丙型肝炎病毒结构蛋白E1在昆虫细胞中的表达及定位

本文在大肠杆菌中表达了与GST融合无跨膜区的丙型肝炎病毒(Hepatitis C Virus,HCV)E1蛋白,并通过免疫兔制备了兔抗E1的抗血清.然后利用Bac-to-Bac杆状病毒表达系统构建了含有HCV结构蛋白E1基因的重组杆状病毒vAcHCVE1.通过Western blot分析,E1蛋白在Sf9细胞中表达分子量大小为30kDa大于预测的20kDa,表明存在翻译后修饰如糖基化等.通过Confocal显微镜观察当感染48h后E1蛋白定位在细胞质和细胞膜上.     

HCV结构蛋白在昆虫细胞中的表达及病毒样颗粒的组装

本文利用Bac-to-Bac杆状病毒表达系统构建了含有丙型肝炎病毒(Hepatitis C Virus,HCV)结构蛋白编码基因的重组杆状病毒vAcHCVsp1,并获得了HCV结构蛋白在昆虫细胞Sf21中的表达.HCV mRNA转录和蛋白质表达时相分析表明,感染后16 h HCV结构蛋白编码基因开始转录,72 h达最高峰;蛋白质表达则是在感染后48h开始,72 h达到高峰.电镜观察表明vAcHCVsp1感染的Sf21细胞96 h时在细胞质中可见很多空泡,空泡中可见50nm的球形颗粒,为HCV结构蛋白组装的病毒样颗粒.     

HCV结构蛋白在昆虫细胞中的表达及病毒样颗粒的组装

本文利用Bac-to-Bac杆状病毒表达系统构建了含有丙型肝炎病毒(Hepatitis C Virus,HCV)结构蛋白编码基因的重组杆状病毒vAcHCVspl,并获得了HCV结构蛋白在昆虫细胞Sf21中的表达。HCV mRNA转录和蛋白质表达时相分析表明,感染后16h HCV结构蛋白编码基因开始转录,72h达最高峰;蛋白质表达则是在感染后48h开始,72h达到高峰。电镜观察表明vAcHCVspl感染的Sf21细胞96h时在细胞质中可见很多空泡,空泡中可见50nm的球形颗粒,为HCV结构蛋白组装的病毒样颗粒。     

鸡白细胞介素18成熟蛋白在昆虫细胞/杆状病毒系统中的表达

将编码鸡的白细胞介素 1 8(chickeninterleukine 1 8,ChIL 1 8)成熟蛋白的基因亚克隆到杆状病毒转移载体pMelBacB上 ,构建真核转移载体pMelBacBChIL 1 8,经限制性内切酶消化、ChIL 1 8特异引物PCR鉴定和确证性序列测定 ,证明目的基因正确克隆到载体的预期位点。将纯化的pMelBacBChIL 1 8质粒与杆状病毒DNA(Bac N BlueTM DNA)共转染sf9昆虫细胞 ,经四轮蓝斑筛选纯化 ,获得了重组杆状病毒 ,命名为rBaculovirusChIL 1 8。提取病毒染色体DNA ,经ChIL 1 8特异引物和重组杆状病毒特异引物PCR鉴定 ,证明获得了纯化的重组杆状病毒。用该重组病毒接种sf9昆虫细胞 ,收获接种后不同时间的细胞进行SDS PAGE电泳。结果表明ChIL 1 8基因在昆虫细胞中获得了表达 ,表达的重组蛋白分子量约为 2 3kDa。应用在大肠杆菌原核表达系统中表达的重组蛋白制备的兔抗ChIL 1 8多克隆抗体进行Westernblot分析 ,表明本研究真核系统表达的ChIL 1 8成熟蛋白和前期原核系统表达的ChIL 1 8成熟蛋白均具有生物学活性。     

HCV E2区基因在原核系统中的表达

用提取的重组表达载体pET-E2转化BL21(DE3)感受态细胞,经IPTG诱导,再进行SDS-PAGE,可得到有一条约34 kDa的表达带,与理论推测的蛋白分子量一致,通过Western-blot鉴定,证明此带即为目的蛋白带.该产物有一个六聚组氨酸尾,主要以包涵体形式存在;计算机扫描分析考马斯亮兰染色后的蛋白胶显示目的蛋白占整个菌体蛋白的36%以上,经Ni-柱纯化的E2蛋白纯度可达95%以上;以纯化的E2蛋白为抗原,用ELISA方法检测了20份抗HCV阴阳性血清,结果表明15份抗HCV阳性血清中检出5份E2抗体阳性血清,而5份抗HCV阴性血清中没有检测到E2抗体.     

HCV E2区基因在原核系统中的表达

用提取的重组表达载体pET-E2转化BL21（DE3）感受态细胞,经IPTG诱导,再进行SDS－PAGE,可得到有一条约34kDa的表达带,与理论推测的蛋白分子量一致,通过Western-blot鉴定,证明此带即为目的蛋白带。该产物有一个六聚组氨酸尾,主要以包涵体形式存在;计算机扫描分析考马斯亮兰染色后的蛋白胶显示：目的蛋白占整个菌体蛋白的36％以上,经Ni-柱纯化的E2蛋白纯度可达95％以上;以纯化的E2蛋白为抗原,用ELISA方法检测了20份抗HCV阳性血清,结果表明15份抗HCV阳必血清中检出5份E2抗体阳性血清,而5份抗HCV阴性血清中没有检测到E2抗体。     

尼帕病毒融合蛋白、受体结合蛋白的表达及特异性高免血清制备

尼帕病毒膜融合蛋白F和受体结合蛋白G在病毒感染和诱导机体产生保护性免疫中起重要的作用。通过PCR扩增获得尼帕病毒F1和G基因片段(均去掉信号肽和跨膜区),克隆至原核表达载体,IPTG诱导大肠杆菌表达目的蛋白,Western blot表明重组F1、G蛋白与兔抗尼帕病毒血清具有良好的反应原性;同时将F1和G基因克隆至经改造过的杆状病毒表达载体,获得了含有目的基因的重组杆状病毒,接种sf9单层细胞,间接免疫荧光检测表明F1、G蛋白在杆状病毒中正确表达,并与抗尼帕病毒血清具有良好的反应原性。以纯化原核表达的F1、G蛋白免疫兔获得了抗F1和抗G重组蛋白的特异血清,Western blot和间接免疫荧光检测表明所制备的血清具有特异性。试验所表达的抗原和制备的特异血清可用于尼帕病的诊断。     

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。     

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。     

丙型肝炎病毒RNA多聚酶在昆虫细胞中的表达

HCV NS5B基因片段克隆入BAC-TO-BACTM重组杆状病毒表达系统的pFASTHTc载体质粒,转化DH10BACTM感受态细菌获得重组的Bacmid质粒,将重组Bacmid质粒转染Sf9细胞,获得的重组杆状病毒可表达目的蛋白.免疫印迹和体外活性检测表明,所表达蛋白为HCV NS5B蛋白,具有多聚酶活性.     

Glycoprotein E1 of hog cholera virus expressed in insect cells protects swine from hog cholera.

The processing and protective capacity of E1, an envelope glycoprotein of hog cholera virus (HCV), were investigated after expression of different versions of the protein in insect cells by using a baculovirus vector. Recombinant virus BacE1[+] expressed E1, including its C-terminal transmembrane region (TMR), and generated a protein which was similar in size (51 to 54 kDa) to the size of E1 expressed in swine kidney cells infected with HCV. The protein was not secreted from the insect cells, and like wild-type E1, it remained sensitive to endo-beta-N-acetyl-D-glucosaminidase H (endo H). This indicates that E1 with a TMR accumulates in the endoplasmic reticulum or cis-Golgi region of the cell. In contrast, recombinant virus BacE1[-], which expressed E1 without a C-terminal TMR, generated a protein that was secreted from the cells. The fraction of this protein that was found to be cell associated had a slightly lower molecular mass (49 to 52 kDa) than wild-type E1 and remained endo H sensitive. The high-mannose units of the secreted protein were trimmed during transport through the exocytotic pathway to endo H-resistant glycans, resulting in a protein with a lower molecular mass (46 to 48 kDa). Secreted E1 accumulated in the medium to about 30 micrograms/10(6) cells. This amount was about 3-fold higher than that of cell-associated E1 in BacE1[-] and 10-fold higher than that of cell-associated E1 in BacE1[+]-infected Sf21 cells. Intramuscular vaccination of pigs with immunoaffinity-purified E1 in a double water-oil emulsion elicited high titers of neutralizing antibodies between 2 and 4 weeks after vaccination at the lowest dose tested (20 micrograms). The vaccinated pigs were completely protected against intranasal challenge with 100 50% lethal doses of HCV strain Brescia, indicating that E1 expressed in insect cells is an excellent candidate for development of a new, safe, and effective HCV subunit vaccine.     

Efficient assembly and release of SARS coronavirus-like particles by a heterologous expression system

Virus-like particles (VLPs) produced by recombinant expression of the major viral structural proteins could be an attractive method for severe acute respiratory syndrome (SARS) control. In this study, using the baculovirus system, we generated recombinant viruses that expressed S, E, M and N structural proteins of SARS-CoV either individually or simultaneously. The expression level, size and authenticity of each recombinant SARS-CoV protein were determined. In addition, immunofluorescence and FACS analysis confirmed the cell surface expression of the S protein. Co-infections of insect cells with two recombinant viruses demonstrated that M and E could assemble readily to form smooth surfaced VLPs. On the other hand, simultaneous high level expression of S, E and M by a single recombinant virus allowed the very efficient assembly and release of VLPs. These data demonstrate that the VLPs are morphological mimics of virion particles. The high level expression of VLPs with correct S protein conformation by a single recombinant baculovirus offers a potential candidate vaccine for SARS.     

Catalase effect on cell death for the improvement of recombinant protein production in baculovirus-insect cell system

Baculovirus expression vector system (BEVS) in host insect cells is a powerful technology to produce recombinant proteins, as well as virus-like particles (VLP). However, BEVS is based on baculovirus infection, which limits the recombinant protein production by inducing insect cell death. Herein a new strategy to enhance cell life span and to increase recombinant protein production was developed. As baculovirus infection induces cellular oxidative stress, the ability of several antioxidants to inhibit cell death was tested during infection. The production of rotavirus structural proteins was used as model to analyse this new strategy. We found that only catalase is able to partially prevent cell death triggered by baculovirus infection and to inhibit lipid peroxidation. An increase in recombinant protein production was coupled with the partial cell death inhibition. In summary, the addition of catalase is a promising strategy to improve recombinant protein production in BEVS, by delaying insect cell death.     

Expression and processing of hepatitis C virus structural proteins in Pichia pastoris yeast

Development of heterologous systems to produce useful HCV vaccine candidates is an important part of HCV research. In this study different HCV structural region variants were designed to express the first 120 aa, 176 aa, 339 aa, and 650 aa of HCV polyprotein, and aa 384 to 521, or aa 384-605 or aa 384-746 of HCV E2 protein fused to the leader sequence of sucrose invertase 2 allowing the secretion of recombinant E2 proteins. Low expression levels were observed for HCV core protein (HCcAg) variants expressing the first 120 aa and 176 aa (HCcAg.120 and HCcAg.176, respectively). Higher expression levels were observed when HCcAg was expressed as a polypeptide with either E1 or E1 and E2 proteins. In addition, HCcAg was processed to produce two antigenic bands with 21 and 23kDa (P21 and P23, respectively) when expressed as a polypeptide with HCV E1 and E2 proteins. Results also suggest E1 processing in the context of HCcAg.E1.E2 polyprotein. On the other hand, E2.521, E2.605, and E2.680 were efficiently excreted to the culture medium. However, the entire E2.746 variant predominantly localized in the insoluble fraction of ruptured cells. Results suggest that the hydrophobic C-terminal E2 region from aa 681 to 746 is critical for intracellular retention of recombinant E2.746 protein in Pichia pastoris cells. Endo H or PNGase F treatment suggests that E2.746 was modified with high-mannose type oligosaccharides in P. pastoris. These data justify the usefulness of P. pastoris expression system to express HCV structural viral proteins which may be useful targets for HCV vaccine candidates.     

Hepatitis C virus core protein is efficiently released into the culture medium in insect cells

Hepatitis C virus (HCV) is a causal agent of the chronic liver infection. To understand HCV morphogenesis, we studied the assembly of HCV structural proteins in insect cells. We constructed recombinant baculovirus expression vectors consisting of either HCV core alone, core-E1, or core-E1-E2. These structural proteins were expressed in insect cells and were examined to assemble into particles. Neither core-E1 nor core-E1-E2 was capable of assembling into virus-like particles (VLPs). It was surprising that the core protein alone was assembled into core-like particles. These particles were released into the culture medium as early as 2 days after infection. In our system, HCV structural proteins including envelope proteins did not assemble into VLPs. Instead, the core protein itself has the intrinsic capacity to assemble into amorphous core-like particles. Furthermore, released core particles were associated with HCV RNA, indicating that core proteins were assembled into nucleocapsids. These results suggest that HCV may utilize a unique core release mechanism to evade the hosts defense mechanism, thus contributing to the persistence of HCV infection.     

Role of N-linked glycans of HCV glycoprotein E1 in folding of structural proteins and formation of viral particles

Envelope proteins E1 and E2 of the hepatitis C virus (HCV) play a major role in the life cycle of a virus. These proteins are the main components of the virion and are involved in virus assembly. Envelope proteins are modified by N-linked glycosylation, which is supposed to play a role in their stability, in the assembly of the functional glycoprotein heterodimer, in protein folding, and in viral entry. The effects of N-linked glycosylation of HCV protein E1 on the assembly of structural proteins were studied using site-directed mutagenesis in a model system of Sf9 insect cells producing three viral structural proteins with the formation of virus-like particles due to the baculovirus expression system. The removal of individual N-glycosylation sites in HCV protein E1 did not affect the efficiency of its expression in insect Sf9 cells. The electrophoretic mobility of E1 increased with a decreasing number of N-glycosylation sites. The destruction of E1 glycosylation sites N1 or N5 influenced the assembly of the noncovalent E1E2 glycoprotein heterodimer, which is the prototype of the natural complex within the HCV virion. It was also shown that the lack of glycans at E1 sites N1 and N5 significantly reduced the efficiency of E1 expression in mammalian HEK293 T cells.     

Expression of Rice Gall Dwarf Virus Outer Coat Protein Gene (S8) in Insect Cells

To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8 gene was subcloned into the pFastBacTM1 vector,to produce the recombinant baculovirus transfer vector pFB-S8.After transformation,pFB-S8 was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector,Bacmid,generating the recombinant bacmid rbpFB-S8.After being infected b...     

人乳头瘤病毒16型E6基因的T—A克隆及在真核细胞中的表达

由于ＨＰＶ１６Ｅ６蛋白能诱导机体保护性免疫反应,可作为基因治疗的靶抗原。用杆状病毒昆虫细胞表达系统制备了ＨＰＶ１６Ｅ６基因工程蛋白,拟用于宫颈癌细胞系小鼠模型抗癌的免疫治疗。用ＰＣＲ技术从ＨＰＶ１６基因组中扩增获得转化基因Ｅ６的完整ＯＲＦ,按ＴＡ策略将其克隆到自行制备的杆状病毒转移载体ｐＶＬ１３９３Ｔ尾载体中,置于杆状病毒ＡｃＭＮＰＶＰｏｌｈ晚期启动子控制之下,用此重组转移质粒ｐＶＬ１３９３Ｅ６与杆状病毒ＤＮＡ共转染昆虫细胞Ｓｆ９,经噬斑筛选获得带有编码Ｅ６蛋白基因的重组杆状病毒株,并在昆虫细胞Ｓｆ９中表达为非融合性Ｅ６蛋白。ＳＤＳＰＡＧＥ电泳分析其分子量约为１８ｋＤ,免疫印迹实验表明,此重组蛋白能被兔抗ＨＰＶ１６Ｅ６抗体所识别。