2型单纯疱疹病毒毒力蛋白ICP34.5的真核表达及其对Ve-ro细胞活性的影响

目的:在非洲绿猴肾细胞(Vero细胞)中表达2型单纯疱疹病毒(HSV-2)毒力蛋白感染细胞多肽34.5(ICP34.5),并检测其对Vero细胞活性的影响。方法:PCR扩增HSV-2的ICP34.5基因,连接至pEGFP-C2载体,并对重组真核表达载体pEGFP-ICP34.5进行双酶切测序验证;将重组子瞬时转染Vero细胞,RT-PCR检测其在mRNA水平的表达,荧光倒置显微镜观察融合蛋白的表达,MTT法检测细胞活性。结果:经双酶切和测序验证表明pEGFP-ICP34.5构建成功,转染细胞后经RT-PCR验证有目的基因的转录,荧光显微镜下观察到融合蛋白在转染的Vero细胞中表达,MTT法检测结果证实重组质粒可以抵消空质粒对细胞的损伤作用。结论:构建了pEGFP-ICP34.5真核表达载体,其能在Vero细胞中高效表达,并能抵消空质粒对细胞的损伤作用。     

单纯疱疹病毒2型潜伏相关转录体RL1的表达及其抗凋亡作用

单纯疱疹病毒2型潜伏相关转录体(LAT)-RL1对放线菌素D诱导的凋亡作用的研究。构建重组pEGFP-RL1质粒,转染Vero细胞,RT-PCR及荧光鉴定重组质粒的表达。放线菌素D诱导Vero细胞凋亡,通过Hochest33342荧光染色观察细胞形态变化,流式细胞术检测细胞凋亡率,JC-1荧光观察膜电位变化,Caspase 3凋亡蛋白检测。RT-PCR和荧光观察表明该真核表达载体能在Vero细胞中高表达。Hochest33342染色转染了pEGFP-RL1的Vero细胞经放线菌素D凋亡诱导后,细胞形态正常。流式结果表明转染重组质粒pEGFP-RL1且经诱导凋亡组与正常对照组凋亡率无差异,而显著低于诱导凋亡组和转染空质粒pEGFP-C2诱导凋亡组。转染重组质粒pEGPF-RL1的细胞JC-1染色后,红色细胞的比例要远大于绿色细胞的比例,而转染空质粒pEGFP-C2被染成绿色细胞的数量较多。Caspase-3结果表明转染了空质粒pEGFP-C2的Vero细胞经诱导凋亡后,活性显著高于转染了pEGFP-RL1经放线菌素D诱导凋亡后的Vero细胞和正常Vero细胞。HSV-2 LAT RL1具有抗放线菌素D诱导的Vero细胞的凋亡作用。     

哺乳动物细胞表达系统研究进展

目前,哺乳动物细胞已成为生产多种生物药物的首选宿主细胞。哺乳动物细胞表达系统可进行翻译后修饰,表达的重组蛋白接近人源构象,因此被大量用于治疗性重组蛋白的生产,如何建立高效的哺乳动物细胞表达系统也受到研究者们的重视。随着基因组学、转录组学、蛋白质组学以及代谢组学研究不断深入,近几年来在优化哺乳动物表达系统方面取得了长足的进步。从高效表达载体构建、宿主细胞改造、高通量筛选、培养基优化等方面阐述哺乳动物细胞表达系统的研究进展,以期为研究者们提供一定的帮助。     

哺乳动物细胞高效表达系统研究进展

哺乳动物细胞高效表达系统是基因工程制药研究中的一个重要内容,而表达载体和宿主细胞是哺乳动物细胞表达系统的2个基本组成部分。近年来通过降低目的基因的位置效应、提高目的基因的转录和翻译水平以及目的基因的拷贝数等方面构建哺乳动物细胞高效表达载体。与此同时,研究也对宿主细胞进行了多方面的改造,使之能更好地适应工业化大规模培养的要求,从而降低细胞培养和产品纯化的成本。本就哺乳动物细胞高效表达载体的构建及宿主细胞的改造等方面的最新进展作一简述。     

iRhom2及其突变基因重组慢病毒的包装和稳定表达细胞系的建立

目的通过重组慢病毒感染,建立iRhom2及其突变基因的Vero细胞稳定细胞表达系,用于iRhom2的功能研究。方法将iRhom2及其突变基因克隆到慢病毒载体Lenti-OE-Flag,构建重组慢病毒载体Lenti-OEiRhom2和Lenti-OE-iRhom2mut,将重组质粒瞬时转染HEK-293T包装细胞,获得重组慢病毒。将重组病毒感染Vero细胞,利用puromycin进行加压筛选,获得二者的重组表达细胞系。结果构建了iRhom2及其突变基因的逆转录病毒载体,并获得了重组逆转录病毒,并利用该病毒获得了二者的稳定表达细胞系。Western-blot实验证实,二者能够在Vero细胞内稳定表达。结论利用重组逆转录病毒感染,成功获得了iRhom2及其突变系的Vero细胞稳定表达株,为进一步研究iRhom2的生物学功能及其机制奠定了良好的基础。     

单纯疱疹病毒2型感染细胞多肽27真核表达质粒的构建及表达

为了构建单纯疱疹病毒2型(HSV-2)感染细胞多肽27(ICP27)真核表达质粒,应用PCR技术从HSV-2 333株的基因组中扩增ICP27基因,并连接至真核表达载体pEGFPC2,对阳性克隆进行菌落PCR、酶切和测序鉴定后,成功构建了重组质粒pEGFPC2-ICP27。用X fect转染试剂盒将重组质粒pEGFPC2-ICP27转染至Vero细胞中,并用RT-PCR及W estern b lot-ting检测其表达情况。结果显示,ICP27基因在Vero细胞中得到正确表达。真核表达质粒pEGFPC2-ICP27的构建成功,为进一步研究ICP27对宿主细胞的影响奠定了基础。     

杆状病毒用于哺乳动物细胞快速高效表达外源基因的研究

现已发现杆状病毒可进入某些培养的哺乳动物细胞,这提示可将杆状病毒作为一种对哺乳动物细胞的新型基因转移载体。对杆状病毒转移载体的改造及对哺乳动物细胞的基因转移方式进行了进一步的研究。以绿色荧光蛋白基因为报告基因,利用Bac-to-Bac系统构建了分别含有正向和反向CMV启动子表达盒的两种重组杆状病毒。可观察到CMV启动子在Sf9细胞中可启动报告基因的表达,但表达效率较低。用重组杆状病毒感染后Sf9细胞的培养上清直接与HepG2细胞作用,以流式细胞术检测基因转移效率及荧光表达强度,发现这两种病毒在相同的感染复数下对HepG2细胞具有相似的基因转移及表达效率。同时,利用流式细胞术进一步研究了直接使用重组杆状病毒感染4d后Sf9细胞的培养上清对哺乳动物细胞进行基因转移的方法。通过对HepG2细胞的实验结果显示,将带毒Sf9细胞培养上清(1.2×10⁷PFU/mL)用哺乳动物细胞培养基1倍稀释后,37℃下孵育靶细胞12h(moi=50),可达到较高的基因转移及表达效率,同时不会对细胞造成明显损伤。将重组杆状病毒与脂质体和逆转录病毒这两种系统对HepG2及CV1细胞的基因转移效率进行了比较,结果发现在同样未经浓缩等特殊处理的条件下重组杆状病毒对这两种细胞的基因转移效率是最高的。因此可以认为,经过适当改造后的Bac-to-Bac重组杆状病毒系统可作为一种对哺乳动物细胞简便高效的基因转移表达载体。     

人尿激酶原cDNA在昆虫杆状病毒真核表达系统中的高效表达

人尿激酶原(pro-urokinase,pro-UK)是一种新型溶栓剂,优于尿激酶,具有血纤维蛋白特异性。为了在昆虫杆状病毒表达系统中高效表达pro-UK,我们在pAc373基础上,插入野生型AcMNPV polyhedrin启动子区-7～1碱基序列,构建了一个高表达转移载体pAcYT。分别经三次克隆将pro-UK cDNA正向插入到转移载体pAc373或PAcYT的BarnHI-KpnI位点上。用LiPofectin将pAcyT-UKDNA或pAc373-UK DNA与AcMNPV DNA共转染到昆虫Sf9细胞中,空斑法筛出重组病毒阳性克隆株。高效表达结果是:1.ELISA法确定重组病毒Sf9细胞分泌表达产物pro-UK为96mg/L培养基上清,平板法测定溶圈活性为1600IU/mL培养基上清;2.亲和层析一步法纯化表达产物,回收率选70％,以上,比活约为60000IU/mg;3.纯化的pro-UK,无论是否经还原处理,其SDS-PAGE图谱均为相同的单一条带,MR 50000;4.Western blot与SDS-PAGE图谱吻合。     

核衣壳蛋白VP39融合TAT转导肽对杆状病毒转导哺乳动物细胞的影响

为了提高昆虫杆状病毒在哺乳动物细胞中转导基因的效率,构建了重组杆状病毒AcRed-tat和AcRed。两者都能在哺乳动物细胞内表达红色荧光蛋白作为报告基因。同时,AcRed-tat带有HIV-1Tat转导肽、病毒主要衣壳蛋白基因vp39及增强型绿色荧光蛋白(egfp)三者的融合基因,并由杆状病毒多角体启动子表达,能够在昆虫细胞中表达该Tat融合蛋白,并掺入子代病毒粒子。而AcRed作为相应的对照病毒,带有多角体启动子表达vp39和egfp的融合基因。2株病毒分别转导哺乳动物细胞后,利用流式细胞仪检测报告基因的表达水平,发现在CHO和Vero细胞中AcRed-Tat介导的报告基因表达水平明显高于AcRed,而在HEK293细胞中2株病毒介导的报告基因表达水平差异不显著。结果表明Tat转导肽可以提高杆状病毒对一部分哺乳动物细胞的转导效率,为改进杆状病毒-哺乳动物细胞转导载体提供了新的思路。     

Stable production of recombinant pro-urokinase by human lymphoblastoid Namalwa KJM-1 cells: Host-cell dependency of the expressed-protein stability

Human pro-urokinase (pro-UK) gene was engineered for expression in mammalian cells. The stability of recombinant pro-UKs produced by two kinds of cells, Chinese hamster ovary (CHO) and human lymphoblastoid Namalwa KJM-1 cells, were compared. The pro-UK expressed in CHO cells in serum-free medium was degraded by cysteine endopeptidase secreted by CHO cells. This endopeptidase was inhibited by pchloromercuribenzonate (PCMB) and leupeptin more efficiently than by aprotinin. On the other hand, the pro-UK expressed in Namalwa KJM-1 cells was not degraded, resulting in the stable production of pro-UK at a rate of 2–3 g/10⁶ cells/day by use of a gene amplification method with dihydrofolate reductase (DHFR) in serum-free medium. Thus, Namalwa KJM-1 cells showed the desired characteristics as a host cell for the production of recombinant proteins. The stability of recombinant proteins produced in heterologous systems may vary depending on the host cells.Abbreviations ABTS 2,2-azino-di-(3-ethylbenzothiazoline) sulfonic acid diammonium salt - AMC 7-amino-4-methylcoumarin - CHO Chinese hamster ovary - DFP diisopropylfluorophosphate - DHFR dihydrofolate reductase - ELISA enzyme-linked immunosorbent assay - MCA 4-methylcoumaryl-7-amide - MTX methotrexate - NEAA non essential amino acid - NEM N-ethylmaleimide - PCMB p-chloromercuribenzonate - PMSF phenylmethanesulfonyl fluoride - pro-UK pro-urokinase     

The expression of essential components for human influenza virus internalisation in Vero and MDCK cells

MDCK and Vero cell lines have been used as substrates for influenza virus replication. However, Vero cells produced lower influenza virus titer yield compared to MDCK. Influenza virus needs molecules for internalisation of the virus into the host cell, such as influenza virus receptor and clathrin. Human influenza receptor is usually a membrane protein containing Sia(α2,6) Gal, which is added into the protein in the golgi apparatus by α2,6 sialyltransferase (SIAT1). Light clathrin A (LCA), light clathrin B (LCB) and heavy clathrin (HC) are the main components needed for virus endocytosis. Therefore, it is necessary to compare the expression of SIAT1 and clathrin in Vero and MDCK cells. This study is reporting the expression of SIAT1 and clathrin observed in both cells with respect to the levels of (1) RNA by using RT-PCR, (2) protein by using dot blot analysis and confocal microscope. The results showed that Vero and MDCK cells expressed both SIAT1 and clathrin proteins, and the expression of SIAT1 in MDCK was higher compared to Vero cells. On the other hand, the expressions of LCA, LCB and HC protein in MDCK cells were not significantly different to Vero cells. This result showed that the inability of Vero cells to internalize H1N1 influenza virus was possibly due to the lack of transmembrane protein receptor which contained Sia(α2,6) Gal.     

Recombinant wild-type and edmonston strain measles viruses bearing heterologous H proteins: role of H protein in cell fusion and host cell specificity

Wild-type measles virus (MV) isolated from B95a cells has a restricted host cell specificity and hardly replicates in Vero cells, whereas the laboratory strain Edmonston (Ed) replicates in a variety of cell types including Vero cells. To investigate the role of H protein in the differential MV host cell specificity and cell fusion activity, H proteins of wild-type MV (IC-B) and Ed were coexpressed with the F protein in Vero cells. Cell-cell fusion occurred in Vero cells when Ed H protein, but not IC-B H protein, was expressed. To analyze the role of H protein in the context of viral infection, a recombinant IC-B virus bearing Ed H protein (IC/Ed-H) and a recombinant Ed virus bearing IC-B H protein (Ed/IC-H) were generated from cloned cDNAs. IC/Ed-H replicated efficiently in Vero cells and induced small syncytia in Vero cells, indicating that Ed H protein conferred replication ability in Vero cells on IC/Ed-H. On the other hand, Ed/IC-H also replicated well in Vero cells and induced small syncytia, although parental Ed induced large syncytia in Vero cells. These results indicated that an MV protein(s) other than H protein was likely involved in determining cell fusion and host cell specificity of MV in the case of our recombinants. SLAM (CDw150), a recently identified cellular receptor for wild-type MV, was not expressed in Vero cells, and a monoclonal antibody against CD46, a cellular receptor for Ed, did not block replication or syncytium formation of Ed/IC-H in Vero cells. It is therefore suggested that Ed/IC-H entered Vero cells through another cellular receptor.     

Xfect介导重组质粒pEGFP-C2/LAT-ORF3高效转染Vero细胞的方法及其条件的优化

旨在用Xfect试剂介导重组质粒pEGFP-C2/LAT-ORF3转染Vero细胞,以期获得转染效率较高的方法,并检测目标片段的表达,从而为进一步研究HSV-2 LAT基因及其ORF3片段的生物学功能奠定基础。用Xfect试剂介导重组质粒pEGFP-C2/LAT-ORF3转染Vero细胞,48 h后观察荧光表达情况,并计算不同转染方法分别在有无血清及质粒与Xfect Polymer比例不同时的转染效率。用RT-PCR检测ORF3片段在Vero细胞中的表达。结果显示,采用贴壁转染法,在有血清及质粒与Xfect Polymer比例为5μg/2μL时转染效率较高;RT-PCR可以获得目的条带,证明ORF3片段在Vero细胞中得到表达。本试验获得的优化条件可以显著提高Xfect Polymer对Vero细胞的转染效率;以绿色荧光蛋白作为标签鉴定目的基因在细胞中表达的方法切实可行。     

单纯疱疹病毒2型潜伏相关转录体ORF1的表达及其抗凋亡作用

旨在研究单纯疱疹病毒2型潜伏相关转录体 (LAT) 开放读码框1 (ORF1) 对放线菌素D诱导的凋亡作用的影响。以HSV-2 333基因组为模板PCR扩增ORF1片段,构建重组质粒pEGFP-ORF1,转染Vero细胞,RT-PCR鉴定ORF1的表达。放线菌素D诱导Vero细胞凋亡,通过荧光显微镜观察凋亡小体,Hochest33258荧光染色观察细胞形态变化,MTT检测细胞活性,流式细胞术检测细胞凋亡率。双酶切和测序确认pEGFP-ORF1构建成功,RT-PCR表明该真核表达载体能在Vero细胞中高效表达。转染了pEGFP-ORF1的Vero细胞经放线菌素D凋亡诱导后,Hochest33258染色显示细胞形态正常。MTT结果表明转染了重组质粒pEGFP-ORF1的Vero细胞经放线菌素D凋亡诱导后Vero细胞活性与未经任何处理的正常对照组相比,无显著差异 (P＞0.05),但高于放线菌素D诱导凋亡的Vero细胞组及与转染空质粒pEGFP-C2且放线菌素D诱导凋亡的Vero细胞组,差异具有统计学意义 (P＜0.05)。流式结果表明,转染重组质粒pEGFP-ORF1且经放线菌素D诱导凋亡组与正常对照组凋亡率差异不显著 (P＞0.05),而显著低于放线菌素D诱导凋亡组和转染空质粒pEGFP-C2且经放线菌素D诱导凋亡组 (P＜0.05)。HSV-2 LAT ORF1具有抗放线菌素D诱导的Vero细胞的凋亡作用。     

脂质体法和电穿孔法在转染Cos-7,Vero和Namalwa细胞中的比较

用脂质体法和电穿孔法分别转染Cos-7,Vero和Namalwa细胞。发现脂质体法在转染效率和操作方便方面比电穿孔法优越,而电穿孔法对细胞种类的适应性方面比脂质体法广。电穿孔法能转染Cos-7、Namalwa和Vero细胞,而用脂质体法只能转染Cos-7和Vero细胞。     

脂质体法和电穿孔法转染哺乳动物细胞研究

用脂质体法和电穿孔法分别转染Cos-7,Vero和Namalwa细胞.发现脂质体法在转染效率和操作方便方面比电穿孔法优越,而电穿孔法对细胞种类的适用性方面似乎比脂质体法广. 结果表明,电穿孔法能转染Cos-7,Namalwa和Vero细胞,而用脂质体法只能转染Cos-7和Vero细胞.     

Expression of human erythropoietin cDNA in human lymphoblastoid Namalwa cells: the inconsistency of a stable expression level with transient expression efficiency

Recombinant plasmids for the expression of human erythropoietin (EPO) cDNA in Namalwa cells were constructed. From the results of the EPO expression efficiency in transiently transfected cells, it was found that the simian virus 40 (SV40) early promoter directs EPO synthesis more efficiently in Namalwa cells than does the long terminal repeat promoter of Rous sarcoma virus and that the 3'-noncoding sequence including splice junction and polyadenylation site derived from the rabbit beta-globin gene are more effective than those of the SV40 early gene. However, in stable transformants, no simple relationship was found between the expression level of EPO cDNA and the structure of the introduced expression vectors.     

Epitope-tagging vectors for the expression and detection of recombinant proteins in mycobacteria

New tools are required to study the growing number of uncharacterised genes derived from genome sequence projects that are specific to bacterial pathogens such as Mycobacterium tuberculosis. We have developed a series of vectors that permit the specific detection of recombinant proteins expressed in mycobacterial species. Gene expression in these vectors is driven by the strong hsp60 promoter of Mycobacterium bovis BCG and detection of expressed products is facilitated by C-terminal fusion of residues 409-419 of the human c-myc proto-oncogene. Using the M. tuberculosis Ag85B as a reporter of gene expression, we demonstrate that the vectors permit the specific detection of recombinant products expressed in the host species M. bovis BCG. BCG over-expressing Ag85B was a potent inducer of Ag85B-specific T cells in immunised mice, indicating that the C-terminal c-myc tag did not alter the characteristics of the recombinant protein. The versatility of the epitope-tagging vectors was demonstrated by the efficient secretion and detection of recombinant products in BCG. The vectors described in this study will facilitate the expression of foreign proteins in mycobacterial host systems.