一种新的丙型肝炎病毒单链丝氨酸蛋白酶基因的构建、表达与鉴定

根据丙型肝炎病毒 (HCV)丝氨酸蛋白酶晶体结构特点 ,设计并构建了一种新的单链型丝氨酸蛋白酶分子 .该分子由辅因子NS4A的核心序列、柔性连接子GSGS和NS3丝氨酸蛋白酶结构域组成 .利用设计的 3条引物 ,通过 2轮PCR获得单链丝氨酸蛋白酶基因 ,插入原核表达载体pQE30中 ,转化大肠杆菌M15 ,获得重组克隆 .经低剂量诱导和低温培养 ,目的基因获得高水平可溶表达 .以金属螯合层析法纯化的重组蛋白纯度达 95 %以上 .间接ELISA法检测 98份血清证实 ,该蛋白具有良好的抗原性和特异性 ;以重组蛋白底物NS5ab和单链丝氨酸蛋白酶建立了简便、实用的丝氨酸蛋白酶体外活性检测系统 ;以该系统观察了PMSF和EDTA对蛋白酶活性的影响 .结果表明 ,PMSF能够抑制蛋白酶的酶切活性 ,而EDTA不能抑制酶的活性 .单链型HCV丝氨酸蛋白酶的成功表达以及体外活性检测系统的建立 ,为丝氨酸蛋白酶抑制剂的研制奠定了物质基础 .     

猪繁殖与呼吸综合征病毒Nsp2蛋白原核表达纯化及其蛋白酶活性分析

摘要：【目的】表达并纯化猪繁殖与呼吸综合征病毒非结构蛋白2（Nsp2）,分析Nsp2的蛋白酶活性。【方法】本研究通过PCR分别扩增nsp2基因的N端和C端,利用原核表达载体pET21a(+)表达Nsp2蛋白的N端和C端(即Nsp2-N 和 Nsp2-C),通过Ni-NTA琼脂糖亲和层析和凝胶过滤的方法纯化两个重组蛋白。预测Nsp2-N含有半胱氨酸蛋白酶结构域,本研究利用western blot检测其顺式酶切蛋白酶活性;并人工合成潜在的十肽底物,利用体外多肽酶切实验检测其反式酶切蛋白酶活性。成功获得Nsp2     

人β肌动蛋白基因原核表达载体的构建及其表达和纯化

目的:构建带GST标签的人β肌动蛋白(β-actin)基因的原核表达产物,纯化出GST-β-actin融合蛋白,为探究β-actin的各项生理功能做准备。方法:以人乳腺文库为模板,利用PCR技术扩增β-actin基因,将其连接到带有GST标签的载体上,经鉴定正确的重组质粒转化大肠杆菌Rossate感受态细胞,小量诱导表达后,利用GST-Sepha-rose 4B亲和珠纯化GST-β-actin融合蛋白,经SDS-PAGE和Western印迹检测。结果:目的基因经PCR技术得以扩增,将其与带GST标签的载体连接后再经双酶切鉴定及测序后确认构建成功;转化大肠杆菌Rossate感受态后获得小量诱导表达,纯化出GST-β-actin融合蛋白,并证实其有生物活性。结论:构建了人β-actin的原核表达载体,并获得了GST-β-actin融合蛋白。     

人GST-14-3-3σ融合蛋白的原核表达、纯化及活性检测

目的:构建人14-3-3σ基因的原核表达载体,获得其原核表达产物,并对融合蛋白进行纯化及活性检测。方法:采用PCR技术从人乳腺文库中扩增14-3-3σ基因编码序列,将其克隆到pGEX-KG载体中,重组质粒转化大肠杆菌Rossate后表达重组蛋白,利用GST-Sepharose 4B亲和珠对原核表达产物进行纯化,并通过SDS-PAGE和Western印迹检测融合蛋白的表达,采用GST pull-down技术检测已纯化的蛋白与已知体外相互作用蛋白AKT之间的相互作用。结果:从人乳腺文库中扩增获得约750 bp的DNA片段,并成功克隆至pGEX-KG载体上,经双酶切鉴定得到与预期片段大小相符的外源基因插入片段,测序与目的基因序列完全一致;在Rossate菌株中诱导表达出相对分子质量约52 000的目的蛋白,SDS-PAGE和Western印迹结果表明融合蛋白表达成功,并纯化得到GST-14-3-3σ融合蛋白;通过GST pull-down技术检测证实GST-14-3-3σ融合蛋白可以和AKT在体外结合,并证实其具有生物学活性。结论:获得了原核表达的活性较好的GST-14-3-3σ蛋白,为后续研究细胞周期蛋白调控机制奠定了实验基础。     

丙型肝炎病毒核心蛋白基因的克隆与表达

以含有丙型肝炎病毒核心蛋白基因的质粒pJLA502-C为模板,用PCR方法重新扩增克隆了核心蛋白基因,在扩增基因的上下端分别增加了NCOⅠ及SalⅠ酶切位点。将克隆的基因酶切后插入表达载体pBV221内,转化大肠肝菌DH_(5α),获得表达非融合核心蛋白的工程菌,42℃热诱导5hr,表达蛋白占菌体蛋白总量的15％。经包涵体纯化及分子筛纯化等,获得核心蛋白,经ELISA及Western Blotting分析表明有较好的抗原性和特异性。     

丙型肝炎病毒NS3蛋白酶在酵母系统中的可溶性表达

利用毕赤酵母系统表达具有催化活性的丙型肝炎病毒 (HCV)NS3蛋白酶 .将PCR直接扩增的病毒NS3丝氨酸蛋白酶基因和重组的带有辅酶的单链NS3 NS4A蛋白酶基因 ,分别插入表达载体pPICZαA的EcoRⅠ和XbaⅠ克隆位点 ,转化毕赤酵母GS115 ,可溶性表达NS3蛋白酶和单链NS3 4A蛋白酶 ;ELISA法测定表达蛋白酶的抗原性 ;原核高效表达载体pBVIL1表达酶切底物NS5A B片段 ,体外与蛋白酶共同温育 ,SDS PAGE鉴定蛋白酶催化活性 .载体测序结果表明 ,重组载体pPICZαA NS3和pPICZαA NS3 4A中的目的基因序列插入正确 ;SDS PAGE结果显示 ,培养物上清中存在分泌型目的蛋白带 ;ELISA结果证实 ,表达蛋白与HCV阳性血清有结合活性 ;蛋白酶与底物NS5A B片段不同作用时间的SDS PAGE ,看到约 2 4kD处底物条带的分解 .说明用毕赤酵母表达系统成功地表达了可溶性HCVNS3和单链NS3 4A蛋白酶 ;两种结构形式的蛋白酶在体外系统中都有催化活性 ,同时也都具有抗原性 .该研究为大量和方便地获得有催化活性的HCVNS3蛋白酶提供了有效途径 .     

具有免疫反应性的猪瘟病毒多表位基因在大肠杆菌中的克隆表达和鉴定

将猪瘟病毒不同抗原表位基因串联构成重组基因BT21,化学合成后克隆至pMD18-T载体中,然后再将BT21串联基因片段插入原核表达载体pGEX-6P-1。经酶切和测序鉴定后,构建的重组质粒pGEX—BT21转化大肠杆菌BL21（DE3）,通过IPTG诱导表达,表达产物进行SDS—PAGE分析,用G1utathione Se-pharose4B亲和层析法纯化目的蛋白和Western blot分析其免疫学活性。结果表明：融合蛋白GST—BT21以可溶形式表达,分子量约为33kDa,与预期大小相符,纯化的重组蛋白可被猪瘟病毒阳性血清所识别,具有良好的免疫学活性。从而为进一步研究该融合蛋白的免疫特性和功能奠定了基础。     

GST-His双标签原核表达载体的构建及应用

目的：在原有带有GST标签的pGEX-KG载体上添加His标签,构建双标签原核表达载体,以提高纯化后的融合蛋白的纯度。方法：双酶切pGEX-KG载体,将同样带有双酶切位点编码His标签的DNA序列酶切后与其连接、转化大肠杆菌DH5α、鉴定阳性克隆并测序,并将编码雌激素受体B（ERβ）的片段构建到该载体上,分别利用GST标签和His标签对ERβ蛋白进行2次纯化。结果：构建了GST-His双标签原核表达载体,将ERβ编码片段克隆入该载体中,在原核生物中得到表达;分别用GST和His抗体进行Westernblot分析,均可检测到GST-His-ERβ融合蛋白的表达;利用此双标签载体纯化得到了纯度较高的ERβ蛋白。结论：GST-His双标签原核表达载体的构建对提高目的蛋白纯度具有重要意义。     

克隆、表达及纯化核糖体蛋白S6用于体外激酶活性检测

目的:构建40S核糖体蛋白S6的原核表达载体,表达并纯化S6蛋白,将其作为底物用于S6激酶(S6K)的体外活性测定。方法:采用RT-PCR方法从人胚肾细胞HEK293中获取S6 cDNA,将扩增产物克隆至大肠杆菌表达载体中,进行酶切及测序鉴定;IPTG诱导GST-S6融合蛋白在大肠杆菌中表达,用谷胱甘肽亲和层析纯化GST-S6,免疫沉淀法检测该蛋白是否可作为底物用于S6K的体外激酶活性测定。结果:酶切及测序鉴定表明构建了S6原核表达载体,并表达及纯化出GST-S6融合蛋白,相对分子质量为55×103。该蛋白可用于S6K的体外激酶活性测定,特异性强。结论:S6蛋白的克隆、表达与纯化成功,可用于S6K的体外激酶活性测定,为研究S6K的功能奠定了基础。     

大鼠丝氨酸或半胱氨酸蛋白酶抑制剂B2的表达、纯化及抗体制备

目的:原核表达、纯化大鼠丝氨酸或半胱氨酸蛋白酶抑制剂B2(SERPINB2),并制备其多克隆抗体.方法:设计扩增大鼠Serpinb2全长基因的特异引物,通过PCR扩增出该基因片段,测序正确后插入含GST基因的原核表达载体pGEX-KG中,以IPTG诱导表达,并经谷胱甘肽琼脂糖珠纯化融合蛋白;用纯化的蛋白免疫小鼠制备多克...     

GB virus B and hepatitis C virus NS3 serine proteases share substrate specificity.

GB virus B (GBV-B) is a recently discovered virus responsible for hepatitis in tamarins (Saguinus species). GBV-B belongs to the Flaviviridae family and is closely related to the human pathogen hepatitis C virus (HCV). Nonstructural protein 3 (NS3) of HCV has been shown to encompass a serine protease domain required for viral maturation. GBV-B and HCV share only about 30% of the amino acid sequence within the NS3 protease domain. The catalytic triad is conserved, and the residue Phe-154, presumed to be a crucial amino acid for determining the S1 specificity pocket of the HCV NS3 protease, is also conserved. We have expressed a synthetic gene encoding the GBV-B NS3 protease domain in Escherichia coli and have characterized the purified recombinant protein for its activity on HCV substrates. We have shown that the NS3 region of the GBV-B genome actually encodes a serine protease that, despite the low sequence homology, shares substrate specificity with the HCV NS3 protease.     

Inhibitor binding induces active site stabilization of the HCV NS3 protein serine protease domain

Few structures of viral serine proteases, those encoded by the Sindbis and Semliki Forest viruses, hepatitis C virus (HCV) and cytomegalovirus, have been reported. In the life cycle of HCV a crucial role is played by a chymotrypsin-like serine protease encoded at the N-terminus of the viral NS3 protein, the solution structure of which we present here complexed with a covalently bound reversible inhibitor. Unexpectedly, the residue in the P2 position of the inhibitor induces an effective stabilization of the catalytic His-Asp hydrogen bond, by shielding that region of the protease from the solvent. This interaction appears crucial in the activation of the enzyme catalytic machinery and represents an unprecedented observation for this family of enzymes. Our data suggest that natural substrates of this serine protease could contribute to the enzyme activation by a similar induced-fit mechanism. The high degree of similarity at the His-Asp catalytic site region between HCV NS3 and other viral serine proteases suggests that this behaviour could be a more general feature for this category of viral enzymes.     

Hepatitis C viral NS3-4A protease activity is enhanced by the NS3 helicase

Non-structural protein 3 (NS3) is a multifunctional enzyme possessing serine protease, NTPase, and RNA unwinding activities that are required for hepatitis C viral (HCV) replication. HCV non-structural protein 4A (NS4A) binds to the N-terminal NS3 protease domain to stimulate NS3 serine protease activity. In addition, the NS3 protease domain enhances the RNA binding, ATPase, and RNA unwinding activities of the C-terminal NS3 helicase domain (NS3hel). To determine whether NS3hel enhances the NS3 serine protease activity, we purified truncated and full-length NS3-4A complexes and examined their serine protease activities under a variety of salt and pH conditions. Our results indicate that the helicase domain enhances serine protease activity, just as the protease domain enhances helicase activity. Thus, the two enzymatic domains of NS3-4A are highly interdependent. This is the first time that such a complete interdependence has been demonstrated for a multifunctional, single chain enzyme. NS3-4A domain interdependence has important implications for function during the viral lifecycle as well as for the design of inhibitor screens that target the NS3-4A protease.     

Control of innate immune signaling and membrane targeting by the Hepatitis C virus NS3/4A protease are governed by the NS3 helix α0

Hepatitis C virus (HCV) infection is sensed in the host cell by the cytosolic pathogen recognition receptor RIG-I. RIG-I signaling is propagated through its signaling adaptor protein MAVS to drive activation of innate immunity. However, HCV blocks RIG-I signaling through viral NS3/4A protease cleavage of MAVS on the mitochondrion-associated endoplasmic reticulum (ER) membrane (MAM). The multifunctional HCV NS3/4A serine protease is associated with intracellular membranes, including the MAM, through membrane-targeting domains within NS4A and also at the amphipathic helix α(0) of NS3. The serine protease domain of NS3 is required for both cleavage of MAVS, a tail-anchored membrane protein, and processing the HCV polyprotein. Here, we show that hydrophobic amino acids in the NS3 helix α(0) are required for selective cleavage of membrane-anchored portions of the HCV polyprotein and for cleavage of MAVS for control of RIG-I pathway signaling of innate immunity. Further, we found that the hydrophobic composition of NS3 helix α(0) is essential to establish HCV replication and infection. Alanine substitution of individual hydrophobic amino acids in the NS3 helix α(0) impaired HCV RNA replication in cells with a functional RIG-I pathway, but viral RNA replication was rescued in cells lacking RIG-I signaling. Therefore, the hydrophobic amphipathic helix α(0) of NS3 is required for NS3/4A control of RIG-I signaling and HCV replication by directing the membrane targeting of both viral and cellular substrates.     

The serine protease domain of hepatitis C viral NS3 activates RNA helicase activity by promoting the binding of RNA substrate

Nonstructural (NS) protein 3 is a DEXH/D-box motor protein that is an essential component of the hepatitis C viral (HCV) replicative complex. The full-length NS3 protein contains two functional modules, both of which are essential in the life cycle of HCV: a serine protease domain at the N terminus and an ATPase/helicase domain (NS3hel) at the C terminus. Truncated NS3hel constructs have been studied extensively; the ATPase, nucleic acid binding, and helicase activities have been examined and NS3hel has been used as a target in the development of antivirals. However, a comprehensive comparison of NS3 and NS3hel activities has not been performed, so it remains unclear whether the protease domain plays a vital role in NS3 helicase function. Given that many DEXH/D-box proteins are activated upon interaction with cofactor proteins, it is important to establish if the protease domain acts as the cofactor for stimulating NS3 helicase function. Here we show that the protease domain greatly enhances both the direct and functional binding of RNA to NS3. Whereas electrostatics plays an important role in this process, there is a specific allosteric contribution from the interaction interface between NS3hel and the protease domain. Most importantly, we establish that the protease domain is required for RNA unwinding by NS3. Our results suggest that, in addition to its role in cleavage of host and viral proteins, the NS3 protease domain is essential for the process of viral RNA replication and, given its electrostatic contribution to RNA binding, it may also assist in packaging of the viral RNA.     

Polynucleotide modulation of the protease, nucleoside triphosphatase, and helicase activities of a hepatitis C virus NS3-NS4A complex isolated from transfected COS cells.

The hepatitis C virus (HCV) nonstructural 3 protein (NS3) is a 70-kDa multifunctional enzyme with three known catalytic activities segregated in two somewhat independent domains. The essential machinery of a serine protease is localized in the N-terminal one-third of the protein, and nucleoside triphosphatase (NTPase) and helicase activities reside in the remaining C-terminal region. NS4A is a 54-residue protein expressed immediately downstream of NS3 in the viral polyprotein, and a central stretch of hydrophobic residues in NS4A form an integral structural component of the NS3 serine protease domain. There is no evidence to suggest that the two domains of NS3 are separated by proteolytic processing in vivo. This may reflect economical packaging of essential viral replicative components, but it could also mean that there is functional interdependence between the two domains. In this study, a full-length NS3-NS4A complex was isolated after expression and autoprocessing in transiently transfected COS cells. The protein was used to examine the effects of polynucleotides on the NTPase, helicase, and protease activities. Unlike the previously reported behavior of a separately expressed NS3 helicase domain, the full NS3-NS4A complex demonstrated optimal NTPase activity between pH 7.5 and 8.5. All three NS3-NS4A activities were modulated by polynucleotides, with poly(U) having the most remarkable effect. These findings suggest that the domains within NS3 may influence the activity of one another and that the interplay of HCV genomic elements may regulate the enzyme activities of this complex HCV replicase component.     

The design of a potent inhibitor of the hepatitis C virus NS3 protease: BILN 2061--from the NMR tube to the clinic

The virally encoded serine protease NS3/NS4A is essential to the life cycle of the hepatitis C virus (HCV), an important human pathogen causing chronic hepatitis, cirrhosis of the liver, and hepatocellular carcinoma. Until very recently, the design of inhibitors for the HCV NS3 protease was limited to large peptidomimetic compounds with poor pharmacokinetic properties, making drug discovery an extremely challenging endeavor. In our quest for the discovery of a small-molecule lead that could block replication of the hepatitis C virus by binding to the HCV NS3 protease, the critical protein-polypeptide interactions between the virally encoded NS3 serine protease and its polyprotein substrate were investigated. Lead optimization of a substrate-based hexapeptide, guided by structural data, led to the understanding of the molecular dynamics and electronic effects that modulate the affinity of peptidomimetic ligands for the active site of this enzyme. Macrocyclic beta-strand scaffolds were designed that allowed the discovery of potent, highly selective, and orally bioavailable compounds. These molecules were the first HCV NS3 protease inhibitors reported that inhibit replication of HCV subgenomic RNA in a cell-based replicon assay at low nanomolar concentrations. Optimization of their biopharmaceutical properties led to the discovery of the clinical candidate BILN 2061. Oral administration of BILN 2061 to patients infected with the hepatitis C genotype 1 virus resulted in an impressive reduction of viral RNA levels, establishing proof-of-concept for HCV NS3 protease inhibitors as therapeutic agents in humans.     

Inhibiting viral proteases: challenges and opportunities

Inhibitor design against viral targets must take into account the peculiar characteristics of viral biology-in particular, the plasticity of their replicative machinery. This includes maturational cleavage of the polyprotein, which is mediated by virally encoded proteases. Designing against a movable target is particularly challenging, but at the same time it offers new opportunities. Here we describe our experience with the NS3/4A (NS: nonstructural) serine protease of human hepatitis C virus (HCV). By extensive use of combinatorial peptide libraries, various inhibitor types were generated, including product inhibitors, serine traps, P-P' inhibitors, and prime side inhibitors. The latter represent a first case for a serine protease. A key finding, derived from structural studies utilizing these inhibitors, was that NS3 is an induced-fit protease, requiring both the NS4A cofactor protein and the substrate to fully activate its catalytic machinery. In the absence of cofactor and/or substrate, NS3 exists in solution as a large conformational ensemble, which can be matched by a correspondingly large set of peptide inhibitors, each one stabilizing a given conformer. In the perspective of inhibiting viral proteases in general, we suggest that combinatorial ligand ensembles may be a powerful tool, to contrast the adaptive potential of the viral quasispecies.     

Signal peptide peptidase cleavage of GB virus B core protein is required for productive infection in vivo

Chronic infection by hepatitis C virus (HCV) is a leading cause of liver disease for which better therapies are urgently needed. Because a clearer understanding of the viral life cycle may suggest novel anti-viral approaches, we studied the role of host signal peptide peptidase (SPP) in viral infection. This intramembrane protease cleaves within a C-terminal signal sequence in the viral core protein, but the molecular determinants of cleavage and whether it is required for infection in vivo are unknown. To answer these questions, we studied SPP processing in GB virus B (GBV-B) infection. GBV-B is the closest phylogenetic relative of HCV and offers an accurate surrogate model for HCV infection. We demonstrate that SPP also processes GBV-B core protein and that a serine residue in the hydrophobic region of the signal sequence (present also in HCV) is critical for efficient SPP cleavage. The small size of the serine side chain combined with its ability to form intra- and interhelical hydrogen bonds likely contributes to recognition of the signal sequence as a substrate for SPP. By introducing mutations with differing effects on SPP processing into an infectious GBV-B molecular clone, we demonstrate that SPP processing of the core protein is required for productive infection in primates. These results broaden our understanding of the mechanism and requirements for SPP cleavage and reveal a functional role in vivo for intramembrane proteolysis in host-pathogen interactions. Moreover, they identify SPP as a potential therapeutic target for reducing the impact of HCV infection.