新型大肠杆菌融合表达系统表达水蛭素Ⅲ基因研究

选用大肠杆菌Ｌ－门冬酰胺酶（ＡＳＰｓⅡ）高效表达质粒ｐＫＡ作为融合表达载体,将水蛙素Ⅲ（ＨＶ３）人工合成编码基因插入 ｐＫＡ质粒的 ＡＳＰｓⅡ编码基因 ａｎｓＢ中,使 ＡＳＰｓ Ⅱ１Ｎ端的 ８９个氨基酸残基通过甲硫氨酸残基与 ＨＶ３形成融合蛋白,从而构建成 ＡＳＰｓⅡ－ＨＶ３融合蛋白表达质粒 ｐＫＡＨ,通过基因操作将该质粒的低拷贝数复制原长更换成 ｐＵＣ高拷贝数复制原点,进而构建成ＡＳＰｓⅡ－ＨＶ３融合蛋白高效表达载体 ｐＵＫＡＨ。该质粒在大肠杆菌 ＪＭ１０５宿主细胞中表达后,融合蛋白（１５． ６ ｋＤ）占细菌总蛋白 １０ ５％,该融合蛋白经 ＣＮＢｒ切割释放出活性水蛭素分子,抗凝血活力达约 ２０ ＡＴＵ／ｍｌ培养液,为以后进一步研究打下了基础。     

人α2b型干扰素的基因克隆及其在大肠杆菌中的表达提高

应用ＰＣＲ的方法，从人的染色体片段制备人α２ｂ干扰素的ｃＤＮＡ，将其克隆到ｐＲＣ２３表达载体上，构建成重组表达质粒ｐＭＺＩ２Ａ和ｐＭＺＩ２Ｂ，并转化大肠杆菌ＤＨ５α组建成工程菌。ｐＭＺＩ２Ａ和ｐＭＺＩ２Ｂ的区别在于后者的α２ｂ基因５＇－卢始密码ＡＴＧ上游编制了一段ＳＤ顺序，与ｐＬ启动子上连接的ＳＤ顺序组成双ＳＤ顺序。表达重组质粒中由于不含ｃＩｔＳ基因，所以采取３７℃连续培养后比较两者的抗病毒活性，     

用裸RNA免疫小鼠产生抗天然HIV-1包膜糖蛋白单克隆抗体

作者介绍了使用西门利克森林病毒（ＳＦＶ）这一新型表达系统产生具有天然构型的１型人类免疫缺陷症病毒（ＨＩＶ－１）包膜（Ｅｎｖ）糖蛋白，以及用裸ＳＦＶ－ｇｐ１６０ＲＮＡ免疫小鼠制备抗Ｅｎｖ单克隆抗体（ＭｃＡｂ）和对１２ＨＺ杂交瘤细胞系进行鉴定的结果。用聚合酶链反应（ＰＣＲ）扩增全长ＨＩＶ－１ＨＸＢ２ｅｎｖ基因并将其插入ｐＳＦＶ－１载体的ＢａｍＨＩ位点，构建表达载体ＰＳＦＶ－ｇｐ１６０．纯比后采用电穿孔技术将ＳＦＶ－ｇｐｌ６０ＲＮＡ转染ＢＨＫ－２１细胞。以蛋白印迹法、ＳＤＳ－ＰＡＧＥ及免疫荧光法检测Ｈ…     

丙肝病毒非结构4区和5区抗原的克隆表达及纯化鉴定

通过分析丙肝病毒（ＨＣＶ）的非结构４区（ＮＳ４）和５区（ＮＳ５）蛋白的氨基酸序列，推测确定抗原决定簇位点。用ＲＴ－ＰＣＲ技术从中国丙肝病人血甭中扩增克隆含抗原决定簇基因的ＮＳ４及ＮＳ５基因，将该丙段基因分别克隆至质粒表达载体ｐＥＴ２８ａ（＋）内，转化大肠杆菌ＢＬ２１（ＤＥ３），成功了高儿表达ＮＳ４和ＮＳ５蛋白的工程菌，ＩＰＴＧ诱导后两蛋白在工程菌内的表达量分别约占菌体蛋白的３３％和２８％。经Ｓｅｐ     

GM-CSF基因增强HCV非结构蛋白DNA疫苗诱导的细胞免疫应答

作者分别构建了编码丙型肝炎病毒（ＨＣＶ）非结构蛋白ＮＳ３、ＮＳ４和ＮＳ５的质粒ｐＴＶ－ＮＳ３４５、编码粒细胞－巨噬细胞－集落刺激因子（ＧＭ－ＣＳＦ）的质粒ｐＴＶ－ＧＭＣＳＦ以及编码ＮＳ３４５和ＧＭ－ＣＳＦ的双顺反子质粒ｐＴＶ－ＮＳ３４５／ＧＭＣＳＦ，分别将这三种质粒转染ＣＯＳ－７细胞，用免疫沉淀法检测ＨＣＶＮＳ３４５蛋白的表达，用ＥＬＩＳＡ检测培养上清中的ＧＭ－ＣＳＦ。将Ｂｕｆｆａｌｏ大鼠分为４组，第１、２、４组分别肌注ｐＴＶ－ＮＳ３４５、ｐＴＶ－ＮＳ３４５／ＧＭＣＳＦ和对照载体ｇＴＶ ４００μ…     

6例河南株HGV NS5基因变异的研究

目的 探讨河南省庚型肝炎病毒基因的变异特征。方法 利用逆转录－巢式－聚合酶链反应（ＲＴ－Ｎｅｓｔ－ＰＣＲ）扩增ＨＧＶ ＮＳ５区序列;将ＰＣＲ扩增产物克隆入Ｔ载体,进行测序和分析。结果 河南株ＨＧＶ ＮＳ５核酸和氨基酸序列与ＧｅｎＢａｎｋ中ＨＧＶ对应位置序列的同源笥分别为８９％￣９８．４％和９５％￣９８．４％;河南株有独特变异点（２３位Ａｌｇ→Ｇｌｙ,１１９位Ｐｈｅ→Ｌｅｕ,１５３位Ａｓｐ→Ｇｌｕ）     

亚硫酸氢钠裂解6－甲氧基－2－萘乙酮肟

亚硫酸氢钠裂解６－甲氧基－２－萘乙酮肟赵文超，沙耀武（清华大学化学系，北京１０００８４）ＣＬＥＡＶＡＧＥＯＦ６－ＭＥＴＨＯＸＹ－２－ＡＣＥＴＯＮＡＰＨＴＨＯＮＯＸＩＭＥＢＹＳＯＤＩＵＭＢＩＳＵＬＦＩＴＥ￥ＺＨＡＯＷｅｎ－Ｃｈａｏ；ＳＨＡＹａｏ－Ｗｕ（...     

争光霉素复合物中两种新组份的研究

从争光霉素产生菌轮枝链霉菌平阳新变种（Ｓｔｒｅｐｔｏｍｙｃｅｓｖｅｒｔｉｃｉｌｌｕｓｖａｒ．Ｐｉｎｇｙａｎ－ｇｅｎｓｉｓｎ．ｓｐ．）的发酵产物争光霉素复合物中分离得到了二种新组份Ｚ－９０１和Ｚ－９０２，通过ＵＶ、ＩＲ、ＣＤ、ＦＡＢＭＳ、１Ｈ－ＮＭＲ、１３Ｃ－ＮＭＲ、１３Ｃ－１ＨＣＯＳＹ等光谱测定，确定了它们的结构，证明它们是二个新的化合物，分别称为ＤｅｐｙｒｕｖａｍｉｄｅＢＬＭＡ５（Ｚ－９０１）和Ｄｅａｍｉｄｅ－Ｄｅｐｙｒｕｖａ－ｍｉｄｅＢＬＭＡ５（Ｚ－９０２）。     

60例肝活检组织中原位杂交及免疫组化研究

为了解在丙型肝炎病毒感染后机体组织中病毒复制与血清学改变是否协同一致及病理诊断丙型肝炎的临床意义，应用了丙型肝炎病毒（ＨＣＶ）核心区段的生物素探针，及病毒核酸的３′和５′末端非结构区（ＮＳ３和ＮＳ５）的单克隆抗体，对６０例血清学抗ＨＣＶ阴性的石蜡包埋的肝活检组织进行原位杂交及免疫组化检测，并同时检测其中乙型肝炎病毒（ＨＢＶ）核酸及ＨＢｓＡｇ、ＨＢｃＡｇ的表达。结果ＨＣＶＲＮＡ的检出率为３０％，ＮＳ３及ＮＳ５分别为４８．８％和５８．３％；ＨＢＶＤＮＡ及ＨＢｓＡｇ、ＨＢｃＡｇ检出率分别为５１．６％和４１．７％。ＨＣＶＲＮＶ的阳性信号为感染细胞浆内分布为主的棕色颗粒出现；ＨＣＶＮＳ３和ＮＳ５抗原的阳性信号亦为棕色细颗粒状，分布于感染的细胞浆中。实验结果可以说明，在丙型肝炎病毒感染的临床诊断中，组织病理学的原位杂交及免疫组化可起到血清学检测中难以取代的作用。     

由烯制备反式1，2－碘乙酸酯

由烯制备反式１，２－碘乙酸酯ＢｅｄｅｋａｒＡＶ等［ＳｙｎＣｏｍｍｕｎ，１９９４，２４：２２９９］碘加至烯烃（－ＣＨ＝ＣＨ－）和醋酸铅三水合物的乙酸溶液中，于室温反应５～６ｈ，可得反式１，２－碘乙酸酯［－ＣＨＩ－ＣＨ（ＯＡｃ）－］，６例收率５３～９０％...     

2a型丙型肝炎病毒非结构基因5区(NS5)全长克隆的构建

目的 构建2a型丙型肝炎病毒(HCV)非结构基因(NS)5区全长克隆质粒。方法 用聚合酶链反应(PCR)从HCV NS5A和NS5B质粒中扩增出NS5A和NS5BPCR片段，再采用融合PCR技术扩增出全长NS5的cDNA片段，克隆后RFLP和序列分析鉴定重组子。结果 融合PCR获得约2．8kb全长NS5序列，同源性分析表明：与HC-J6的核苷酸同源性为91．2％，推导的氨基酸同源性为95．5％。结论 利用融合PCR技术简单方便，利用该技术获得了2a型HCV NS5全长序列，构建了全长基因克隆质粒。     

丙型肝炎病毒IRES基因T载体克隆及序列分析

目的构建含丙型肝炎病毒（HCV）核糖体插入位点（IRES）序列的基因克隆,为以后的亚克隆和抑制肝炎病毒作用的研究提供实验材料。方法以含HCV全长基因的质粒为模板,用PCR技术扩增出HCV的IRES序列,将扩增产物IRES基因插入到PMD18T载体后转化DH5α,筛选阳性克隆,抽提重组质粒并进行PCR及酶切鉴定,再行序列分析。结果经PCR获得355bp含限制性内切酶位点的阳性产物,T载体克隆、PCR及酶切鉴定和序列分析后证实,克隆片段与GeneBank中该基因的序列同源性为99%。结论该实验成功构建了含HCV IRES基因序列的T载体克隆,提示该克隆是用作亚克隆和抑制肝炎病毒研究的理想克隆。     

HCV Core基因真核表达载体构建及其对HeLa细胞自噬的影响

目的 构建丙型肝炎病毒（HCV）核心蛋白（Core）真核表达载体,并研究其对人宫颈癌 HeLa细胞自噬功能的影响。方法 以 HCV复制子质粒 pJFH1为模板,PCR法扩增 HCV Core 基因片段,经纯化回收后与 PLVX-IRESZsGreen1载体用同源重组法相连,构建 PLVX-IRES-ZsGreen1-Core重组质粒,并经测序验证其序列的正确性。转染宫颈癌 HeLa细胞,并设 PLVX-IRES-ZsGreen1-Core重组质粒转染组、PLVX-IRES-ZsGreen1空载体转染对照组以及未转染质粒空白对照组,通过免疫印迹法验证 HCV Core蛋白在 HeLa细胞中的表达,并用免疫荧光（IF）和免疫印迹检测自噬生物标记物微管相关蛋白 1轻链 3（LC3）的表达水平。结果 获得 PLVX-IRES-ZsGreen1-Core重组质粒,测序结果表明构建的重组质粒序列完全正确,HCV Core重组蛋白可在 HeLa细胞中有效表达;免疫荧光和免疫印迹结果表明,HCV Core过表达细胞 LC3焦点和 LC3 Ⅱ蛋白表达水平明显增多,PLVX-IRES-ZsGreen1-Core重组质粒转染组的细胞 LC3 Ⅱ水平（1.069±0.049）高于 PLVX-IRES-ZsGreen1空载体转染对照组（0.776±0.047）,差异有统计学意义（P＜0.05）。结论 成功构建了 PLVX-IRES-ZsGreen1-Core 重组质粒,HCV Core 蛋白过表达可诱导 HeLa 细胞自噬。     

Polyfunctional CD8(+) T cells are associated with the vaccination-induced control of a novel recombinant influenza virus expressing an HCV epitope

In hepatitis C virus (HCV) infection, CD8(+) T cell responses have been shown to be important in viral clearance. Examining the efficacy of CD8(+) T cell vaccines against HCV has been limited by the lack of an HCV infectious model in mice and the differences between MHC restriction in humans and mice. Using HLA-A2 transgenic HHD mice, we demonstrate that intranasally delivered Pam2Cys-based lipopeptides containing HLA-A2-restricted HCV epitopes can induce polyfunctional CD8(+) T cell responses in several organs including the liver. To examine the activity of these responses in an infectious context, we developed a recombinant influenza virus that expresses the NS5B(2594-2602) epitope from non-structural protein 5B of hepatitis C virus (PR8-HCV(NS5B)). We showed that mice inoculated with a lipopeptide containing the NS5B epitope had reduced viral loads following challenge with the PR8-HCV(NS5B) virus. This reduction was associated with the induction of NS5B(2594-2602)-specific IFN-γ and TNF-α co-producing CD8(+) T cells. The T cell receptor usage in the NS5B(2594-2602) response was found to exhibit a Vβ8.1/8.2 bias that was characterized by a narrow repertoire and a common CDR3β motif. This work has identified CD8(+) T cell functions induced by lipopeptides that are associated with viral control and demonstrate the potential of lipopeptide-based vaccines as candidates for treatment of HCV infection.     

Current perspective of HCV NS5B inhibitors: a review

Hepatitis C virus (HCV) infection has emerged as one of the most significant disease to affect humans. Despite its large medical and economical impact, there are no vaccines or efficient therapies without major side effects. The HCV non-structural protein 5B (NS5B) is the RNA-dependent RNA polymerase responsible for the complete copy of the RNA viral genome and is a target of choice for the development of anti-HCV drugs. Although many small molecules have been identified as allosteric inhibitors of NS5B, very few are active in clinical applications. Developments in the field have prompted us to review the research work on HCV NS5B polymerase inhibitors, especially their structure activity relationships and molecular modeling studies. This review will focus on the journey of drug discovery of HCV NS5B inhibitors covering both nucleoside and non-nucleosides.     

Recent advances in discovery and development of promising therapeutics against hepatitis C virus NS5B RNA-dependent RNA polymerase

Lack of highly effective and safe therapeutics for hepatitis C virus (HCV) infection provides an opportunity as well as a challenge to discover novel and potent anti-HCV drugs. HCV NS5B RNA-dependent RNA polymerase (RdRp) is responsible for viral genome replication and thus constitutes a valid target for therapeutic intervention. To date, numerous HCV NS5B RdRp inhibitors have been discovered. This review focuses on the recent advances in discovery, mechanism of action studies and biological characterization of several distinct classes of potent inhibitors for NS5B RdRp. The clinical efficacy and developmental status of several promising compounds are also outlined.     

丙型肝炎病毒基因免疫重组真核表达质粒构建的初步研究

目的:利用真核表达载体pCDNA3.1(+)与HCV核心区基因重组,为进一步表达蛋白打基础。方法:首先从1例HCV感染者血清中用反转录-PCR法获得全长的核心区基因并测序,继而将其克隆到原核表达载体pQE-30上并使之在大肠杆菌中得到表达,然后将核心区基因与真核表达载体pCDNA3.1(+)重组,构建重组体。结果:HCV核心区基因为HCV型,将其克隆到原核表达载体pQE-30上并使之在大肠杆菌中得到表达,分子量为22KD,表达量占菌体蛋白的8.7%。然后将核心区基因与真核表达载体pCDNA3.1(+)重组,构建了pCDNAHCVc191和pCD-NAHCVc-hIL2两种重组体。结论:pCDNAHCVc191和pCDNAHCVc-hIL2两种重组体,尤其后者是核心区基因与人白介素-2基因融合,可引发更好的基因免疫效果。上述工作奠定了整个HCV基因免疫研究的基础。     

Development and characterization of a replicon-based phenotypic assay for assessing HCV NS4B from clinical isolates

The hepatitis C virus (HCV) NS4B inhibitors have shown potent inhibition of HCV replication in vitro. To assess the effect of viral diversity on the susceptibility to NS4B inhibitors, genotype (GT)-specific GT1a and GT1b replicon shuttle vectors were designed and created for cloning HCV NS4B genes from clinical isolates. For the GT1b NS4B shuttle vector, the S2204I adaptive mutation was introduced in NS5A to improve replication due to the replacement of the K1846T adaptive mutation in NS4B with NS4B from the clinical isolates. In addition to the adaptive mutations, a newly identified Huh-7 cell line, Huh-7-1C, which is highly permissive for both GT1a and GT1b replication, was used to further enhance the replication levels. HCV NS4B gene from clinical isolates was amplified and inserted into the corresponding GT1a and GT1b modified lab strain chimeric replicons. GT1a and GT1b chimeric replicons expressing diverse NS4B genes from corresponding subtypes of clinical isolates replicated at highly efficient levels for phenotypic analysis. Due to natural variation in their amino acid residues in NS4B, these isolates displayed varying drug susceptibilities to an NS4B inhibitor. In mixed populations with wild-type, the sensitivity of resistance detection of NS4B resistant mutants H94R and V105M was between 20% and 80%. The chimeric shuttle vectors can be used to characterize the activity of antiviral drugs targeting NS4B from diverse natural clinical isolates and aid in the development of novel compounds against HCV NS4B.     

Replication efficiency of chimeric replicon containing NS5A-5B genes derived from HCV-infected patient sera

A transient subgenomic replicon-based shuttle vector system has been developed to investigate how genetic heterogeneity affects HCV replication efficiency. Individual NS5A or NS5B genes or cassettes containing both NS5A and NS5B genes were amplified from "quasispecies" pools derived from HCV genotype 1a or 1b patient sera using RT-PCR and cloned into their respective shuttle vectors. All shuttle vectors containing NS5A or NS5A-5B genes were constructed with the S2204I "adaptive" mutation because replicons lacking the S2204I mutation replicated poorly. Gene sequences of the quasispecies pools within either genotype 1a or 1b patient samples ranged from 94 to 95% in identity. The replication capacity of 1b shuttle vectors containing patient-derived NS5A or NS5B genes averaged 67 and 75%, respectively, relative to the laboratory-optimized 1b replicon. In contrast, the replication efficiencies of both 1a and 1b shuttle vectors containing patient-derived NS5A-5B gene cassettes averaged around 2% relative to the respective laboratory-optimized replicon. All patient-derived replicons were tested in a transient assay for their sensitivity to either interferon-alpha (IFN-alpha) or to the polymerase inhibitor A-782759. Despite the differences in replication efficiency, IC(50) values measured for most of the patient-derived replicons were equivalent to the respective values measured in the control laboratory strain replicons. These results demonstrate that patient sequence heterogeneity affects replication efficiency whenever patient-derived NS5A-5B genes are inserted into the laboratory-optimized replicon. The findings also demonstrate the utility of the shuttle vector system to test patient-derived gene sequences for sensitivity to IFN-alpha and to small molecule inhibitors.     

用杆状病毒双穿梭载体表达系统表达HCV结构基因及HCV病毒样颗粒装配的初步观察

利用长RT-PCR技术，一次性克隆出HCV的结构基因C、E1、E2及部分5’UTR，经测序，验证了其正确性。将克隆基因插入杆状病毒双穿梭载体系统的转移载体，经转座并转染sf9细胞，得到重组病毒Bmhce，经SDS-PAGE和ELISA测定，表明HCV结构基因在重组病毒感染的SO细胞中表达；另外，电镜观察发现在重组病毒感染的SO细胞质的空泡(vesicle)中有HCV样颗粒的存在，说明HCV结构基因表达产物在SO细胞中组装出病毒样颗粒。