因的质粒转入ＨｅＬａ细胞内,用流式细胞仪分析Ｖｐｒ诱导的Ｇ２停滞和细胞致死性;用荧光显微镜观察ＧＦＰ－Ｖｐｒ融合蛋白的荧光确定其核定位功能。结果 Ｖｐｒ具有核定位功能,Ｖｐｒ和ＶｐｒＸ蛋白都具有细胞     

汉滩病毒囊膜糖蛋白G2与核蛋白氨基端在昆虫细胞中的融合表达

目的：在Bac-to-Bac杆状病毒表达系统中，融合表达汉滩病毒的囊膜糖蛋白G2与核蛋白氨基端。方法：构建含有汉滩病毒G2S0．7嵌合基因的表达载体pFBDHTa—G2S0．7，并转化DH10Bac感受态菌。利用其含有的细菌Tn7转座系统，将嵌台基因重组至杆状病毒穿梭质粒hacmid中，并筛选含有G2S0．7嵌合基因的重组杆状病毒，在昆虫细胞中表达该融合蛋白。对表达产物用间接免疫荧光、ELISA和免疫印迹进行检测。结果：构建了的含G2S0．7嵌合基因的重组杆状病毒，感染昆虫细胞后，可表达相应大小的融合蛋白。该蛋白可被抗汉滩病毒核蛋白及糖蛋白G2的特异性单克隆抗体(mAb)所识别。结论：利用杆状病毒表达系统，成功地表达同时具有核蛋白及糖蛋白G2生物学活性的融合蛋白G2S0．7，为进一步研究其免疫学特性奠定了基础。     

Antigenic characterization of bovine ephemeral fever rhabdovirus G and GNS glycoproteins expressed from recombinant baculoviruses

Recombinant baculoviruses expressing the BEFV envelope glycoprotein G and non-structural glycoprotein G(NS) were constructed. The G protein expressed in insect cells was located on the cell surface and induced spontaneous cell fusion at mildly acidic pH. The expressed G protein reacted with MAbs to continuous and conformational neutralization sites (G1, G2, G3b and G4), but not to conformational site G3a. The expressed G(NS) protein was also located on the cell surface but did not exhibit fusogenic activity. The G(NS) protein reacted with polyclonal antiserum produced from vaccinia-virus-expressed recombinant G(NS) but did not react with G protein antibodies. A His(6)-tagged, soluble form of the G protein was expressed and purified by Ni(2+)-NTA chromatography. The purified G protein reacted with BEFV-neutralizing MAbs to all continuous and conformational antigenic sites. The highly protective characteristics of the native BEFV G protein suggest that the secreted, baculovirus-expressed product may be a useful vaccine antigen.