利用杆状病毒表达幽门螺杆菌cagA基因

幽门螺杆菌cagA基因克隆到杆状病毒表达系统的pBlueBacHis2A转移载体中,将重组质粒pBlueBacHis2A-CagA与亲本病毒Bac-N-blue DNA共转染Sf9细胞,以空斑法纯化获得的重组杆状病毒.经PCR法鉴定后进行扩增培养,SDS-PAGE和Western bolt检测结果证实所表达的蛋白为CagA蛋白,间接ELISA分析表明,表达产物可与Hp感染者血清发生特异性的免疫反应.     

甲型H1N1流感病毒HA基因在昆虫细胞中的截短表达

目的:利用Bac-to-Bac Baculovirus Expression System表达重组HA蛋白,Western blot及IFA方法鉴定其表达。方法:采用PCR方法扩增A/California/04/2009(H1N1)HA基因,将其克隆到pFastBacHT A载体上,重组质粒pFastBacHT-HA经双酶切及测序鉴定正确后,转化阳性重组载体进入E.coli DH10Bac感受态细胞中,通过Bluo-gal蓝白斑筛选、PCR鉴定获得重组转座子rBacmid-HA。从重组转座子中提取rBacmid-HA质粒DNA转染sf 9昆虫细胞,制备重组杆状病毒。重组杆状病毒感染sf 9细胞表达重组蛋白,Western blot及IFA鉴定重组蛋白表达情况。结论:成功构建了甲型H1N1流感病毒HA基因的昆虫杆状病毒表达载体,该表达载体转染昆虫细胞后制备的重组杆状病毒病毒滴度较高,重组杆状病毒表达的重组蛋白经Western blot 及IFA 鉴定后具有良好的免疫反应原性。     

幽门螺杆菌cagA克隆表达

克隆表达4株幽门螺杆菌的cagA基因,以方便地获得大鼠CagA蛋白和重组表达质粒,为临床诊断CagA阳性幽门螺杆菌感染,以及进一步研究不同类型CagA功能及其与疾病关系提供材料。PCR扩增幽门螺杆菌的cagA基因,克隆至PinPoint^TMXa-1T载体,酶切鉴定连接方向,IPTG诱导正向连接克隆表达CagA融合蛋白并进行SDS－PAGE和Western blots鉴定。结果显示PCR扩增得到3.5-3.8kb的CagA基因,PCR及酶切鉴定得到正向连接的重组克隆,SDS－PAGE及Western blots证实正向连接的重组克隆表达CagA融合蛋白。构建了4种cagA的重组表达质粒,通过转化同一宿主菌可研究不同CagA的功能和致病性差异;通过亲和层析纯化融合蛋白可获大量CagA蛋白,用于血清学诊断CagA阳性幽门螺杆菌感染,及不同抗原性CagA与疾病之间的关系。     

丙型肝炎病毒NS5A基因在昆虫细胞中的表达及其分布研究

应用PCR方法从含有丙肝病毒全部非结构蛋白基因的质粒pBAC25中扩增出全长的NS5A基因DNA片段(约1.34kb),PCR扩增NS5A基因片段克隆到转移载体pBlueBacHisA中.重组转移质粒pBlueBacHis5A DNA与野生型杆状病毒(AcNPV)DNA共转染SF-9昆虫细胞,通过空斑纯化获得带有NS5A基因的重组病AcNS5A.对重组病毒基因组DNA进行酶切和PCR鉴定,证实HCV NS5A基因已插入重组病毒基因组中.AcNS5A感染SF-9细胞后,在细胞中表达出一条64kD的蛋白,用Western-blot分析,结果表明这种蛋白与抗HCV HS5A特异性抗体发生强烈反应,说明NS5A基因已在细胞中得到表达,应用免疫荧光技术与免疫组化技术进一步研究NS5A蛋白在昆虫细胞中不同时间的表达情况及其分布,结果表明,NS5A蛋白在AcNS5A重组病毒感染细胞24h后主要分布在细胞质膜上,而在48h后则同时分布于细胞质膜和细胞核内,在72h则完全布满整个细胞,我们认为NS5A蛋白定位于质膜和细胞核中,暗示着在病毒复制过程中NS5A蛋白可能参与病毒RNA在质膜上复制和细胞基因表达的调控.     

16型人乳头瘤病毒衣壳蛋白在真核细胞中的表达

为了构建HPV16型晚期蛋白重组杆状病毒,并使其在昆虫细胞中获得高效表达.首先构建2株重组杆状病毒转移质粒,分别携带人乳头瘤病毒晚期基因L1及L1和L2,再用线性化的杆状病毒DNA与该重组杆状病毒转移质粒共转染sf9昆虫细胞进行同源重组,获得2株重组杆状病毒.经鉴定该重组病毒中有目的基因存在且可表达所编码的L1或L2晚期蛋白.结果表明HPV16型晚期蛋白在昆虫细胞中获得成功表达,为HPV16型预防性基因工程亚单位疫苗的研制和诊断试剂的研究开发奠定了基础.     

以杆状病毒为载体在鸡原代骨骼肌细胞中表达IBDV VP2基因

摘要：【目的】本研究旨在构建在鸡原代骨骼肌细胞中表达IBDV病毒VP2基因的重组杆状病毒。【方法】从IBDV适应细胞毒中提取RNA,用RT-PCR技术扩增VP2基因,将其克隆到自主构建的杆状病毒转移载体的CMV启动子之下,通过Bac-to-Bac系统获得VP2重组Bacmid,并将其转染Sf9昆虫 细胞,获得了VP2重组杆状病毒。重组病毒经扩增后以50个MOI感染鸡原代骨骼肌细胞,接种72h后裂解细胞收获蛋白。【结果】蛋白样品经SDS-PAGE和Western blot证实VP2蛋白获得表达,分子量约48kDa,与预测蛋白大小一致,且能被IBDV阳性血清所识别。【结论】重组杆状病毒可以有效地将VP2基因导入鸡原代细胞,并在CMV的启动下表达具有抗原性的VP2蛋白,本研究为研制IBDV及其他重要禽类传染病的杆状病毒载体疫苗奠定了基础。     

昆虫杆状病毒表达系统的研究进展与应用

昆虫杆状病毒表达载体系统具有安全性好、重组蛋白表达量高、能同时表达多个基因、重组蛋白翻译后加工完整等特点,因而得到了广泛的应用。随着重组杆状病毒构建技术的不断发展,昆虫杆状病毒表达载体系统的操作在逐渐简化,重组杆状病毒获得的效率也在不断提高。昆虫细胞培养技术的改进和转基因昆虫细胞系的发展,进一步推动了昆虫杆状病毒表达载体系统在商品化药物、治疗性抗体、生物农药研发和基因治疗中的应用。尽管仍存在着重组蛋白降解的问题,但随着分子生物学技术的发展,对杆状病毒载体的研究与改造也会更加深入,未来昆虫杆状病毒表达载体系统的应用将更为广泛。     

猪瘟病毒E2基因在昆虫细胞/杆状病毒中的表达

目的利用昆虫细胞/杆状病毒系统表达猪瘟病毒(CSFV)E2蛋白,用于E2蛋白功能、开发CSF新型疫苗以及建立相关血清学诊断方法等研究。方法采用RT-PCR扩增CSFV E2基因,将PCR产物克隆到pGEM-T-Easy载体,将该基因插入到pFast-BacHT A载体中,构建重组转座载体后转化DH10Bac感受态细胞,获得重组Bacmid质粒后转染sf9昆虫细胞,传毒3代,对表达蛋白进行Western-blot及免疫组化鉴定。结果成功克隆CSFVE2基因,其核苷酸序列为1119 bp。SDS-PAGE电泳结果显示表达E2蛋白相对分子质量约为43×103,Western-blot和免疫组化结果证实表达蛋白能够被CSFV标准阳性血清识别。结论在Bac-to-Bac杆状病毒系统中的成功表达了CSFV E2蛋白,与CSFV标准阳性血清具有较好的反应性。     

幽门螺杆菌东、西方株CagA的序列差异及其对胃癌细胞生长与凋亡的影响

【背景】幽门螺杆菌(Helicobacter pylori,H.pylori)是胃癌的主要致病因素,其分泌的细胞毒素相关基因A蛋白(Cytotoxin associated gene A,CagA)是目前已知唯一能被H.pylori注入胃上皮细胞并模拟细胞内蛋白发挥作用的癌蛋白,参与胃癌的发生发展。【目的】比较H.pylori东亚株和西方株CagA结构差异,初步探讨H.pylori-CagA对胃癌细胞增殖与凋亡的影响。【方法】对H.pylori东亚株和西方株CagA的核酸及氨基酸序列进行生物信息学分析,构建含东亚株和西方株cagA基因的真核表达载体,转染胃癌细胞AGS,用Western blot法检测CagA蛋白的表达,用CCK8法测定细胞的生长曲线,流式细胞术检测细胞凋亡。【结果】生物信息学分析发现H.pylori东亚、西方菌株CagA的核酸序列和氨基酸序列均存在特征性差异。构建了含东亚、西方菌株cagA基因的表达载体[命名为GZ7/cagA(东亚株)和26695/cagA(西方株)]。与空载体组比较,GZ7/cagA和26695/cagA转染组均表达CagA蛋白,两组比较表达量无显著性差异,GZ7/cagA转染组细胞生长显著增加,而26695/cagA转染组细胞生长显著降低(P0.05)。GZ7/cagA转染组、26695/cagA转染组细胞的凋亡率分别为7.23±0.96及9.17±1.40,均高于空载体组(5.03±0.63),差异有统计学意义(P0.05)。【结论】东亚株与西方株CagA之间有结构和功能的差异,东亚株CagA能促进细胞增殖,而西方株CagA却抑制细胞增殖,但两者均能促进细胞凋亡。     

幽门螺杆菌CagA蛋白N端片段的表达、纯化及其与磷脂酰丝氨酸的亲和力检测

目的:表达和纯化幽门螺杆菌不同菌株的CagA蛋白N端片段,检测其与磷脂酰丝氨酸(PS)的相互作用及亲和力。方法:用PCR方法从幽门螺杆菌3个菌株中扩增出CagA蛋白N端基因,并连接到表达载体pET-28a上;转化大肠杆菌BL21,经IPTG诱导可溶性表达CagA蛋白N端880残基片段;经镍柱亲和纯化后,利用PLOA法检测CagA蛋白与PS的相互作用。结果:构建了3种幽门螺杆菌菌株cagA基因的原核表达质粒pET-28a/cagAJ99、pET-28a/cagA11637及pET-28a/cagASS1,并在大肠杆菌中获得可溶性表达,SDS-PAGE和Western印迹证实得到目标融合蛋白,亲和纯化得到高纯度CagA蛋白。PLOA结果表明,CagA蛋白与PS有明显的相互作用。结论:3种幽门螺杆菌菌株CagA蛋白与PS之间存在相互作用,且不同的CagA与PS有不同的亲和力。     

Helicobacter pylori stimulates gastric epithelial cell MMP-1 secretion via CagA-dependent and -independent ERK activation

Because the mechanisms of Helicobacter pylori-induced gastric injury are incompletely understood, we examined the hypothesis that H. pylori induces matrix metalloproteinase-1 (MMP-1) secretion, with potential to disrupt gastric stroma. We further tested the role of CagA, an H. pylori virulence factor, in MMP-1 secretion. Co-incubation of AGS cells with Tx30a, an H. pylori strain lacking the cagA virulence gene, stimulated MMP-1 secretion, confirming cagA-independent secretion. Co-incubation with strain 147C (cagA(+)) resulted in CagA translocation into AGS cells and increased MMP-1 secretion relative to Tx30a. Transfection of cells with the recombinant 147C cagA gene also induced MMP-1 secretion, indicating that CagA can independently stimulate MMP-1 secretion. Co-incubation with strain 147A, containing a cagA gene that lacks an EPIYA tyrosine phosphorylation motif, as well as transfection with 147A cagA, yielded an MMP-1 secretion intermediate between no treatment and 147C, indicating that CagA tyrosine phosphorylation regulates cellular signaling in this model system. H. pylori induced activation of the MAP kinase ERK, with CagA-independent (early) and dependent (later) components. MEK inhibitors UO126 and PD98059 inhibited both CagA-independent and -dependent MMP-1 secretion, whereas p38 inhibition enhanced MMP-1 secretion and ERK activation, suggesting p38 negative regulation of MMP-1 and ERK. These data indicate H. pylori effects on host epithelial MMP-1 expression via ERK, with p38 playing a potential regulatory role.     

Sequence Analysis of East Asian cagA of Helicobacter pylori Isolated from Asymptomatic Healthy Japanese and Thai Individuals

CagA, especially East Asian type, is one of the most important virulence factors of Helicobacter pylori, which is believed to contribute to the gastric cancer development. There is extreme sequence heterogeneity on 3' region of cagA gene, demonstrated by the sequence analysis of cagA of H. pylori strains isolated from gastric disease patients. However, whether such heterogeneity of the cagA gene sequence is related to the pathogenicity of H. pylori in the gastric cancer development is not certain. Therefore, in this study, the 3' region of cagA sequences isolated from asymptomatic healthy individuals in Japan and Thailand, which show high and low gastric cancer prevalence, respectively, were analyzed and compared with those from patients with gastric cancer. The CagA sequences analysis in 21 and 12 H. pylori DNA samples obtained from Japanese and Thai individuals, respectively, by the molecular phylogenetic method showed that the sequences were more conserved in the Thai individuals (concordance rates among Thai sequences, 93.9-100%) than in the Japanese individuals (concordance rates among Japanese sequences, 82.8-100%) as shown by unrooted neighbor-joining (N-J) consensus trees constructed with the sequence between Asn869 and Ala967 in CagA. CagA sequences in gastric cancer patients were obtained from published data; analysis of these sequences revealed that CagA sequences from almost all Thai individuals were concentrated in one branch. In contrast, CagA sequences from Japanese individuals were uniformly distributed throughout the N-J consensus tree. These results suggest that the difference in the CagA sequences between asymptomatic healthy Japanese and Thai individuals may be linked to the incidence of gastric cancer in Japan and Thailand.     

Helicobacter pylori inhibits expression of heat shock protein 70 (HSP70) in human epithelial cell line. Importance of Cag A protein.

Heat shock proteins (HSP) are crucial for the maintenance of cell integrity under normal cell growth and at pathophysiological conditions such as colonization of gastric mucosa by Helicobacter pylori (Hp). The effect of Hp on mRNA expression for HSP70 in the gastric epithelial cells in vitro has been little studied and remains inconclusive. In this study we attempted to determine the alterations in gene expression for HSP70 induced by two live strains of Hp in the epithelial MKN7 cells. The following Hp strains were employed; 1) Hp strain expressing cagA and vacA, and 2) cagA and vacA negative Hp strain without or with addiction of exogenous recombinant protein CagA. MKN7 cells were incubated in a standard medium RPMI 1640 supplemented with 10% fetal bovine serum at 37 degrees C with 5% CO2 and humidified atmosphere under basal condition or in a presence of Hp (1 x 10(9) CFU per dish) without or with the recombinant CagA (10 microg/ml of RPMI 1640 medium). After 3 h, 24 h and 48 h of incubation with Hp and in some experiments with the prolonged incubation time up to 72 h, the cells were harvested, the total cellular RNA was isolated and the expression of mRNA for HSP70 was determined by RT-PCR. The incubation of the MKN cells with CagA protein alone failed to affect significantly the expression of HSP70. In contrast, the strain Hp (cagA+, vacA+) inhibited in time-dependent manner the expression of mRNA for HSP70. When the MKN7 cells were coincubated with Hp (cagA+, vacA+) and exogenous CagA, the significant inhibition of the signal intensity for HSP70 mRNA was observed at 3 h and 24 h of incubation and these effects were followed by complete disappearance of the signal for HSP70 mRNA at 48 h. The incubation of MKN7 with Hp (cagA-, vacA-) also significantly attenuated the expression of HSP70 mRNA with the most pronounced inhibitory effect observed at 72 h of incubation with this Hp strain. Addition of the recombinant CagA to Hp (cagA-, vacA-) completely suppressed the expression of HSP70 at 48 h and 72 h after the end of incubation periods. We conclude that 1) both, Hp (cagA+, vacA+) and Hp (cagA-, vacA-) inhibit expression of HSP70 in MKN7 human gastric epithelial cells independently of the presence or absence of cagA gene, and that 2) recombinant CagA protein may exert biological activity in vitro via acceleration of inhibitory effect of Hp negative for Cag A and VacA on HSP70 expression in epithelial cells infected with this bacteria.     

Identification of Helicobacter pylori and the cagA genotype in gastric biopsies using highly sensitive real-time PCR as a new diagnostic tool

The CagA protein is one of the virulence factors of Helicobacter pylori, and two major subtypes of CagA have been observed, the Western and East Asian type. CagA is injected from the bacteria into gastric epithelial cells, undergoes tyrosine phosphorylation, and binds to Src homology 2 domain-containing protein-tyrosine phosphatase SHP-2. The East Asian type CagA binds to SHP-2 more strongly than the Western type CagA. Here, we tried to distinguish the CagA type by highly sensitive real-time PCR with the objective of establishing a system to detect H. pylori and CagA subtypes from gastric biopsies. We designed primers and probe sets for Western or East Asian-cagA at Western-specific or East Asian-specific sequence regions, respectively, and H. pylori 16S rRNA. We could detect the H. pylori 16S rRNA gene, Western and East Asian-cagA gene from DNA of gastric biopsies. The sensitivity and specificity for H. pylori infection was 100% in this system. In Thai patients, 87.8% (36/41) were cagA-positive; 26.8% (11/41) were Western-cagA positive and 53.7% (22/41) were East Asian-cagA positive, while 7.3% (3/41) reacted with both types of cagA. These results suggest that this real-time PCR system provides a highly sensitive assessment of CagA type as a new diagnostic tool for the pathogenicity of H. pylori infection.     

Evolution of cagA oncogene of Helicobacter pylori through recombination

Helicobacter pylori is a gastric pathogen that infects half the human population and causes gastritis, ulcers, and cancer. The cagA gene product is a major virulence factor associated with gastric cancer. It is injected into epithelial cells, undergoes phosphorylation by host cell kinases, and perturbs host signaling pathways. CagA is known for its geographical, structural, and functional diversity in the C-terminal half, where an EPIYA host-interacting motif is repeated. The Western version of CagA carries the EPIYA segment types A, B, and C, while the East Asian CagA carries types A, B, and D and shows higher virulence. Many structural variants such as duplications and deletions are reported. In this study, we gained insight into the relationships of CagA variants through various modes of recombination, by analyzing all known cagA variants at the DNA sequence level with the single nucleotide resolution. Processes that occurred were: (i) homologous recombination between DNA sequences for CagA multimerization (CM) sequence; (ii) recombination between DNA sequences for the EPIYA motif; and (iii) recombination between short similar DNA sequences. The left half of the EPIYA-D segment characteristic of East Asian CagA was derived from Western type EPIYA, with Amerind type EPIYA as the intermediate, through rearrangements of specific sequences within the gene. Adaptive amino acid changes were detected in the variable region as well as in the conserved region at sites to which no specific function has yet been assigned. Each showed a unique evolutionary distribution. These results clarify recombination-mediated routes of cagA evolution and provide a solid basis for a deeper understanding of its function in pathogenesis.     

Translocation of the Helicobacter pylori CagA protein in gastric epithelial cells by a type IV secretion apparatus

Helicobacter pylori is one of the most common bacterial pathogens, infecting about 50% of the world population. The presence of a pathogenicity island (PAI) in H. pylori has been associated with gastric disease. We present evidence that the H. pylori protein encoded by the cytotoxin-associated gene A ( cagA ) is translocated and phosphorylated in infected epithelial cells. Two-dimensional gel electrophoresis (2-DE) of proteins isolated from infected AGS cells revealed H. pylori strain-specific and time-dependent tyrosine phosphorylation and dephosphorylation of several 125–135 kDa and 75–80 kDa proteins. Immunoblotting studies, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), cell fractionation and confocal microscopy demonstrated that one of the 125–135 kDa proteins represents the H. pylori CagA protein, which is translocated into the host cell membrane and the cytoplasm. Translocation of CagA was dependent on functional cagA gene and virulence ( vir ) genes of a type IV secretion apparatus composed of virB4 , virB7 , virB10 , virB11 and virD4 encoded in the cag PAI of H. pylori . Our findings support the view that H. pylori actively translocates virulence determinants, including CagA, which could be involved in the development of a variety of gastric disease.