高致病性禽流感病毒H5N1亚型核蛋白NP真核表达载体的构建、表达与鉴定

目的构建高致病性禽流感病毒H5N1亚型核蛋白(NP)的真核表达载体,并在哺乳动物细胞中表达、鉴定其编码重组蛋白NP。方法采用RT-PCR法扩增NP基因,T-A克隆到pMD18-T载体中构建pMD18-T-NP质粒。经PCR、双酶切鉴定后,双酶切阳性质粒与pXJ40-HA载体,电泳后胶回收,连接目的片段,构建pXJ40-HA-NP质粒,经PCR、双酶切、测序分析鉴定为阳性的质粒即为NP蛋白的真核表达载体。转染293T细胞后,采用免疫印迹法(Western blot)鉴定重组NP蛋白的表达。结果成功构建了高致病性禽流感病毒H5N1亚型NP基因的真核表达载体,并在293T细胞中成功表达出分子量为56kD的重组蛋白。结论成功构建的NP蛋白真核表达载体为进一步研究其功能,研发高致病性禽流感病毒H5N1亚型的诊断、治疗方法和疫苗奠定了基础。     

甲型流感病毒SwH1N1血凝素蛋白HA1真核表达载体的构建与表达

目的构建甲型流感病毒SwH1N1血凝素蛋白(HA1)的真核表达载体,并表达其编码蛋白HA1。方法利用RT-PCR技术扩增HA1基因,克隆至pMD18-T Simple Vector中构建pMD18-T-HA1质粒。双酶切pMD18-T-HA1与PXJ40后回收并连接回收片段,构建PXJ40-HA1真核表达载体,鉴定后转染293T细胞,用免疫印迹法(western-bolt)鉴定重组HA1蛋白的表达。结果实验成功构建HA1基因的真核表达载体,并在真核细胞中表达出分子量为40kD的重组蛋白。结论成功构建的甲型流感病毒SwH1N1HA1基因的真核表达载体,可为后期的流感快速检测及基因工程疫苗的制备奠定良好的基础。     

甲型流感病毒H1N1亚型NS1蛋白真核表达载体的构建与表达

[目的]构建甲型流感病毒H1N1亚型NS1蛋白真核表达载体,并在293T细胞中表达.[方法]采用RT-PCR技术,从甲型流感病毒H1N1毒株提取的病毒总RNA中,扩增NS1全长基因,将其克隆至pMD18-T Vector中构建pMD18-T-NS1质粒,双酶切pMD18-T-NS1与PXJ40-HA后,构建真核表达载体PXJ40-HA-NS1,经酶切及测序鉴定后将质粒转染到293T细胞中,通过免疫印迹法鉴定NS1蛋白的表达.[结果]酶切、测序证明重组真核表达载体PXJ40-HA-NS1构建成功,免疫印迹法可见NS1基因编码蛋白表达.[结论]成功构建甲型流感病毒H1N1亚型NS1蛋白真核表达载体PXJ40-HA-NS1,并在293T细胞中传染表达,该表达载体的构建为后期建立稳定表达NS1蛋白的细胞模型和NS1蛋白功能研究提供了材料.     

H5N1亚型禽流感病毒聚合酶酸性蛋白真核表达载体的构建与表达

目的构建甲型禽流感病毒H5N1亚型聚合酶酸性蛋白(PA)的真核表达载体,并表达其编码蛋白PA。方法采用RT-PCR法扩增PA基因,克隆至pMD18-T载体中构pMD18-T-PA质粒。双酶切pMD18-T-PA质粒与pXJ40-HA质粒后,胶回收并连接目的片段,构建真核表达载体pXJ40-HA-PA,鉴定后转染293T细胞。采用免疫印迹法(Western blot)鉴定重组PA蛋白的表达。结果成功构建了禽流感病毒H5N1亚型PA基因的真核表达载体,并在真核细胞中成功表达出分子量为75kD的重组蛋白。结论成功构建禽流感病毒H5N1亚型PA基因的真核表达载体,为后期进一步研究PA蛋白的功能奠定了基础。     

Dectin-1融合蛋白真核表达载体的构建及鉴定

目的 构建小鼠Dectin-1融合蛋白真核表达质粒并进行真核表达初步研究.方法 运用RT-PCR技术扩增小鼠腹腔巨噬细胞Dectin-1基因的细胞外碳水化合物识别域(CRD),与真核表达载体pSecTag2C进行双酶切之后进行连接反应,构建重组融合表达载体,转染入293细胞中,目的 蛋白在细胞培养基中分泌表达,表达产物经蛋白浓缩、纯化后经SDS-PAGE、Western印迹鉴定.结果 获得重组真核表达载体pSecTag2C-Dectin-1,测序结果 证明质粒DNA序列完全正确,并在转染入293细胞后检测到目的 蛋白的表达.结论 成功构建重组真核表达载体pSecTag2C-Dectin-1,为进一步纯化蛋白研究真菌检测方法 及Dectin-1与真菌相互作用机制、功能等奠定了基础.     

H5N1禽流感病毒血凝素和神经氨酸酶基因在杆状病毒中的共表达

目的构建共表达H5N1禽流感病毒血凝素(HA)和神经氨酸酶(NA)蛋白的杆状病毒表达系统。方法利用PCR方法分别扩增A/Hubei/1/2010(H5N1)病毒的HA和NA基因,克隆至经改造带有对虾白斑综合病毒(WSSV)早期启动子iel的mpFastBac Dual载体,构建mpFast Bac-HA-NA表达质粒,转化DH10Bac感受态细胞,用获得的Bacmid-HA-NA穿梭质粒转染sf9细胞,获得重组杆状病毒Bac-HA-NA;提取重组杆状病毒Bac-HA-NA的DNA并使用PCR方法检测;利用Western blot检测HA和NA表达,并分别检测重组杆状病毒Bac-HA-NA红细胞凝集能力和神经氨酸酶活性。结果经PCR鉴定,构建的质粒Bacmid-HA-NA正确;Western blot检测表明Bacmid-HANA能在sf9细胞中有效表达HA和NA蛋白,生物活性检测表明Bac-HA-NA能引起红细胞凝集并具有神经氨酸酶活性。结论获得了能同时表达禽流感病毒HA和NA的重组杆状病毒Bac-HA-NA,为进一步研究流感疫苗奠定了基础。     

寨卡病毒非结构蛋白1的真核表达纯化和免疫反应性鉴定

目的 在HEK293F细胞中表达寨卡病毒(Zika Virus,ZKV)的非结构蛋白1(Nonstructural Protein 1,NS1),并分离纯化得到重组ZKNS1以及验证其免疫反应性。方法 根据GenBank中的ZKNS1序列合成基因,在基因上下游分别引入信号肽和组氨酸标签,通过NotI和XbaI双酶切将基因克隆至pcDNA31(+)载体;将重组载体转染HEK293F细胞,重组蛋白用镍亲和柱层析纯化;免疫印迹法(Western blot)鉴定重组ZKNS1与寨卡病毒阳性人血清的免疫反应性。结果 成功构建了ZKNS1的真核表达载体pcDNA31(+)-ZKNS1;在HEK293F细胞中重组ZKNS1以分泌形式表达于细胞培养上清中,经纯化后获得了纯度较高的分子量约为46kDa的重组蛋白;Western blot结果显示重组ZKNS1与寨卡病毒阳性人血清能发生特异性结合。结论 成功构建ZKNS1的真核表达载体,获得了免疫反应性较高的纯化的重组ZKNS1,为进一步建立ELISA检测方法及制备单克隆抗体奠定了基础。     

ERCC1密码子118单核苷酸多态性转染细胞模型建立

目的 建立切除修复交叉互补集团1(ERCC1)基因单核苷酸多态性(SNP)转染细胞模型,为体外研究基因多态性功能提供平台.方法Gateway定向克隆技术构建ERCC1表达载体,包含不同ERCC1 codon 118 SNP表达载体转染中国仓鼠卵巢细胞(CHO)缺陷型UV20细胞,蛋白免疫印迹法(western blot)检测ERCC1蛋白表达,焦磷酸短序列测序PSQ检测ERCC1 118 SNP基因型.结果酶切鉴定及测序分析表明ERCC1表达载体构建正确,各转染细胞表达绿色荧光蛋白并可在G418培养基选择生长;ERCC1在UV20细胞中表达分子量为38 kDa蛋白,ERCC1SNP基因型正确,2种转染细胞ERCC1的SNP基因型不同,分别为C/C或T/T.结论 ERCC1 codon 118 SNP细胞转染模型构建成功,为体外研究ERCC1 codon 118 SNP不同基因型提供了实验技术平台.     

人Notch受体胞内区基因N2ICD克隆及真核表达

目的构建真核重组质粒N2ICD/p CMV-Tag4并转染HEK 293T细胞进行表达。方法根据GenBank上Notch2的NICD序列设计引物,采用逆转录聚合酶链反应(RT-PCR)的方法从人宫颈癌细胞Hela细胞扩增人Notch2受体胞内区基因(N2ICD),酶切和测序鉴定后克隆至携带FLAG标签的真核表达载体pCMV-Tag4并进行瞬时和稳定转染HEK 293T细胞,荧光定量PCR(Q-PCR)和蛋白印迹(WB)检测目的蛋白的表达。结果通过RT-PCR克隆获得人N2ICD基因并成功构建重组质粒N2ICD/pCMV-Tag4,目的蛋白N2ICD在HEK 293T细胞中获得表达。结论成功构建稳定表达N2ICD的HEK 293T细胞株,为进一步探讨Notch2受体的功能奠定基础。     

H7N9禽流感病毒HA2基因真核表达与免疫原性初步研究

目的利用真核表达载体构建H7N9禽流感病毒血凝素刺突茎部(HA2)的真核表达质粒,在293F细胞中表达HA2蛋白,并初步探讨其免疫原性。方法根据pMD18-T-HA中的序列设计H7N9禽流感病毒HA2扩增引物,在下游引物中引入胰酶酶切位点;目的基因经特异性酶切位点克隆入自身带有Fc标签的pFUSE-IgG1-Fc1载体,构建重组质粒HA2-Fc;将重组质粒转染293F细胞,通过间接免疫荧荧光(IFA)和免疫印迹法(WB)鉴定HA2蛋白的表达和免疫原性。结果成功构建H7N9禽流感病毒HA2基因真核表达质粒HA2-Fc,并在293F细胞中表达出分子量大约为50kDa的重组蛋白。IFA和WB显示该蛋白与抗H7N9病毒鼠多抗具有良好的免疫反应。结论成功构建表达HA2亚单位的真核表达系统,重组蛋白具有较高的免疫原性,为筛选H7N9广谱疫苗候选分子、广谱中和抗体,及深入研究其致病机理和免疫机制奠定基础。     

GSTM1及GSTT1和GSTP1基因多态性对尿中1-羟基芘水平的影响

目的 研究GSTM1、GSTT1和GSTP1基因多态性对多环芳烃接触工人尿中1-羟基芘(1-OHP)水平的影响.方法 分别选取2个炼焦厂共447名多环芳烃职业接触工人(接触组)和某线材厂220名非职业接触工人(对照组)作为研究对象,采用高效液相色谱法测定尿中1-OHP水平,采用线性回归统计模型分析GSTM1和GSTT1缺失型及GSTP1 I105V位点的多态性对不同人群尿中1-OHP水平的修饰作用.结果 接触组工人尿中1-OHP浓度为4.61 μmol/mol Cr,明显高于对照组(0.34μmol/mol Cr),差异有统计学意义(P＜0.05).接触类别和吸烟分别是影响尿中1-OHP水平的主要因素,在控制各混杂因素的影响后,线性回归分析显示,接触组尿中1-OHP水平和GSTP1 I105V位点多态性有关(单基因分析,P=0.012;多基因分析,P=0.011),对总体样本,单基因模型和多基因模型均显示,尿中1-OHP水平可能和GSTT1缺失型多态有关(P=0.055),多基因交互作用分析显示,GSTT1和GSTP1基因多态对接触组尿中1-OHP水平具有交互作用.结论 谷胱甘肽硫转移酶(GSTs)基因的多态性对接触多环芳烃工人尿中1-OHP水平有影响.

Abstract：

Objective To investigate the modification of GSTM1, GSTT1 and GSTP1 gene polymorphisms on urinary 1-hydroxypyrene (1-OHP) excretions in workers under different exposure levels. Methods Four hundred and forty-seven occupationally exposed workers from two coking plants and 220 control workers from a wire rod plant were genotyped to analyze the modification of GSTM1, GSTT1 and GSTP1 gene polymorphisms on urinary 1-OHP excretions. Results The urinary 1-OHP concentration in exposed group was much higher than that in control group (4.61 vs 0.34 μmol/mol Cr, P＜0.05). Occupational exposure levels and cigarette smoking were of the dominating factors affecting 1-OHP excretions in urine. After controlling potential confounders, decreased excretion of urinary 1-OHP was associated with GSTP1 I105V AG + GG genotype in coke oven workers (single-gene model, P=0.012; multi-gene model, P=0.011 ) and with GSTT1 null type in the analysis including all subjects (P=0.055 in both single-gene and multi-gene models). GSTT1 and GSTP1 were interacted on the urinary concentrations of 1-OHP. Conclusion Urinary 1-OHP concentrations can be modified by GSTM1, GSTT1 and GSTP1 gene polymorphisms, indicating that these genes are involved in the metabolism of polycyclic aromatic hydrocarbons.     

多药耐药及相关蛋白基因抗砷作用

目的 研究多药耐药基因(MDRI)、多药耐药相关蛋白基因(MRP1)在长期砷暴露的细胞获得对砷的耐受过程中的作用,为抗砷机制的研究提供基础依据.方法 采用抑制剂维拉帕米(Verapamil)、MK571,在单一水平和联合水平上阻断MDR1和MRP1基因发挥作用,通过四甲基偶氮噻唑蓝(MTT)法检测细胞的生存率及半数致死量(IC50);通过原子荧光法(AFS)分别检测给予Vempamil、MK571的实验组和对照组在不同时间点抗砷细胞内砷的含量.结果 在2,4,8,16,32,64 μmol/L砷浓度下,给予抑制剂的抗砷细胞生存率分别为(73.5±0.02)%,(54.6±0.07)%,(44.2±0.05)%,(20.5±0.09)%,(13.5±0.1)%,(7.6±0.05)%,明显低于同步对照组的生存率(93.5±0.05)%,(71.5±0.02)%,(49.2±0.03)%,(38.8±0.08)%,(37.6±0.06)%,(19.5±0.04)%.Verapamil作用下IC50为8.8 μmol/L,MK571作用下IC50为6.22 μmol/L;同时给于2种抑制剂时,逆转作用更为明显,IC50为5.23μmol/L;MK571作用下,抗砷细胞内砷含量增加明显,至10 h时砷含量达到最大值1.62 ng,明显高出Verapmil组和对照组,差异有统计学意义(P<0.05).结论 MDR1、MRP1基因在抗砷细胞获得耐药性过程中起重要作用.     

工作场所空气中1,1-二氯-1-硝基乙烷的气相色谱测定法

目的 建立工作场所空气中1,1-二氯-1-硝基乙烷的气相色谱测定方法.方法 活性炭管采集工作场所空气中的1,1-二氯-1-硝基乙烷,以二硫化碳解吸后毛细管柱气相色谱法测定.结果 方法的测定范围为4.0～858.2μg/ml;回归方程Y=283X-1076,相关系数r=0.9999;最低检出浓度为0.4 mg/m3(以采集3L空气样品计);不同浓度测定的相对标准偏差(RSD)为1.8%～4.1%(批内精密度试验的RSD分别为2.9%、1.8%和2.0%;批间精密度试验的RSD分别为4.1%、3.5%和3.2%);解吸效率为88.5%～90.6%;穿透容量大于0.7 mg;采样效率100%;样品在室温下至少可保存7 d.结论 方法的各项指标均符合GBZ/T210.4-2008<职业卫生标准制订指南-工作场所空气中化学物质测定方法>要求,可用于工作场所空气中1,1-二氯-1-硝基乙烷的测定.     

Co-circulation of pandemic 2009 H1N1, classical swine H1N1 and avian-like swine H1N1 influenza viruses in pigs in China

The pandemic A/H1N1 influenza viruses emerged in both Mexico and the United States in March 2009, and were transmitted efficiently in the human population. They were transmitted occasionally from humans to other mammals including pigs, dogs and cats. In this study, we report the isolation and genetic analysis of novel viruses in pigs in China. These viruses were related phylogenetically to the pandemic 2009 H1N1 influenza viruses isolated from humans and pigs, which indicates that the pandemic virus is currently circulating in swine populations, and this hypothesis was further supported by serological surveillance of pig sera collected within the same period. Furthermore, we isolated another two H1N1 viruses belonging to the lineages of classical swine H1N1 virus and avian-like swine H1N1 virus, respectively. Multiple genetic lineages of H1N1 viruses are co-circulating in the swine population, which highlights the importance of intensive surveillance for swine influenza in China.     

Detection of CYP1A1 gene polymorphism using designed RFLP and distributions of CYP1A1 genotypes in Japanese

Isoleucine (Ile)-valine (Val) polymorphism, which is caused by a point mutation from A to G in exon 7, is reported to be associated with an elevated risk of lung cancer among Japanese. Because CYP1A1 catalyzes bioactivation of environmental procarcinogens, such as benzo[a]pyrene, it is very important to study the clinical meaning of Ile-Val polymorphism using an epidemiological study. In an epidemiological study, easy, economical, rapid and reliable identification of the CYP1A1 genotype is necessary. The present study shows that the new method, designed restriction fragment length polymorphism (designed RFLP), can detect Ile-Val polymorphism of CYP1A1 The Ile-Val polymorphism detected using this new method was consistent with that found by the allele-specific PCR amplifications (ASA) method in six cases tested. This new method detected Ile-Va1 polymorphism of CYP1A1 using 240 healthy Japanese who lived in the northern Kyusyu region. The frequency of the genotypes was as follows: Ile/Ile, 159 (66.2%); Ile/Val, 65 (27.1%); Val/Val, 16 (6.7%). The frequency of the Ile gene was 0.798 and that of the Val gene, 0.202. There was no difference in Ile-Val polymorphism based on sex or age. Racial differences influenced the distribution of this polymorphism, but Japanese regional differences did not. Since this new method, designed RFLP, is rapid, reliable and suitable for large-scale screening of polymorphisms, it may be used routinely to detect Ile-Val polymorphism of CYP1A1 Furthermore, it will help to evaluate the relationship between CYP1A1 polymorphism and individual sensitivity to xenobiotics that may affect the incidence of lung cancer.     

CYP1A1和CYP1B1基因多态性与RPL易感性

目的 探讨CYP1A1、CYP1B1基因多态性与复发性流产(RPL)遗传易感性关系,为预防和治疗该病提供新靶点.方法 本研究采用等位基因特异性PCR (As-PCR)和聚合酶链反应-限制性片断长度多态性(PCRRFLP)方法,针对CYP1A1基因MspI酶切位点和CYP1B1 L432V多态位点,检测81例患有原因不明RPL病例组和98名有生育史健康女性对照组之间差异.结果 RPL组和对照组CYP1A1 MspI位点3种基因型m1/m1、m1/m2、m2/m2分布频率差异无统计学意义(x2=0.335,P＞0.05);CYP1B1 L432V多态位点3种基因型C/C、C/G、G/G在病例组和对照组分布差异有统计学意义(x2=7.467,P＜0.05);2组间C、G等位基因分布差异有统计学意义(x2=9.129,P=0.003);G/G、C/G基因型与C/C基因型比较,RPL危险度分别提高2.620、1.954倍;等位基因G使RPL危险性增加2.038倍.结论 CYP1B1 L432V突变基因型增加RPL发病风险,尚不能认为CYP1A1基因MspI位点多态性与RPL易感性有关.     

Protective efficacy of an inactivated Eurasian avian-like H1N1 swine influenza vaccine against homologous H1N1 and heterologous H1N1 and H1N2 viruses in mice

Eurasian avian-like H1N1 (EA H1N1) swine influenza viruses are prevalent in pigs in Europe and Asia, but occasionally cause human infection, which raises concern about their pandemic potential. Here, we produced a whole-virus inactivated vaccine with an EA H1N1 strain (A/swine/Guangxi/18/2011, SW/GX/18/11) and evaluated its efficacy against homologous H1N1 and heterologous H1N1 and H1N2 influenza viruses in mice. A strong humoral immune response, which we measured by hemagglutination inhibition (HI) and virus neutralization (VN), was induced in the vaccine-inoculated mice upon challenge. The inactivated SW/GX/18/11 vaccine provided complete protection against challenge with homologous SW/GX/18/11 virus in mice and provided effective protection against challenge with heterologous H1N1 and H1N2 viruses with distinctive genomic combinations. Our findings suggest that this EA H1N1 vaccine can provide protection against both homologous H1N1 and heterologous H1N1 or H1N2 virus infection. As such, it is an excellent vaccine candidate to prevent H1N1 swine influenza.