Association of the Platelet Glycoprotein Ia C807T Gene Polymorphism With Risk of Myocardial Infarction in Chinese

Objectives To investigate the relationship of the GPIa C807T dimorphism to the risk of myocardial infarction （MI） in Chinese. Methods We did a case-control study including 100 patients and 110 controls with same race. An allele-specific polymerase chain reaction （PCR） was used for genotyping of C807T polymorphism. Results An apparent association was found between the T807 allele and MI among individuals younger than the mean age of 60 years （odds ratio, 2. 49 ; 95 % confidence interval, 1.08 - 6.22 ）. The T807 allele remained an independent risk factor for MI when age, sex, smoking, hypertension, diabetes, bodymass index, LDL-cholesterol and HDL-cholesterol were adjusted by logistic regression. Conclusions GPIa T807 appears to be an independent risk factor for MI.

Cloning and Sequence Analysis of Glycoprotein D Gene of Bovine Herpesvirus-1 Strain Luojing

By means of PCR,the gene encoding gD of bovine herpesvirus-1 (BHV-1) strain Luojing was amplified,cloned and sequenced.The nucleotide sequence of this gD gene was (1 251 bp,)encoding 417 amino acids.Comparied with the published P8-2 strain,the homology of the necleotide sequence is 99.92%,and that of the deduced amino acid sequence is 100%.The results indicated that gD of BHV-1 was highly conservative.

Cloning and Sequence of Glycoprotein H Gene of Duck Plague Virus

The glycoprotein H （gH） gene homologue of duck plague virus （DPV） was cloned by degenerate polymerase chain reaction （PCR） and sequenced. It was located immediately downstream from the thymidine kinase gene （TK）. In addition, the 3＇-end of the gene homologue to herpesvirus UL21 was located downstream from the gH gene. DPV gH gene open reading frame （ORF） was 2 505 bp in length and its primary translation product was a polypeptide of 834 amino acids long. It possessed several characteristics of membrane glycoproteins, including an N-terminal hydrophobic signal sequence, an external domain containing eight putative N-linked glycosylation sites, a C-terminal transmembrane domain, and a charged cytoplasmic tail. Comparison with other herpesvirus revealed identities of 20.2, 25.1, 23.0, 23.0, 26.5 and 26.0% with the gH counterparts of the human herpesvirus virus 1 （HSV1）, equine herpesvirus 4 （EHV4）, bovine herpesvirus 1 （BHV1）, pseudorabies virus （PRV）, gallid herpesvirus 2 （GHV2） and gallid herpesvirus 3 （GHV3）, respectively.

ZP1基因突变在空卵泡综合征中的研究进展

空卵泡综合征(empty follicle syndrome)发病机制尚不清楚,越来越多的研究聚焦于遗传因素,尤其是调控卵母细胞发育的相关基因。其中,透明带糖蛋白1(zona pellucida glycoprotein 1,ZP1)是透明带基质结构完整性的关键组成部分。由ZP1基因突变造成的透明带结构和功能的缺陷常会导致卵母细胞成熟障碍、脆性增加,进而造成反复取卵失败。现已鉴定出多种导致空卵泡综合征表型的ZP1基因突变。综述不同部位ZP1基因突变引起空卵泡综合征表型及其作用机制。

Detection and Genetic Characterization of Rabies Virus from Human Patients

Saliva and blood were collected from two patients who had not received post exposure prophylaxis in the cities of Wenzhou and Xinning respectively. Both patients were confirmed as positive for rabies by detection of rabies virus specific nucleoprotein antibodies in the sera by Western Blot. However, rabies virus specific RNA was only identified in the saliva collected from the patient in Wenzhou. Furthermore, the isolate Zhejiang Wz0 (H) was obtained by inoculating one-day-old suckling mice. Both nucleoprotein (N) and glycoprotein (G) genes from the isolate were amplified by RT-PCR and sequenced. Phylogenetic analysis indicated that the isolate belonged to classic rabies virus, and shared a higher homology with the street viruses from dogs in the main endemic areas in China and the street virus from dogs in Indonesia than with other known strains. Further comparison of the deduced amino acid sequences between the isolate and the vaccine strains used in China showed that the virus had a higher level of homology with the vaccine strain CTN than with the other vaccine strains (3aG, PV, PM and ERA). In particular, amino acid residues substitutions located in antigenic site Ⅲ in the G protein, which could react with the neutralizing antibodies, were observed. These results suggested that the virus belonged to the classic rabies virus, and both N and G genes diverged from the current vaccine strains used in China at either the nucleotide or the amino acid level.

The Effect of Different Interventional Treatment on P-Glycoprotein in Different Histopathological Types and Grades of Primary Hepatocellular Carcinoma

To study the effect of the different interventional treatment on P Glycoprotein (Pgp) in different histopathological types of primary hepatocellular carcinoma (PHC), 98 surgically and histologically verified PHC specimens were obtained. The patients included 57 patients treated by surgical resection alone and 41 patients receiving second stage surgical resection after four kinds of interventional treatment. SABC immunohistochemical staining with a monoclonal antibody against human Pgp was used to observe the Pgp in all specimens. The positive rate of Pgp was 100 % in group of chemotherapy alone ( P <0.05), 62.5 % in group of chemotherapy combined with iodized oil ( P >0.05), 46.6 % in group of chemotherapy combined with iodized oil and spongia gelatini absorbens (Sga) ( P >0.05), 18.18 % in group of chemotherapy combined with Ethanol iodized oil and Sga ( P <0.05) and 52.63 % in group of surgical resection alone. The positive rate of Pgp varied with different histopathological types, with rate of clear cell PHC being the lowest, and that of poorly differentiated or undifferentiated PHC the highest. The positive rate of Pgp was increased as pathological grades increased. Overexpression of Pgp may be responsible for the intrinsic and acquired drug resistance of PHC. Multidrug resistance (MDR) varied with different histological types. Therapy of PHC should be tailored according to individual. Local chemotherapy combined with ethanol iodized oil and Sga embolization may become a new way to overcome MDR of PHC.

Expression and Characterization of HIV-1 Envelope Glycoprotein in Pichia Pastoris

To obtain a sufficient amount of glycoprotein for further studying the structure and function of HIV-1 envelope glycoprotein, amplified and modified HIV-1 envelope glycoprotein gene which recombined subtypes（850 amino acids） from Guangxi in China was inserted into Pichia pastoris expression vector pPICZαB; then the recombinant plasmid was transported into the yeast cells to induce the expression of Env protein with methanol. The results of SDS-PAGE and Western blot indicate that the envelope glycoprotein could be expressed in Pichia pastoris with productions of a 120000 glycoprotein and a 41000 glycoprotein, which showed satisfactory immunogenicity by indirect ELISA.

Overexpression of P-glycoprotein in hepatocellular carcinoma and its clinical implication

INTRODUCTIONMost advanced hepatocellular garcinoma (HCC) isinsensitive to most anticancer drugs which might berelated to the high frequency of expression of themultidrug resistance-1(MDR1) gene and itsproduct,P-glycoprotein (p-gp).p-gp expressionmay also be concerned with tumor progression anddifferentiation.In the present study。

Disease control by regulation of P-glycoprotein on lymphocytes in patients with rheumatoid arthritis

The main purpose of treatment of rheumatoid arthritis(RA) with disease modifying antirheumatic drugs(DMARDs) is to control activation of lymphocytes,although some patients do not respond adequately to such treatment. Among various mechanisms of multidrug resistance, P-glycoprotein(P-gp), a member of ATP-binding cassette transporters, causes drugresistance by efflux of intracellular drugs. Certain stimuli,such as tumor necrosis factor-α, activate lymphocytes and induce P-gp expression on lymphocytes, as evident in active RA. Studies from our laboratories showed spontaneous nuclear accumulation of human Y-boxbinding protein-1, a multidrug resistance 1 transcription factor, in unstimulated lymphocytes, and surface overexpression of P-gp on peripheral lymphocytes of RA patients with high disease activity. The significant correlation between P-gp expression level and RA disease activity is associated with active efflux of drugs from the lymphocyte cytoplasm and in drugresistance.However, the use of biological agents that reduce P-gp expression as well as P-gp antagonists(e.g., cyclosporine) can successfully reduce the efflux of corticosteroids from lymphocytes in vitro, suggesting that both types of drugs can be used to overcome drug-resistance and improve clinical outcome. We conclude that lymphocytes activated by various stimuli in RA patients with highly active disease acquire P-gpmediated multidrug resistance against corticosteroids and probably some DMARDs, which are substrates of P-gp. Inhibition/reduction of P-gp could overcome such drug resistance. Expression of P-gp on lymphocytes is a promising marker of drug resistance and a suitable therapeutic target to prevent drug resistance in patients with active RA.

Liposome-mediated Functional Expression of Multiple Drug Resistance Gene in Human Bone Marrow CD34^+ Cells

Summary: The expression and functional activity of multiple drug resistance (MDR1) gene in human normal bone marrow CD34+ cells was observed. Human normal bone marrow CD34+ cells were enriched with magnetic cell sorting (MACS) system, and then liposome-mediated MDR1 gene was transferred into bone marrow CD34+ cells. Fluorescence-activated cell sorter was used to evaluate the expression and functional activity of P-glycoprotein (P-gp) encoded by MDR1 gene. It was found that the purity of bone marrow CD34+ cells was approximately (91±4.56) % and recovery rate was (72.3±2.36) % by MACS. The expression of P-gp in the transfected CD34+cells was obviously higher than that in non-transfected CD34+ cells. The amount of P-gp in non-transfected CD34+ cells was (11.2±2.2) %, but increased to (23.6±2.34) % 48 h after gene transfection (P<0.0l). The amount of P-gp was gradually decreased to the basic level one week later. The accumulation and extrusion assays showed that the overexpression of P-gp could efflux Rh-123 out of cells and there was low fluorescence within the transfected cells. The functional activity of P-gp could be inhibited by 10 μg/ml verapamil. It was suggested that the transient and highly effective expression and functional activity of P-gp could be obtained by liposome-mediated MRD1 transferring into human normal bone marrow CD34+ cells.

Expression of multidrug resistance 1 gene and C3435T genetic polymorphism in peripheral blood of patients with intractable epilepsy

BACKGROUND： Increased expression of multidrug resistance 1 （MDR1） mRNA in peripheral blood of patients with intractable epilepsy is not due to epilepsy drugs, but epilepsy behavior. Monitoring MDR1 expression in peripheral blood is a target for MDR1 gene evaluation. OBJECTIVE： To investigate the influence of antiepileptic drugs and seizures on MDR expression in intractable epilepsy, and to analyze the genetic polymorphisms of C3435T in the MDRl gene. DESIGN, TIME AND SETTING： Factorial designs and comparative observations at the experimental center of the Affiliated Hospital of Qingdao Medical College, Qingdao University between October 2003 and October 2004. PARTICIPANTS： A total of 120 subjects were recruited from the epilepsy clinical department of the Affiliated Hospital of Qingdao Medical College. Four groups （n = 30） were classified according to statistical factorial design： intractable epilepsy, treatment response, no treatment, and normal control groups. METHODS： One-step semi-quantitative reverse-transcription polymerase chain reaction technology was used to test expressions of the MDR1 gene in 120 subjects. C3435T polymorphisms in intractable epilepsy group and normal control groups were analyzed by polymerase chain reaction-restriction fragment length polymorphism. MAIN OUTCOME MEASURES： Expression of MDR1 mRNA in the four groups, and C3435T genetic polymorphisms in intractable epilepsy and normal control groups. RESULTS： MDRl gene expression was increased in the intractable epilepsy group, due to the factor seizures, but not the antiepileptic drugs. However, the interaction between the two factors was not statistically significant. Of the 30 subjects in the intractable epilepsy group, the following genotypes were exhibited： 3 （10%） C/C genotype, 9 （30%） C/T genotype, and 18 （60%） T/T genotype at the site of C3435T, while 4 （13%）, 10 （33%）, and 16 （53%） subjects were determined to express these genotypes in the normal control group, respectively. C and T allele frequency were 25% and 75% in the intractable epilepsy group, and 30% and 70% in the normal control group, respectively. However, there was no statistical difference between the groups. CONCLUSION： Results demonstrated that seizures, not antiepileptic drugs, induced MDR1 gene expression in intractable epilepsy. Genetic polymorphisms of C3435T in the MDR1 gene did not contribute to the development of multidrug resistance in patients with intractable epilepsy.

马疱疹病毒8型TaqMan实时荧光定量PCR检测方法的建立

为了建立一种能够快速检测马疱疹病毒8型(EHV-8)的方法,本试验以EHV-8的全基因组序列为模板,针对糖蛋白B(gB)基因的保守序列,进行对比分析,设计EHV-8的特异性引物和探针,优化反应条件,建立EHV-8 TaqMan实时荧光定量PCR检测方法,并对该方法的敏感性、特异性和重复性进行验证,使用该方法进行临床样本检测。结果显示,建立的EHV-8 TaqMan实时荧光定量PCR检测方法对EHV-8 DNA模板的最低检测限为1.1×10^(2)copies/μL,敏感性高;EHV-8与EHV-1、EHV-4、马流产沙门氏菌和肠产毒性大肠杆菌均无交叉反应,特异性强;批内重复性试验和批间重复性试验均表明该方法重复性好。对132份临床样本的检测结果显示,阳性检出率为10.61%,基因测序正确。由此可见,本试验建立的EHV-8 TaqMan实时荧光定量PCR检测方法能够满足EHV-8的检测需求,且该方法敏感性高、特异性强、重复性好,为EHV-8的进一步研究提供了有效的辅助检测手段。

DDAH基因多态性影响格列苯脲治疗妊娠期糖尿病效果及其相关性分析

目的:探讨DDAH基因多态性对格列苯脲治疗妊娠期糖尿病(Gestational diabetes mellitus,GDM)疗效的影响及其与人类软骨糖蛋白-39(YKL-40)、视黄醇结合蛋白4(Retinol binding protein,RBP4)水平的相关性研究。方法:选择山西省儿童医院妇幼保健院于2019年1月至2022年1月收治的346例GDM患者为研究对象,根据DDAH rs1241321位点基因多态性分为AA基因型组、AG基因型组及GG基因型组。三组患者给予格列本脲联合门冬胰岛素治疗8周后对比三组患者的血糖指标、临床疗效及治疗前后血清YKL-40、RBP4表达水平差异;并通过相对风险度(OR)分析DDAH1基因与格列苯脲降糖效应之间的相关性。结果:格列本脲联合门冬胰岛素治疗后,AA基因型组餐后血糖指标2-hPG、HOMA-IR、FPG与血清YKL-40、RBP4浓度均高于GG基因型组、AG基因型组,且AG基因型组高于GG基因型组,差异具有统计学意义(P<0.001)。相对风险度(OR)分析结果显示DDAH1基因rs1241321位点基因型(AA、AG、GG)与YKL-40、RBP4下降水平之间存在显著相关性(OR=0.516,95%CI=0.262~0.846,P=0.012;OR=0.456,95%CI=0.184~0.827,P=0.023)。结论:DDAH基因多态性可能通过内皮一氧化氮合酶(NOS)/DDAH通路改善胰岛素敏感性,影响血清YKL-40、RBP4表达水平,导致格列本脲联合门冬胰岛素治疗GDM在降糖效果及临床疗效上存在差异。

我国流行的4株狂犬病街毒株G基因序列及分子流行病学研究

目的探讨我国流行的狂犬病毒(RV)基因差异,评价现有疫苗的适用性。方法通过RT-PCR并对其产物测序后获得4株RV街毒株的G基因序列以及G与M和L基因的区间序列。利用计算机分析软件比较4株RV与已经发表的毒株的核苷酸和推导的氨基酸序列。结果糖蛋白(GP)完整的编码基因序列长度为1575 bp。GP氨基酸序列同源性明显高于相应的核苷酸序列(除Mokola外),4株街毒与CTN的同源性高于aG株。GP不同区段比较显示,膜外区同源性高于膜内区。G-M区间核苷酸序列长度为5 bp;除广西4外,其余毒株100％同源。GL区间核苷酸序列长度为2,4 bp,越1与肥东同源性为100％。结论4株RV均属于基因I型。广西的毒株之间存在不同的来源。街毒与疫苗株之间基因存在差异。4株RV与SAD-B19进化途径相近,与PV株相差较远。越1与肥东株亲缘关系极近,可能是由同一毒株演化而来。

新城疫病毒分离株的蚀斑纯化及影响蚀斑形成的主要因素

从西北地区 1979～ 1999年发生新城疫的鸡群中分离和收集的 15株新城疫病毒 ( NDV) ,经蚀斑纯化 ,共得到 11个克隆毒株 ;对其进行了蚀斑形成能力的测定 ,证明蚀斑形成能力与毒力成正比 ;探讨了温度对蚀斑形成能力的影响 ,证明 NDV蚀斑形成能力在 4 1℃比 37℃强 ,表现为蚀斑出现早、蚀斑大、蚀斑形成单位 ( PFU)高 ,同时在 4 1℃时细胞生长状况良好 ,不易污染 ,故认为在 4 1℃进行 NDV的蚀斑纯化值得推荐。

鸡传染性支气管炎病毒中国地方分离株M基因的分子特征

本实验根据已经发表的鸡传染性支气管炎病毒 (IBV)M蛋白基因序列设计并合成一对引物 ,利用RT_PCR扩增得到了IBV新疆分离株LX4株 (HA价为 2 7)M基因 678bp的片段 ,将该片段克隆到PUC18载体上 ,通过对所得到的重组质粒进行酶切分析、PCR鉴定 ,证明得到了含有目的基因片段的阳性重组质粒。采用Sanger’s双脱氧末端终止法对插入片段进行核苷酸序列测定 ,获得了IBV_LX4株M基因的核苷酸序列 ,利用DNASIS分析软件 ,将它与GENBANK中发表的 15株国外参考毒株相比较 ,发现核苷酸的同源性 (除D14 66和DE0 72外 )为 85 % ～92 % ,氨基酸的同源性为 83 % ～92 % ,与国内参考株 (H5 2_GD)相比分别为 90 %和 92 % ,确证我们得到的克隆片段为IBV -LX4株的M基因。且含有两个糖基化位点 ,9个高度保守的半胱氨酸 ,三个跨膜区域 。

中国不同来源狂犬病病毒野毒株G基因主要功能区的序列分析

对我国不同来源的狂犬病病毒野毒株８２０２、ＢＲＶ、ＭＲＶ的Ｇ基因４０５～１１４６位核苷酸序列，进行了逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）扩增、克隆和序列测定，并应用计算机对这３个毒株的测定序列和已发表的中国人源毒株（ＣＧＸ８９）的相应序列进行了分析比较。结果表明，这４个中国不同来源狂犬病病毒的Ｇ基因同源性较低，８２０２与ＢＲＶ的核苷酸同源性只有７９．５％，与ＭＲＶ的氨基酸同源性亦只有８２．２％；同一地区自不同动物分离的ＭＲＶ株与ＢＲＶ株其亲缘关系较远。４个野毒株的糖基化位点和主要的诱导中和抗体产生的抗原决定簇内某些氨基酸亦不同，从而证明我国存在亲缘关系较远的狂犬病野毒株。

广西12株狂犬病野毒株的g基因序列测定与分析

对广西12株狂犬病病毒株g基因全序列进行测序分析,结果显示:广西毒株属于I型,可分为3个群,即Ⅰ群、Ⅱ群和Ⅲ群。GX01、GX08、GX09、GX014、GX091、GX195、GX260、GXLA8株为Ⅰ群,GX219、GX074、GXBM3株为Ⅱ群,GXN119株为Ⅲ群。Ⅰ群的广西毒株g基因核苷酸的同源性为97.6%～99.9%,氨基酸同源性在97.7%～100%之间;Ⅱ群的核苷酸的同源性为98.2%～99.0%,氨基酸的同源性在98.5%～99.2%之间。糖蛋白在主要的抗原位点GI、GⅡ区以及与中和抗原有关的36、263、367位氨基酸没有变异,GⅢ区上只有Ⅱ群在332位缬氨酸变异为异亮氨酸,G蛋白上的氨基酸主要在-2、-5、-13、-14、-15、-16、90、96、132、140、156、168、170、204、241、249、253、264、289、332、382、427、436、445、463、474位共26个氨基酸发生了变异,这些氨基酸的变异具有群的特异性。

传染性支气管炎病毒S_1基因DNA免疫质粒的构建及其免疫原性

为探讨ＤＮＡ免疫防制传染性支气管炎（ＩＢ）的可能性，以克隆的Ｍａｓｓｃｈｕｓｅｔｅ型类Ｍ４１地方分离ＱＤ株Ｓ１基因为免疫原基因，引入终止密码后插入ＳＶ４０启动子、增强子下游和ＳＶ４０ｐｏｌｙＡ信号上游，构建了Ｓ１基因ＤＮＡ免疫表达质粒ｐＳＶＱＤＳ１。将大量扩增并经聚乙二醇纯化的ｐＳＶＱＤＳ１溶于ＰＢＳ，以３００μｇ／只的剂量肌肉注射免疫小鼠，于４周后采集分离免疫小鼠血清进行鸡胚病毒中和试验。结果表明，ｐＳＶＱＤＳ１能够诱导产生针对传染性支气管炎病毒（ＩＢＶ）Ｍ４１株的中和抗体，具有良好的免疫原性。

河南牛、鼠源狂犬病毒G基因主要功能区的比较分析

本研究对从我国河南牛狂犬病疫区分离的狂犬病病毒牛源毒ＢＲＶ和鼠源毒ＭＲＶ的糖蛋白（Ｇ）基因膜外主要功能区进行反转录—聚合酶链反应（ＲＴ－ＰＣＲ）扩增、克隆、测序和两者的比较分析。分别得到了每个毒株Ｇ基因膜外区７４２ｂｐ的ｃＤＮＡ片段（位于Ｇ基因４０５～１１４６号碱基）和推导的氨基酸序列，它含有可以诱导产生中和抗体的多个抗原决定簇和决定毒力及与神经细胞受体结合位点。计算机的比较分析表明两者在这一基因片段的同源性很低，核苷酸和氨基酸序列的同源性分别为８１．１％和８５．４％，主要抗原决定簇ＡｇⅡ的２０２位和ＡｇⅢ的３３２位氨基酸两者不同，并且ＭＲＶ在２０２位形成糖基化位点。研究结果证明ＢＲＶ和ＭＲＶ是亲缘关系较远的毒株，甚至有型或亚型的差别，两者的核苷酸和氨基酸序列均有较大差异，ＢＲＶ不可能是ＭＲＶ在短时间内演化而来的毒株。