Susceptibility to AcMNPV and expression of recombinant proteins by a novel cell clone derived from a <Emphasis Type="Italic">Trichoplusia ni</Emphasis> QAU-BTI-Tn9-4s cell line

It is well known that Tn5B1-4 (commercially known as the High Five) cell line is highly susceptible to baculovirus and provides superior production of recombinant proteins when compared to other insect cell lines. But the characteristics of the cell line do not always remain stable and may change upon continuous passage. Recently an alphanodavirus, named Tn5 Cell Line Virus (or TNCL Virus), was identified in High Five cells in particular. Therefore, we established a new cell line, QB-Tn9-4s, from Trichoplusia ni, which was determined to be free of TNCL virus by RT-PCR analysis. In this paper, we describe the development of a novel cell clone, QB-CL-B, from a low passage QB-Tn9-4s cell line and report its susceptibility to AcMNPV, and the level of recombinant protein production. This cell clone was similar to its parental cells QB-Tn9-4s and Tn5B1-4 cells in morphology and growth rate; although it also showed approximately the same responses to AcMNPV infection and production of occlusion bodies, there were higher levels of recombinant protein production in comparison to QB-Tn9-4s (parental cells) and High5 cells.     

Establishment, Growth kinetics, and Susceptibility to AcMNPV of Heat Tolerant Lepidopteran Cell Lines

Lepidopteran heat-tolerant (ht) cell lines have been obtained with sf-9, sf-21 and several Bombyx cells. They have a distinct karyotype, membrane lipid composition, morphology and growth kinetics from the parental cell lines. In this paper, we report the development of ht cell lines from other insect species and examination of their growth characteristics and virus susceptibility. Adaptation of cell lines sf-9, BTI-TN-5B1-4 (High5) and BTI-TN-MG1 (MG1) to 33℃ and 35℃ was carried out by shifting the culture ...     

肌动蛋白在AcMNPV向细胞外运输过程中作用的初步研究

本实验利用AcMNPV(Autographa californica multiple nuclear polyhedrosis virus,AcMNPV)的bac-to-bac系统构建了两种重组病毒,即含GFP-actin融合基因的重组病毒AcMNPV-GFP-actin和含GFP基因的重组病毒AcMNPV-GFP。用这两种重组病毒分别感染Sf9细胞,以AcMNPV-GFP感染的Sf9细胞为对照,用共聚焦激光扫描显微镜观察了绿色荧光在病毒感染过程中的分布情况。由于肌动蛋白和绿色荧光蛋白是共定位的,所以绿色荧光的分布情况就是肌动蛋白的分布情况。实验中观察发现,AcMNPV-GFP感染的Sf9细胞中的绿色荧光,在整个感染过程中是弥散分布的,而AcMNPV-GFP-actin感染Sf9细胞后24172h这段时间内,肌动蛋白最初聚集在细胞核内,随后逐渐由细胞核向细胞质转移,最后完全聚集于细胞膜。根据实验结果,推测肌动蛋白可能参与了AcMNPV出芽型病毒粒子(BV)由细胞核向细胞质运输以及从细胞膜排出的过程。     

Establishment, Growth kinetics, and Susceptibility to AcMNPV of Heat Tolerant Lepidop teran Cell Lines

Lepidopteran heat-tolerant(ht)cell lines have been obtained with sf-9,sf-21 and several Bombyx cells.They have a distinct karyotype,membrane lipid composition,morphology and growth kinetics from the parental cell lines.In this paper,we report the development of ht cell lines from other insect species and examination of their growth characteristics and virus susceptibility.Adaptation of cell lines sf-9,BTI-TN-5131-4(High5)and BTI-TN-MG1(MG 1)to 33℃ and 35℃ was carried out by shifting the culture temperature between 28℃ and higher temperatures by a gradual stepwise increase in temperature.The process of adaption to a higher culture temperature was accomplished over a period of 2 months.The cell lines with the temperature adaption were designated as sf9-ht33,sf9-ht35,High5-ht33,High5-ht35,MG1-ht33,MG1-ht35.These cell lines have been subcultured over 70 passages.Adaption to high temperatures was confirmed by a constant population doubling time with individual cell lines.The population doubling time of heat adapted cell lines were 1-4 h less than these of parental cell lines.Cell shapes did not show obvious change,however,the cell size of sf9-ht cells was enlarged and those of High5 and MG1 ht cells were reduced after heat adaption.When the cell lines were infected with Autographa californica nuclear polyhedrosis virus(AcMNPV)at 28℃,33℃,35℃ and 37℃,production of budded virus and occlusion bodies in each cell line was optimum at its own adapted temperature.     

丙型肝炎病毒功能基因在CHO细胞中的表达研究

丙型肝炎病毒(HCV)与宿主细胞因子的相互作用已经成为国内外研究的热点和难点。近期研究已经证实HCV的感染对宿主多种途径中基因的转录均能产生影响。为了进一步研究究竟是HCV中的哪些功能基因在与特定细胞因子的相互作用中起主导作用,构建了分别含有HCV Core、E1、E2、p7、NS2、NS3、NS4A、NS4B、NS5A和NS5B基因的真核表达质粒,分别转入宿主细胞CHO-K1中,在G418的选择压力下筛选获得稳定表达HCV单个蛋白的细胞系(10株)。PCR和RT-PCR可分别从稳定细胞系中检测到相应的HCV基因的DNA和mRNA,冻存和复苏不会造成HCV基因的丢失。Western-blot检测到稳定细胞系中表达E1,E2和NS5B蛋白,说明HCV基因在CHO-K1中已经形成稳定表达。薄层层析(TLC)结果显示,含有不同HCV基因的稳定传代细胞系中,UDP-葡萄糖神经酰胺葡萄糖基转移酶(UGCG)活性均发生了不同程度的变化,其中E2和p7的表达使胞内UGCG的活性提高了约1倍,NS2和NS5A则使UGCG的酶活提高了约0.6倍。该稳定细胞系的建立为研究病毒与宿主因子的相互作用及药物筛选奠定了基础。     

稳定表达HLA-A33蛋白细胞系的建立

旨在构建能稳定表达HLA-A33蛋白的细胞系,并观察其在细胞中的表达水平.首先克隆取得HLA-A33基因,并将其插入慢病毒载体,经酶切和测序鉴定,确定载体构建正确.通过慢病毒系统感染正常RD细胞,将HLA-A33基因整合进RD细胞的基因组.提取构建细胞系的基因组做PCR鉴定,并通过免疫荧光和蛋白免疫印迹法检测,结果显示,HLA-A33基因成功整合入RD细胞基因组中,且在重组细胞系中成功表达.该细胞系可为A33等位基因与HBV感染后的慢性化以及EV71感染的相关研究提供试验参考.     

不同HCV基因型C蛋白在HepG2细胞引起基因表达改变的初步研究

目的:探讨丙型肝炎病毒(HCV)不同基因型C蛋白在HepG2细胞中的基因表达。方法:分别构建能在HepG2细胞表达HCV-1b、HCV-2a和HCV-4d等3种基因型C蛋白的重组体,将Affymetrix公司人基因芯片HG-U133A和HG-U133B用于本研究。结果:3种C蛋白均可引起不同基因上调和下调改变。3种C蛋白表达如两两相比,有若干相同基因表达改变;如三者相比,有PPM1A、TNNI2、ZNF236、FSCN1基因表达出现相同改变。结论:HCV不同基因型C蛋白所引起的基因表达谱各有特征,主要涉及分子转运、信号转导、致病或癌基因等,这对从基因表达层面认识HCVC蛋白的功能及HCV致病机制均有重大帮助。     

高转移潜力食管癌细胞株的建立及其生物学特征

目的建立具有高转移潜力食管癌细胞株并研究其生物学特征。方法将食管癌细胞系EC109细胞悬液异位移植到SCID小鼠胃壁,约3个月后或动物濒临死亡时处死,行病理学解剖,将肉眼可见的纵隔淋巴结转移瘤块接种于SCID鼠皮下扩增,然后取小鼠皮下瘤组织块进行细胞培养,得到性状稳定的细胞株NMC109后,用MTT法分析细胞生长曲线,Western bloting法检测与细胞分裂增殖能力密切相关的TopoⅡα表达,酶谱法检测MMP-2和MMP-9的活性,划痕实验和Transwell体外移动实验检测细胞的移动能力。结果与母本细胞EC109相比,所获得的细胞株NMC109其增殖能力和TopoⅡα表达明显增强,MMP-9的活性明显升高,移动能力明显增强。结论获得了具有高转移潜力的食管癌细胞株。     

A suspended cell line from Trichoplusia ni (Lepidoptera): Characterization and expression of recombinant proteins

A suspended cell line from Trichoplusia ni embryos was established, and its susceptibility to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infection was investigated. This cell line had characteristics distinct from the BTI‐Tn5Bl‐4 cell line (Tn5Bl‐4) from T. ni in growth, and showed approximately the same responses to AcMNPV infection, production of occlusion bodies, and levels of recombinant protein expression. No clumps were observed at maximum cell density at late‐log phase in shake‐flask or T‐flask cultures, and thus the cells represent a useful new contribution for baculovirus research. The cells consist of two major morphological types: approximately 70% spindle‐shaped cells and 30% round cells. The cell line was highly susceptible to virus infection and produced around 107 AcMNPV occlusion bodies per cell, on average. Production of β‐galactosidase and secreted alkaline phosphatase was high with 3.97 ± 0.13 × 10⁴IU/mL and 3.48 ± 0.40 IU/mL, respectively. This cell line may be applicable for studies of scale‐up production of viruses or baculovirus‐insect cell expression. We also believe the new line can be a source for cell clones with higher production of virus and recombinant proteins compared to the parent or other existing cell lines such as Tn5Bl‐4.     

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.     

抗菌肽Cec4a的重组表达和抗菌活性研究*

目的:构建Cec4a的原核重组表达体系,通过诱导表达、酶切纯化获得重组蛋白,并检测产物的抗菌活性。方法:基于Cec4a的序列设计引物,克隆Cec4a基因的DNA片段。利用原核表达载体(pCold-SUMO)构建重组原核表达质粒,并将其转化到大肠杆菌C41(DE3)等感受态细胞,使用IPTG进行诱导表达。通过Ni-NTA亲和层析柱纯化,获得含有His-SUMO标签的重组Cec4a融合蛋白。在SUMO蛋白酶酶切后,再次使用Ni-NTA亲和层析纯化,得到目的蛋白,最后用鲍曼不动杆菌(ATCC19606)作为指示菌检测表达产物的抗菌活性。结果:成功构建pCold-SUMO-Cec4a原核表达质粒,测序分析其序列与预期结果一致。Cec4a融合蛋白表达量为42.8mg/L,纯化后的Cec4a重组蛋白对鲍曼不动杆菌的MIC为4 μg/mL。结论:通过原核表达,并经Ni-NTA亲和层析纯化,获得了具有抗菌活性的重组蛋白Cec4a,为研究Cec4a的生物活性、抗菌机制及应用奠定了基础。     

可诱导的稳定表达Spata3细胞系的建立及特征

spata3是一个在睾丸中特异性表达的基因,可能与精子发生或生精细胞凋亡相关.为了进一步研究Spata3的功能,将spata3克隆入经修饰的pcDNA5/FRT/TO表达载体,应用Flp-InTMT-RExTM-293 细胞系作为拗飨赴? 成功地构建了可被四环素或 Doxycline 诱导的稳定表达 Flp-InTMT-RExTM-sptat3 的细胞系.该细胞系在spata3基因 的3'端有2×FLAG tag和2×Histag,在缺乏可利用的spata3或其抗体的情况下,也能够很容易地应用商品化的FLAG抗体检测到spata3全长蛋白的表达.这种可诱导的稳定表达Flp-InTMT-Rex TM-spata3 的细胞系的建立,不仅有利于spata3的分析鉴定和功能研究,而且对于其他蛋白质的分离纯化和功能研究也有很好的借鉴作用.     

重组hS100A6蛋白的原核表达及其对人骨肉瘤细胞系143B的生物学作用

目的:制备重组hS100A6蛋白,并研究其对人骨肉瘤细胞系143B的生物学作用。方法:构建pGST-HRV3C-hS100A6质粒,经转化至E.coli BL21,IPTG诱导表达融合蛋白GST-HRV3C-hS100A6,经超声破菌,谷胱甘肽-琼脂糖4B球珠纯化,GST-HRV3C酶切,再纯化,Western blot鉴定,分光光度法蛋白定量。以人骨肉瘤细胞系143B为研究对象,MTT检测细胞增殖,Hoechst检测细胞凋亡,Transwell检测细胞迁移和侵袭,Western blot检测hS100A6对β-catenin表达的影响。结果:成功制备重组hS100A6蛋白,测得该蛋白产量约为4mg/L菌液。30μg/ml重组hS100A6促进143B细胞的增殖、迁移、侵袭以及β-catenin的表达(P0.05),对143B细胞的凋亡无明显影响(P0.05)。结论:成功制备重组hS100A6蛋白,30μg/ml重组hS100A6蛋白对143B细胞有一定的促进作用。     

锦鲤鳍条组织细胞系的建立及其生物学特性

采用组织块移植培养技术,对来源于锦鲤（Cryprinus carpiod）鳍条组织的细胞进行原代培养,建立了锦鲤鳍条组织细胞系,已稳定传代60多次,命名为Koi-Fin。锦鲤鳍条组织细胞为成纤维样细胞,最佳培养基为MEME,最适血清体积分数为10%,最适培养温度为25 oC,群体倍增时间为43.5 h。该细胞经液氮冷冻保藏12个月后采用台盼蓝染色,约（80.21±5.84）%的细胞具有细胞活性,复苏细胞生长旺盛。细胞染色体分析显示,第16代锦鲤鳍条组织细胞的染色体数目为正常二倍体2n=100,第40代细胞的染色体众数为52。病毒敏感性试验结果表明,Koi-Fin细胞系对锦鲤疱疹病毒（Koi Herpesvirus,KHV）敏感,可产生典型细胞病变效应,病毒滴度为107.86±0.51TCID50/mL。针对锦鲤疱疹病毒胸苷激酶（thymidine kinase,TK）基因设计特异性引物进行PCR检测,可扩增出病毒靶基因片段。     

中和性流行性感冒病毒基因工程抗体在昆虫细胞中的全基因表达、纯化及鉴定

将中和性流行性感冒 (流感 )病毒基因工程抗体IV 2、IV 6的轻链和重链Fd段基因 ,分别克隆入全抗体表达载体 pAC L Fc ,构建成杆状病毒表达载体pAC L Fc Ⅳ 2和 pAC L Fc Ⅳ 6 ,转染昆虫Sf9细胞 ,利用杆状病毒 /昆虫细胞系统实现抗体的分泌型表达 ,表达产物进行亲和层析分离纯化。SDS PAGE电泳和Westernblot法证实有完整免疫球蛋白的表达 ,免疫印迹法证实它们能与流感病毒血凝素蛋白特异性结合。经间接竞争性抑制ELISA法测定 ,抗体与流感病毒抗原结合的解离常数KD 值分别为 2 5× 10 -9M和 3 0× 10 -9M。流感病毒基因工程全抗体经在昆虫细胞中的表达、纯化和抗体特性鉴定 ,获得了两株纯化的全抗体 ,可用于以后的动物模型呼吸道粘膜被动免疫抗感染的研究。     

A Pair of Ligation-Independent Escherichia coli Expression Vectors for Rapid Addition of a Polyhistidine Affinity Tag to the N- or C-Termini of Recombinant Proteins

6×His tag is one of the most widely used affinity fusion tags that facilitates detection and purification of recombinant proteins. However, the location of this tag within a particular type of protein may influence the expression, solubility, and bioactivity of the protein, and the optimal location needs to be determined experimentally. To provide a tool for rapid generation of 6× His tags at the N- or C-terminus of any recombinant protein, we have constructed a pair of Escherichia coli expression vectors—pLIC-NHis and pLIC-CHis—based on the pET30a vector, for ligation-independent cloning (LIC). Construction of this new pair of LIC vectors was accomplished by replacement of the multiple cloning site of pET30a with two specifically designed LIC cloning sites. A target gene derived by PCR with a pair of predesigned primers can be inserted into the LIC site of pLIC-NHis for expression of recombinant proteins fused with the N-terminal sequence MHHHHHHG or into that of pLIC-CHis for expression of recombinant proteins with the C-terminal sequence THHHHHH. Successful expression of two normal mammalian prion proteins and five bacterial proteins in E. coli using this pair of LIC vectors reveals that these vectors are valuable tools for the production of recombinant His-tagged proteins in E. coli.     

毛喉萜抑制人胃癌细胞BGC－823增殖与ras基因表达相关性研究

毛喉萜（ｆｏｒｓｋｏｌｉｎ）对人胄癌细胞ＢＧＣ－８２３增殖有明显抑制作用,具药物剂量和作用时间之依赖性。剂量为２×１０~（－５）ｍｏｌ／Ｌ之毛喉萜使胃癌细胞在软琼脂中形成集落的能力显著降低;癌基因ｃ－Ｈａ－ｒａｓ之表达明显被抑制,细胞核中与ｒａｓ基因上游调控区２．５ｋｂ片段结合的三种蛋白结合能力下降。联系到以同样浓度药物处理胃癌细胞７２ｈ,细胞质、膜与细胞核中蛋白激酶Ｃ（ＰＫＣ）活性均下降的现象,可能ＰＫＣ活性下降与Ｈａ－ｒａｓ基因上游片段２．５ｋｂ结合蛋白之结合能力下降存在相关性,ＰＫＣ可能通过影响ＤＮＡ结合蛋白的磷酸化作用,导致了Ｈａ－ｒａｓ基因表达之被阻抑。而ｒａｓ基因表达下降可能是毛喉萜抑制胃癌细胞增殖的一个重要分子事件。     

嵌合中国分离株基因的丙型肝炎病毒（1b／2a）细胞培养模型的建立

目的：建立嵌合中国分离株基因的丙型肝炎病毒（HCV）细胞培养模型。方法：利用3片段融合PCR的方法将中国HCV河北分离株（1b）的全长包膜蛋白基因引入JFH1（2a）株基因骨架,构建包膜蛋白基因区相互置换的嵌合HCV（1b／2a）全长基因组,经线性化后体外转录获得全长RNA,转染Huh7．5．1细胞系,用免疫荧光及蛋白印迹实验检测。结果：该RNA可以产生具有体外感染活性的嵌合HCV,且感染性可在共同培养的细胞间传播。结论：首次在国内建立了嵌合中国HCV分离株基因的HCV细胞培养体系。     

一个在肺腺癌细胞系中差异表达cDNA片段的定位及表达分析

为了研究和克隆肺癌转移相关候选基因,探讨肺癌发生及转移的分子基础,应用细胞培养,cDNA克隆,Noorthern印记杂交和生物信息学技术分析了在细胞来源相同,但转移能力不同的肺腺癌细胞系AGZY83－a和Anip973中差异表达片段OPB7－1在人不同组织中和不同人肺癌细胞系中的表达情况,并应用RH定位技术对该片段进行了基因定位。表明OPB7－1与已知基因同源性差,该基因在正常人多种组织中有表达,心肌和骨骼肌中高表达,转录本均为3.0kb左右。在不同人肺癌细胞系中存在该基因的表达差异,高转移潜能,低分化及高浸润的细胞系中呈高表达趋势,且表达的片段大小略有差别。提示OPB7－1是一个具有广泛表达为的基因,可能是与肺癌的发生发展相关的新基因。