Construction of transposon-mediated baculovirus vector and expression of green fluorescent protein in insect cells and larvae

A transposon-shuttle vector Hanpvid was constructed by using wild-type genomic DNA fromHeliothis armigera nuclear polyhedrosis virus (HaNPV). It could replicate inE. coli cells as a large plasmid and remain infectious when being induced into insect cells. Hanpvid comprises HaNPV DNA and a transposon cassette which includes a miniF replicon, a kanamycin resistance gene (kan), lacZa and an attachment site for Tn7 (attTn7). Recombinant virus rHa-FaGP was obtained after transposition of a donor plasmid carrying green fluorescent protein gene (gfp) and polyhedrin gene (ocu) into attTn7. SDS-PAGE analysis shows that both gfp and ocu genes were highly expressed inHeliothis armigera cells. Green Hemolymphocytes can be seen under a fluorescent microscope 4 d after recombinant virus rHa-FaGP infected the third-instar larvae. The infected larvae show strong green fluorescence 6 d post infection.     

Identification and expression of a gfpK79R mutant of green fluorescent protein

A mutant strain of E.coli showing strong green fluorescence is obtained during the study of cloning and expression of blue fluorescent protein gene bfp. A recombinant plasmid pHN122 has been isolated. It is demonstrated by enzyme digestion, subcloning and sequence analysis that it contains both of bfp and gfpK79R mutant genes. The results of spectrometric analysis show that GFPK79R expressed by pHN122 is the same as the wild type GFP, but its fluorescent intensity is increased 1.25～2.44-fold.     

乙型肝炎病毒HBX基因的克隆、表达和蛋白的纯化

克隆乙肝病毒X基因,并在大肠杆菌中进行表达和纯化。用PCR方法从结乙肝病毒基因组扩增出HBX基因片段,克隆至pMD18-T载体中。序列测定正确后,将其亚克隆到表达载体pProExHTa并在大肠杆菌BL21中表达。表达蛋白经SDS-PAG及Western-blot分析后,亲和层析法纯化蛋白。结果成功克隆了HBX基因,并对其在E.coli中进行了表达。SDS-PAGE及Western blot分析表明表达产物正确。通过IMAC纯化系统获得17 kD纯化蛋白,与文献报道相符。结果成功获得了纯化的HBX蛋白,为进一步研究HBX蛋白与宿主蛋白之间的相互作用奠定基础。     

Secretory expression of the fusion protein composed of human IL-2 and PreS antigen of hepatitis B virus in mammalian cells

In order to make entire HBV pmSAg secrete from mammalian cells, we conatmcted an eukaryotic expression vector by using leader sequence of human interleukin-2 (IL-2) as secretory signal peptide, and using high hydmphilic amino acids as the linker between IL-2 C end and preSAg N end. As a result, the IL-2preS fusing protein could be secreted from mamalian cells transfected with the reconstructed vector and the expression efficiency was identical to that of natural IL-2. It was considered that the retentive effect of preSlAg could be successfully bypassed. The results not only laid a theoretical and practical foundation for constructing specific gene vaccine against HBV persistent infection, but also supplied experimental evidence for studying modulation of protein secretory expression.     

Studies on the sructure, mechanism of green fluorescent protein and its application

The green fluorescent protein (GFP) from jellyfishAequorea victoria has unique superiority as a kind of biological label. GFP has been widely used in all fields of biology based on the recent studies on its structure, characteristic and mechanism of bioluninescence.     

Expression of green fluorescent protein gene with baculovirus vector in insect cells

The green fluorescence of bioluminescent jellyfishAequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10³ dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus-insect cell expression system. Supported by the National Natural Science Foundation of China Hu Jianhong: born in July, 1972, Master graduate student     

绿色荧光蛋白融合人核糖核酸酶抑制因子逆转录病毒载体pLNOX-EGFP-C1-hri的构建及鉴定

构建表达重组绿色荧光蛋白融合人核糖核酸酶抑制因子的逆转录病毒载体pLNCX—EGFP-c1-hri，为探讨人核糖核酸酶抑制因子抗肿瘤作用机制打下基础．用亚克隆法，将目的片段egfp-c1—hri从表达载体pEGFP-C1-hri克隆到pLNCX上，用酶切筛选得到阳性克隆后，用脂质体法将其转染到小鼠黑色素瘤细胞B16中，用800mg／L G418筛选2周后，在荧光显微镜下检测其表达．双酶切鉴定得到pLNCX-EGFP-C1-hri阳性克隆，荧光显微镜显示绿色荧光在B16细胞质中高效表达．成功构建逆转录病毒表达载体pLNCX—EGFP-C1-hri．     

绿色荧光蛋白融合人核糖核酸酶抑制因子逆转录病毒载体pLNCX-EGFP-C1-hri的构建及鉴定

构建表达重组绿色荧光蛋白融合人核糖核酸酶抑制因子的逆转录病毒载体pLNCX-EGFP-c1-hri,为探讨人核糖核酸酶抑制因子抗肿瘤作用机制打下基础.用亚克隆法,将目的片段egfp-c1-hri从表达载体pEGFP-C1-hri克隆到pLNCX上,用酶切筛选得到阳性克隆后,用脂质体法将其转染到小鼠黑色素瘤细胞B16中,用800 mg/L G418筛选2周后,在荧光显微镜下检测其表达.双酶切鉴定得到pLNCX-EGFP-C1-hri阳性克隆,荧光显微镜显示绿色荧光在B16细胞质中高效表达.成功构建逆转录病毒表达载体pLNCX-EGFP-C1-hri.     

乙型肝炎母婴垂直传播(综述)

综述乙肝母婴传播的途径及其阻断方法。乙肝母婴传播主要通过胎盘、乳汁、羊水及唾液传播。目前主要阻断方法为应用乙肝免疫球蛋白、乙肝疫苗及拉米夫定等治疗     

大鼠谷氨酰胺转运蛋白EGFP-SNAT3融合蛋白的表达与鉴定

SLC38基因家族编码的Na+偶联的中性氨基酸转运蛋白3(SNAT3)是System N中的一员.它在大脑谷氨酸-谷氨酰胺循环以及肝细胞质膜的谷氨酰胺转运等生理过程中发挥着重要作用.为了方便检测SNAT3在细胞膜上的表达与定位,本研究采用PCR扩增和酶切连接技术将增强型绿色荧光蛋白(EGFP)与SNAT3的N末端连接,通过菌液PCR、酶切和DNA测序验证得到pBK-CMV(Δ[1098-1300])-EGFP-SNAT3重组真核表达质粒.用脂质体转染法将构建好的质粒瞬时转染进入人体胚肾细胞(HEK293T cells),利用激光扫描共聚焦显微镜和Western blot技术检测EGFP-SNAT3融合蛋白的表达和亚细胞定位.结果表明,EGFPSNAT3重组质粒在细胞中正确表达并定位于细胞膜上.EGFP-SNAT3重组质粒的成功构建将有助于日后进一步研究SNAT3的结构和功能.     

甲/戊型肝炎病毒重组抗原基因的克隆及表达

 将甲肝病毒vp1编码基因(aa 24~171)和戊型肝炎病毒ORF2基因(aa 431~615)通过一段编码柔性甘氨酸链(Gly4 Ser Gly4)的基因片断连接,获得编码HAV/HEV融合蛋白基因(AEAg342).将该融合基因插入质粒pBV220,构建表达质粒pBV220-AEAg342.将pBV220-AEAg342转化入大肠杆菌BL21中进行表达,表达量约占菌体总量的20%,经离子交换层析纯化,目的蛋白纯度达90%以上.Western blot证实该蛋白能与甲肝及戊肝患者阳性血清发生特异性反映,为进一步开发双价疫苗和联合诊断试剂奠定了一定基础.     

Detecting putative recombination events of hepatitis B virus: An updated comparative genome analysis

An updated collection of 791 human hepatitis B virus (HBV) genomes and 38 non-human primate HBV genomes was analyzed for identifying putative recombination events and their recombinants by using two bioinformatics software tools: Simplot and RDP3 with five algorithms (RDP, GENECONV, MaxChi, Chimaera, and SiScan). A total of 61 recombinants from nine putative recombination events were detected with RDP3, especially the breakpoints of six events which have both two parental sequences that can be determined precisely with Simplot. To our knowledge, 53 recombinants were found for the first time. Our study also suggests that a relatively high recombination frequency occurs in the PreC/C gene region and the position near gene boundaries.     

武汉地区丙型肝炎病毒包膜蛋白E1的生物信息学研究

使用生物信息学的方法,分析武汉地区不同基因型、亚型的丙肝病毒的包膜E1蛋白,预测其二级结构、核苷酸变异性、糖基化位点、亲水性、跨膜区、信号肽、蛋白修饰位点、B细胞抗原.结果显示各HCV的包膜E1蛋白二级结构差距不大;序列始末段有数个氨基酸残基的差距,序列上存在高变异位点,基因型2a的型内一致性最高;序列大量糖基化,有多个糖基化基化位点;序列分布着亲水性区域,基因型1b的分值很高;基因型1b、6a、3b、3a多数亚型有1个跨膜区,基因型2a多数亚型有两个跨膜区;基因型1b、6a、3b、3a无信号肽,基因型2a都有信号肽;蛋白序列上存在多个不同修饰位点和B细胞抗原,各基因型、亚型之间有明显差距,具有较大异质性.该研究为揭示病毒感染机制和研制地区性疫苗提供一定的科学依据.     

TD1修饰绿色荧光蛋白透皮功能的研究

TD1作为一种含有11个氨基酸的短肽,具有良好的促进蛋白类大分子透皮的功能.过去的研究显示TD1可以有效协助胰岛素通过皮肤进入循环并最终降低血糖.在本研究中我们构建了一种TD1 N端修饰的GFP融合蛋白(TGFP). 我们的实验表明, 与TD1与GFP蛋白的混合物相比, TGFP具有更加良好的透皮功能.这一发现为透皮给药研究提供了一条新的途径,并对解释TD1透皮功能具有指导意义.     

丙型肝炎病毒NS3基因真核质粒的构建及其在人肝细胞中的诱导表达

目的：进行丙型肝炎病毒非结构蛋白3基因真核表达载体的构建,并分析其在体外培养的人肝细胞中的表达。方法：从含有丙肝病毒全长基因的重组质粒pBRTM／HCV1-3011表达载体中PCR扩增出HCVNS3基因片段,将其与表达载体pcDNA3．1（-）重组,得到重组的真核表达载体pcDNA3．1（-）／NS3。然后采用阳离子多聚体将其转染人肝细胞QSG7701,以免疫组织化学SP法及Western Blotting检测HCVNS3蛋白的表达。结果：所得到的NS3片段正确,序列正确,所构建的真核质粒成功转染QSG7701细胞并表达蛋白,表达的NS3蛋白相对分子质量为70000。结论：成功构建了丙型肝炎病毒非结构蛋白3基因的真核表达载体pcDNA3．1（-）／NS3,并且该载体在体外培养的人肝细胞中能有效表达特异性HCVNS3蛋白。     

Interaction of hepatitis B virus with tumor suppressor gene p53: its significance and biological function

The mechanism of the interaction of hepatitis B virus (HBV) with tumor suppressor p53 and its role in the hepatocar-cinogenesis have been studied by PCR-directed sequencing, gel shift assays and in situ ultraviolet cross-linking assay. The biological function of the interaction of HBV with p53 gene was investigated by co-transfection of chloramphenicol acetyltransferase ( CAT) reporter gene. p53 and HBV DNA. and quantitative PCR. Among the 16 primary hepatocellular carcinoma (PHC) samples. 13 were HBV-DNA positive. 10 HBxAg positive and 9 p53 protein positive. The p53 gene point mutation was found in 5 samples, one of which had a G to T substitution located at codon 249. After analyzing the HBV genome by a computer program, a p53 response element binding sequence was found in HBV genome at upstream of enhancer I. from 1047 to 1059 nucleotides. This sequence could specifically bind to p53 protein, increase p53 protein accumulation in the PHC cells and stimulate the transactivating activity of p53 and HBV replication . The results also revealed that HBxAg could combine with p53 protein to form a complex in the cells and enhance CAT expression. Immunocytochemical staining showed that p53 protein complex was located in the cytoplasm and the process of p53 entry to nuclei was. in part, blocked. From our results, we conclude that the mutation of p53 gene at codon 249 is infrequent in HBV-associated PHC. the DNA-protein binding between HBV and p53. and the protein-protein binding between HBxAg and p53 might lead to the reduction or inactivation of p53 protein, which in turn resulting in HBV-associated hepatocarcinogenesis.     

乙型肝炎病毒携带者孕妇与正常孕妇妊娠结局比较

目的探索妊娠合并乙肝病毒感染的妊娠结局及孕晚期肌注乙肝免疫球蛋白对预防乙肝病毒感染作用。方法对2003年11月至2004年11月本院孕产妇妊娠结局分析。结果(1)妊娠合并乙肝病毒感染者胎膜早破、胎儿窘迫、剖宫率升高;(2)对乙肝病毒携带者孕晚期肌注乙肝免疫球蛋白新生儿感染为8%,比文献报道低。结论妊娠合并乙肝病毒感染易发生胎膜早破、胎儿窘迫,使剖宫产率升高,孕晚期肌注乙肝免疫球蛋白,对预防乙肝病毒感染有一定作用。     

Spike protein homology between the SARS-associated virus and murine hepatitis virus implies existence of a putative receptor-binding region

Coronavirus has been determined to be the cause of the recent outbreak of severe acute respiratory syndrome (SARS). Human coronavirus 229E had been studied well and its receptor-binding domain was restricted to aa417-547 of S protein. However, this region has no homology with the newly separated SARS-associated virus (Hong Kong isolate CUHK-W1). Then we analyzed the phyiogenesis of S1 subunit of the coronavirus spike protein (SARS-associatedvirus, Hong Kong isolate CUHK-W1). Interestingly, thehighest homology between murine hepatitis virus (MHV)and SARS-associated coronavirus was found. And the important sites (aa62-65 and aa214-216) on the spike proteinof MHV with receptor-binding capacity were highly conservative in comparison with the newly separated SARS-associated virus (the corresponding sites are aa51-54 and aa195-197). These results from bioinformatics analysis mighthelp us to study the receptor-binding sites of SARS-associated virus and the mechanism of the virus entry into the target cell, and design antiviral drugs and potent vaccines.     

增强型绿色荧光蛋白基因真核表达载体的构建

构建增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因的真核表达载体pCDNA3．1( )-EGFP,转染至培养的Hela细胞中成功表达,并发出绿色荧光,证明EGFP一种良好的报告基因和筛选标记．     

齐墩果酸、鞣花酸与拉米夫定体外联合抗HBV作用研究

为了观察拉米夫定（3TC）分别与齐墩果酸（OA）、鞣花酸（EA）联合应用抗乙型肝炎病毒（HBV）的作用,在HepG2．2．15细胞培养液中分别加入适当浓度的3TC、OA、EA、3TC＋OA、3TC＋EA含药培养液,每4d换用含同等药物浓度的新鲜培养液,第8d末取上清液,用ELISA法检测上清液中HbsAg和HbeAg的量,计算各药对HepG2．2．15细胞分泌HbsAg和HbeAg的抑制率,并用金正均Q值法分析联合用药的抗HBV效果．结果表明,3TC与OA合用,对HbeAg的抑制表现为拮抗作用,在低浓度时对HbsAg的抑制为协同作用,其它浓度时均为相加作用．3TC与EA合用,对HbeAg、HbsAg的抑制均呈相加作用,但对HbeAg的相加作用在所取浓度范围内比较稳定（Q值在0．85-0．90之间）,对HbsAg的相加作用则随药物作用浓度的增大而逐渐减弱．