汉、蒙古族人MBL基因ExonⅠ54位密码子点突变频率及其血浆含量相关性研究

目的　调查健康汉、蒙古族人甘露 (聚 )糖结合凝集素 (MBL)基因 5 4位密码子点突变的情况 ,测定健康汉、蒙古族人血浆MBL含量 ,并对健康汉、蒙古族人MBL基因 5 4位密码子点突变频率与其血浆MBL含量进行相关性分析。方法　PCR扩增 ,测定序列并建立MBL基因点突变检测方法 ,即PCR RFLP方法 (BanⅠ酶切 ) ;用ELISA法测血浆MBL含量。结果　 (1)建立了MBLPCR方法 ,该方法特异性高 ,重复性好 ,最低检出量为 16 0pgDNA。 (2 )MBL的限制性内切酶BanⅠ酶切结果 :通过酶切电泳分析结果显示 ,所扩增标本出现下列 3种情况 :①MBL 5 4位密码子野生型纯合子为 2条带 ,对应的相对分子质量为 2 32bp和 93bp ;② 5 4位密码子突变体纯合子只显示 1条约 32 5bp带 ;③ 5 4位密码子野生型突变体杂合子显示 32 5bp、2 32bp和 93bp 3条带。 (3)通过简单数基因计算MBL基因5 4位密码子点突变的频率 ,健康汉、蒙古族人基因突变频率分别为 0 .2 32 1和 0 .175 7。 (4 )血浆MBL含量的测定 :5 6例健康汉族人血浆MBL含量的平均值为 1998.75 0 μg L ,标准差为 15 0 5 .15 2 ;37例健康蒙古族人血浆MBL含量的平均值为 2 5 2 5 .6 76 μg L ,标准差为 195 5 .188。 (5 )健康汉族人血浆MBL含量与其编码基因ExonⅠ 5 4位密码子的点突变频率 ,     

连环蛋白基因功能区截短突变体的构建及其功能

目的 :观察连环蛋白 (βcatenin)基因功能截短体的亚细胞定位及转录活性的变化。方法 :采用反转录PCR克隆全长野生型βcatenin基因。在此基础上 ,分别构建 βcatenin基因N端、C端及Armadillo区缺失突变体的真核基因表达载体和融合绿色荧光蛋白 (EGFP)的表达载体。应用双荧光素酶报告系统 ,在2 93细胞中检测不同功能截短体的转录活性。在倒置荧光显微镜下观察不同功能截短体在COS7细胞中的亚细胞定位。结果 :成功地构建了 βcatenin基因不同功能截短体的真核表达载体及与绿色荧光蛋白基因融合的表达载体。在 2 93细胞中检测到Armadillo区缺失的突变体蛋白丧失了转录活性 ,N端及C端缺失突变体蛋白的转录活性较野生型分别下降了80 %和 90 %。在COS7细胞中观察到C端缺失突变体蛋白主要定位于胞浆中 ,N端缺失突变体蛋白以粗颗粒形式定位于胞核中 ,Armadillo区缺失突变体的定位情况类似野生型 βcatenin ,以细颗粒形式定位于胞核中。结论 :βcatenin基因不同功能区亚细胞定位的不同 ,提示其在转录激活过程中发挥着不同的作用。     

EXT2基因IVS2+1G>A突变致遗传性多发性外生性骨疣

目的　确定一个遗传性外生性骨疣家系的致病基因。方法　应用基因组扫描方法 ,利用 8、11和 19号染色体上的微卫星标记对该家系进行连锁分析 ,确定候选基因 ,然后对候选基因的编码区及外显子与内含子交界处进行测序分析寻找突变 ,并行逆转录 - PCR扩增 m RNA加以证实。结果　该家系致病基因被定位在 11号染色体的 EXT2基因区 ,在 EXT2基因中检测到 1个 IVS2 1G>A(5 36 1G>A)突变 ,该突变与疾病共分离。逆转录 - PCR证实 ,该突变导致编码区的第 316～ 5 36位共 2 2 1个碱基的缺失 ,使 10 6位密码子至 178位密码子及紧随的两个核苷酸的缺失 ,造成移码 ,形成 12 5个氨基酸的截短蛋白。结论　EXT2基因的 IVS2 1G>A突变是导致这个家系发生外生性骨疣的原因。     

原发性肝细胞癌RIT1基因的突变和扩增

目的　探讨 RIT1基因突变和扩增在肝细胞癌 (hepatocellular carcinoma,HCC)的发生情况及其与发病的关系。　方法　采用 PCR直接测序法检测 5 0例原发性 HCC患者的肝癌组织和非癌肝组织RIT1基因在所包含的 6个外显子的全序列寻找突变位点 ;并用荧光定量 PCR法检测 RIT1基因的扩增情况。　结果　在 5 0例肝癌组织中 1例出现第 5外显子编码区 2 4 1位核苷酸 G/ C变异 ,其对应密码子改变为GAG81CAG,编码氨基酸改变为 Glu81Gln,该氨基酸变异位于 GTP结合的保守功能域内 ,该病例的非癌肝组织以及其余 4 9例的肝癌组织和非癌肝组织均未发生此种改变 ;在 5 0例肝癌组织和非癌肝组织中均出现 5′- UTR(起始密码子前 2 1位核苷酸 ) G/ C变异 ;在获得有效扩增数据的 4 3例肝细胞癌患者中 ,11例有 2～2 97倍的 RIT1基因扩增 ,扩增率为 2 5 .6 %。　结论　基因扩增是 RIT1基因在肝细胞癌的激活方式之一 ,可能与肝细胞癌的发病有关 ,而点突变方式可能意义不大。     

一个脂蛋白肾小球病家系中apoE基因的九个碱基缺失

目的 分析1个脂蛋白肾小球病家系的载脂蛋白E(apolipoprotein E,apoE)基因突变.方法 用盐析法提取该家系4名成员和2名正常人的外周血基因组DNA,PCR扩增apoE基因第4外显子、第3外显子以及多态性片段,扩增产物纯化后分别构建到pTA2载体中,克隆载体转化至大肠杆菌DH5α感受态中,经蓝白斑和抗菌素筛选后集菌,提取重组子质粒,EcoRⅠ酶切鉴定是否转化成功,鉴定正确的菌液行双向测序.PCR-限制性片段长度多态性检测apoE基因多态性.结果 家系中先证者及其母亲和姐姐apoE基因第4外显子第484～492位9个碱基(CAAGCTGCG)缺失,为杂合缺失,其导致apoE氨基酸序列第143～145位KLR的3个氨基酸缺失.第3外显子测序未发现异常.apoE基因多态性分析结果与测序结果一致.结论 该家系的脂蛋白肾小球病因可能为apoE基因第484～492位9个碱基(CAAGCTGCG)缺失.     

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。     

IPS-1基因缺失突变体对IFN-β诱生作用及抗HSV-1病毒作用的影响

目的:初步研究缺失300-444位氨基酸突变体△IPS-1对IFN-β诱生作用及抗HSV-1病毒作用的影响,进而确定该部位对IPS-1发挥正常功能的作用.方法:通过重叠延伸PCR法获得缺失300～444位氨基酸的△IPS-1,构建突变重组质粒pEF-BOS-FLAG-△IPS-1,并经酶切及测序鉴定.采用磷酸钙体外转染HEK293T真核细胞.经Western blot检测蛋白的表达;ELISA检测细胞上清中IFN-β分泌量;HSV-1病毒感染分别转染了pEF-BOS-FLAG/IPS-1、pEF-BOS-FLAG/△IPS-1质粒的HEK293T细胞,空斑检测各转染细胞组不同时间段细胞上清的病毒滴度.结果:成功构建了缺失300～444位氨基酸的IPS-1基因重组真核表达载体pEF-BOS-FLAG/△IPS-1;与转染pEF-BOS-FLAG/IPS-1质粒组相比转染了pEF-BOS-FLAG/△IPS-1的细胞在不同时间段其上清中IFN-β分泌量有一定量的减少;空斑测定结果表明转染了pEF-BOS-FLAG/△IPS-1质粒组细胞中病毒滴度明显高于pEF-BOS-FLAG/IPS-1质粒组.结论:△IPS-1与IPS-1相比能使HEK293T细胞IFN-β分泌量明显减少,△IPS-1不能完全抑制HSV-1感染细胞产生的细胞病变效应,该突变体抗HSV-1病毒作用有一定程度的减弱.     

信号蛋白14-3-3 β及其缺失突变体与朊蛋白体外相互作用的初步研究

目的 确定PrP蛋白与14-3-3蛋白是否发生分子间的相互作用,以及相互作用区域.方法 表达纯化PrP和人14-3-3 β及其缺失突变体.通过Pull-down和免疫共沉淀方法研究全长人14-3-3 β及缺失突变体与PrP蛋白是否发生分子间相互作用以及相互作用的区域.结果 14-3-3 β能与PrP23-231发生体外的相互作用,且与PrP相互作用的区域位于14-3-3 β N端第1位至38位氨基酸.结论 首次证实人14-3-3 β与PrP相互作用的区域位于14-3-3 β N端第1位至38位氨基酸.     

全人源抗人干扰素α1b基因工程抗体与抗原的结合表位鉴定

目的鉴定抗原人干扰素α1b(huIFN-α1b)与1株全人源抗体蛋白AIFNa1IgG1的结合表位。方法从正常人分离纯化淋巴细胞并抽提mRNA进行CDNA合成,以CDNA为模板通过PCR扩增得到huIFN-α1b基因,通过重叠延伸PCR法构建huIFN-α1b蛋白29-35位氨基酸残基缺失突变体及点突变体,野生型和突变基因与载体pET30a连接构建重组表达载体,将以上重组表达载体转化大肠杆菌表达宿主E.coli Rosseta(DE3),诱导表达后的huIFN-α1b蛋白及突变体通过Western blot分析其与全人源抗huIFN-α1b基因工程抗体蛋白的结合活性。结果 huIFN-α1b蛋白在E.coli Rosseta(DE3)细胞中成功表达,Western-blot分析表明野生型huIFN-α1b蛋白与抗体结合,29-35位氨基酸缺失与抗体失去结合活性,其中27位的Ser,33位的Asp,34位的Arg,35的His,36位的Asp突变为Gly后,抗体与huIFN-α1b的结合可减弱到不可检测的水平。结论 huIFN-α1b蛋白29-35位氨基酸形成的表位是与抗体AIFNa1IgG1结合的重要表位。     

跨膜型肿瘤坏死因子信号肽氨基酸部分缺失对细胞毒作用影响

目的　探讨跨膜型肿瘤坏死因子 (TM TNF)信号肽在其杀瘤中的作用。方法　用PCR构建突变体及体外转录、翻译蛋白质 ,分别获得TM TNF信号肽胞浆段、胞外与分泌型肿瘤坏死因子(S TNF)连接段以及部分跨膜段缺失即信号肽第 73～ 4 7、 73～ 34、 34～ 1、 2 1～ 1位氨基酸缺失的TM TNF体外翻译蛋白。用MTT法检测其对HL 6 0细胞的杀伤率。结果　 34～ 1、 2 1～ 1位氨基酸缺失可提高TM TNF对HL 6 0细胞的杀伤 , 73～ 4 7位氨基酸缺失对其杀瘤力无明显作用 ,而 73～ 34位氨基酸缺失可使其杀瘤率明显下降。结论　去除信号肽胞外段可提高TM TNF α对HL 6 0细胞的杀伤率 ,而TM TNF α信号肽 4 7～ 34位氨基酸在其杀瘤过程中有重要作用。     

Cloning and sequence analysis of the pfl gene encoding pyruvate formate-lyase from Streptococcus mutans.

We have isolated a sorbitol-negative mutant of Streptococcus mutans GS-5 following random mutagenesis with plasmid pVA891 clone banks. This mutant did not metabolize sorbitol anaerobically but did so aerobically. A 10-kb chromosomal DNA fragment flanking the pVA891 insertion was deleted in this mutant. The corresponding region from the parental strain GS-5 was then recovered by a marker rescue method with Escherichia coli. The pyruvate formate-lyase gene, pfl, was identified within a 3-kb PstI-XbaI fragment located in the middle of the deleted region of the chromosome, and its inactivation in S. mutans produced the same sorbitol-negative phenotype. Nucleotide sequence analysis of the pfl gene revealed a 2.3-kb open reading frame (ORF) preceded by potential ribosome-binding and promoter-like sequences. The ORF specified a putative protein of 775 amino acid residues with a calculated molecular weight of 87,533. The amino acid sequence deduced from the ORF exhibited significant similarity to that of the E. coli pfl gene.     

幽门螺杆菌cagL基因缺失株的建立

目的:构建幽门螺杆菌Ⅳ型分泌系统cagL基因缺失株,为研究cagL基因的功能奠定基础。方法:利用同源重组原理,PCR扩增该基因编码区两侧序列,作为同源臂,构建出带卡那霉素抗性标志的载体,采用电穿孔法将其转化入受体菌中,并经PCR验证后获得了cagL基因缺失株;突变株和野生株分别与胃上皮细胞GES-1共培养后,检测其对CagA蛋白转运的影响。结果:构建cagL基因缺失的自杀质粒,并获得一株cagL基因缺失株;CagA转运实验表明,cagL基因缺失后,导致幽门螺杆菌CagA转运功能的丧失。结论:成功获得了幽门螺杆菌cagL基因缺失株,该基因参与CagA蛋白的转运,是该菌IV型分泌系统中重要组成成分之一。     

Determination of the coding capacity of the BamHI DNA fragment B of apathogenic Herpes simplex virus type 1 strain HFEM by DNA nucleotide sequence analysis.

Herpes simplex virus type 1 (HSV-1) strain HFEM acquired an apathogenic phenotype due to a deletion within the DNA sequences of the BamHI DNA fragment B of the viral genome. In order to investigate the coding strategy of this particular region of the genome of HSV-1 strain HFEM the DNA nucleotide sequence of the BamHI DNA fragment B was determined. This analysis revealed that the BamHI DNA fragment B of HSV-1 strain HFEM comprises 6593 bp, corresponding to the nucleotide positions (np) 113322 to 117088 and np 120643 to 123465 of the genome of HSV-1 strain 17. According to these data the deletion of the genome of HSV-1 strain HFEM occurred between the np 117089 and 120642. The promoter region of the UL56 gene of HSV-1 strain HFEM is a part of the deleted DNA sequences. Therefore, this gene of HSV-1 strain HFEM is affected and cannot be expressed. The first 35 amino acid (AA) residues of the deduced amino acid sequence of the UL56 open reading frame (ORF) were found to be identical to the amino acid sequence of the UL56 genes of HSV-1 strains 17 and F. However, due to a deletion at np 3494 of the BamHI DNA fragment B of HSV-1 strain HFEM the amino acid composition of the predicted UL56 gene of HSV-1 strain HFEM is different from HSV-1 strain 17 between amino acid positions 36 and 233. In addition the deduced amino acid sequence of the IRL (inverted repeat of the long segment) copy of the IE110 gene of HSV-1 strain HFEM was found to be about 342 amino acids shorter than the amino acid sequence of IE110 gene of HSV-1 strain 17 (775 AA). This was based on a point mutation which was detected within the DNA sequences of Exon 3 of this copy of IE110 gene of HSV-1 strain HFEM.     

Mutations in polyomavirus middle T antigen affecting tumorigenesis

P155 is a polyomavirus mlt mutant with normal transforming ability but impaired tumorigenic potential. The mutation, a 12-bp deletion (nucleotides 1348-1359), removes amino acids 372 to 375 from middle T and affects its ability to function in tumorigenesis (C. Gelinas, S. Masse, and M. Bastin, 1984, J. Virol. 51, 242-246). We used deletion loop mutagenesis to introduce point mutations within the wild-type sequence spanned by the P155 deletion. A mutant phenotype resembling that of P155 could be produced by as little as one alanine to valine substitution at residue 373. The mutants were impaired in their ability to induce tumors in rats but they could still transform established cell lines or primary fibroblasts in culture. To define the biochemical defect, we examined the mutant middle T antigen both for association with pp60c-src, the cellular src gene product, as well as its pattern of phosphorylation. No obvious differences explaining the phenotype were observed. The mutant middle T associated with, and activated pp60c-src, but exhibited a slightly altered pattern of phosphorylation, presumably because of additional sites on the middle T protein.     

鼻咽癌细胞株SUNE1中EB病毒LMP1全基因克隆及部分？…

目的 研究广东地区鼻咽癌（ＮＰＣ）细胞株ＳＵＮＥ１中ＥＢ病毒ＬＭＰ１基因的结构特征。方法 利用聚合酶链反应从ＳＵＮＥ１细胞基因组中扩增ＬＭＰ１全长ＤＮＡ,并克隆到ｐｃＤＮＡ３载体中,采用Ｓａｎｇｅｒ双脱氧末端终止法测定ＳＵＮＥ－ＬＭＰ１ ３个外显子及２个内含子覆盖的序列,并与病毒原型Ｂ９５－８－ＬＭＰ１进行比较分析。结果 克隆到ｐｃＤＮＡ３中的ＳＵＮＥ１－ＬＭＰ１全基因长度为２．６ｋｂ,测序长度为     

Searching for the initiating site of the major capsid protein to generate virus-like particles for a novel laboratory mouse papillomavirus

Correctly folded virus-like particles (VLPs) of papillomavirus (PV) display conformationally dependent epitopes that are type specific, maintained on authentic virions, and induce neutralizing antibodies. Alignment of the L1 amino acid (aa) sequences of 84 PVs revealed that the lengths of their N-termini are diverse and that multiple, possible initiation methionine (met) codons exist. The L1 gene of MusPV (MmuPV1), that naturally infects immunodeficient laboratory mouse strain (NMRI-Foxn1^nu/Foxn1^nu), has four met codons at the 1st, 2nd, 28th, and 30th aas from its N-terminus. Of these, the 3rd and 4th mets, that are at the 28th and 30th aa position from the N-termius, respectively, are located at the position where most PVs have their first met. These two mets, located at the 9th and 11th from the YLPP conserved aas of most PVs, should be considered as consensus initiation codons of PV L1s. Three L1 proteins of MusPV, starting from the 2nd, 3rd, and 4th mets, were expressed using a baculovirus expression system and characterized for their ability to self-assemble into VLPs. While MusPV L1 proteins starting from the 2nd met expressed an L1 protein that did not fold into VLPs, the L1s starting from the 3rd and 4th mets generated correct VLPs in abundant quantities. We now conclude that the highest quantity and best quality VLPs are made from the consensus L1 met of MusPV.     

Heparin-binding domain of bovid herpesvirus 1 glycoprotein gIII

Summary Bovid herpesvirus 1 (BHV-1) glycoprotein gIII functions as a major virus attachment protein through binding to a heparinlike moiety on the host cells. To identify the functional domain, a panel of gIII deletion mutants was constructed, expressed in COS-7 cells, and examined for heparin-binding activity. Mutants with deletion of amino acid residues 103–173 and 324–443 bound to heparin as well as full-length gIII, whereas a mutant with residues 172–337 deleted showed no binding to heparin. In another mutant, with residues 172–211 deleted, the activity was reduced by one-third. These data suggest that the amino acid sequence between residues 172 and 323 contains the functional domain of BHV-1 gIII for heparin-binding and that especially the sequence between residues 212–323 includes a critical site for the activity.     

可溶性CD14突变体的构建与表达研究

目的 将人CD14信号转导启动区97－101氨基酸进行丙氨酸置换突变。,构建直核表达质粒,并在COS－7细胞中进行表达。方法 采用两轮聚合酶链反应定点突变方法,对人可溶性CD14推测中的LPS信号转导启动区之一,N端97－101氨基酸用丙氨酸进行置换交换。将突变PCR片段克隆至载体pGEM-T,进行酶切鉴定及序列分析。将突变基因亚克隆至真核表达质粒pClneo中,转染COS－7细胞进行瞬时表达,并用SDS－PAGE和Western blot检测表达蛋白。结果 测序结果证实,CD14的N端97－101氨基酸发生了连续丙氨酸置换突变。SDS－PAGE表明,突变体基因在COS－7细胞中得到瞬时表达。Western blot显示,抗CD14多克隆抗体可与重组表达的突变体蛋白特异性结合。结论 成功地构建并表达CD14 97－101氨基酸丙氨酸置换突变体,为研究CD14信号转导缺失突变体的生物学活性与功能打下了基础。     

O139群霍乱弧菌TLC、CTX、RTX基因簇缺失株的构建及鉴定

目的构建O139群霍乱弧菌TLC、CTX、RTX基因簇缺失株。方法PCR扩增TLC上游片段（TLCup）、RTX下游片段（RTXdown）,克隆入pU18中,再在两者间插入氯霉素抗性（Cm）基因,获得重组质粒pUC18-TLC up—Cm-RTX down。酶切回收TLC up—Cm—RTX down片段,克隆入自杀质粒pDS132,构建重组自杀质粒pDS132-TLC up-Cm—RTX down。通过接合将重组自杀质粒转入O139群霍乱弧菌ZJ199693,20％蔗糖选择培养基（Cm抗性）筛选TLC、CTX、RTX基因缺失株。结果PCR、酶切证明重组自杀质粒pDS132-TLC up-Cm-RTX down构建正确。PCR证实O139群霍乱弧菌ZJ199693TLC、CTX、RTX毒力基因簇成功缺失。结论成功缺失O139群霍乱弧菌ZJ199693TLC、CTX、RTX基因簇．为进一步研究其功能和疫苗构建打下基础。     

竞争性定量RT-PCR技术在t(8;21)AML微小残留病变检测中的应用

目的：建立针对ＡＭＬ－ＥＴＯ融合基因转录本的竞争性定量反向－聚合酶链反应（ＲＴ－ＰＣＲ）体系,并探讨其在ｔ（８;２１）急性髓细胞性白血病（ａｃｕｔｅ ｍｙｅｌｏｉｄ ｌｅｕｋｅｍｉａ,ＡＭＬ）微小残留病变跟踪中的应用价值。方法：采用重叠延伸剪接术（ｓｐｌｉｃｉｎｇ ｂｙ ｏｖｅｒｌａｐｐｉｎｇ ｅｘｔｅｎｔｉｏｎ,ＳＯＥ）获得竞争性ＤＮＡ片段,建立竞争性定量ＰＣＲ方法,并应用于ｔ（８;２１）ＡＭＬ患者的检测。结果：成功建立了竞争性定量ＰＣＲ方法〈并获得不同ｔ（８;２１）ＡＭＬ患者的ＡＭＬ１－ＥＴＯ融合基因转录本的竞争性定量ＰＣＲ结果。结论：以ＳＯＥ方法为基础的竞争性定量ＰＣＲ方法简单、适用;不同生存状态下的ｔ（８;２１）ＡＭＬ患者的ＡＭＬ１－ＥＴＯ融合基因转录本表达量具有明显差异。