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。     

内江猪IL-18基因的克隆及原核表达

以ConA刺激的内江猪外周血单核淋巴细胞(PBMC)为模板,采用RT-PCR技术扩增出猪IL-18全长基因,克隆其成熟蛋白的编码基因(471bp)至pMD18-T克隆载体,经双酶切和测序获得阳性克隆。通过EcoRⅠ/XhoⅠ双酶切及连接反应,构建了pET-32a( )-IL-18原核表达质粒。经双酶切和DNA测序证实重组质粒构建正确,将阳性重组质粒转化大肠杆菌BL21(DE3),IPTG诱导表达,SDS-PAGE,Western-blot证实表达出35kD左右的融合蛋白。经实验确定重组内江猪IL-18蛋白诱导表达的最佳条件为IPTG 0.2mmol/L,30℃诱导培养5h。在最佳表达条件下,pIL-18的相对含量为59.6%。内江猪IL-18的原核表达为重组IL-18的纯化及其生物学功能的进一步研究提供了基础。     

鼠白细胞介素12（mIL—12）在昆虫细胞中的表达

Since human IL-12 is species-specific in its functions and elicits little biological responses from mouse lymphocytes, it is necessary to express recombinant murine IL-12 for the usage in studying the effects of this cytokine in various rodent models. Thereby, we can investigate the role of IL-12 in immune response in vivo and evaluate its potential clinical utility. Thus, we firstly constructed two expression vectors, pVL1393-mp40 and pVL1393-mp35. They were used to co-transfect the insect cells(Sf9) separately with linearized polyhedrosis virus genomic DNA. Two kinds of recombinant viruses AcNPV-mp40 and AcNPV-mp35 were visually screened out, and mp40 and mp35 were co-expressed in the insect cells co-infected by AcNPV-mp40 and AcNPV-mp35. The results of real-time Biomolecular Interaction Analysis (BIA) and Northern blot demonstrated that the recombinant mIL-12 was expressed successfully in the insect cells. The molecular weights of recombinant mp40 and mp35 were 40 KDa and 22 KDa on SDS-PAGE under reducing conditions, respectively. The apparent molecular weight of recombinant mIL-12 is 80 KDa under non-reducing conditions of Western blot. Biological activity of the recombinant product was detected in conditional medium using antibody-capture bioassay. The expression level of recombinant mIL-12 was about 10-15 micrograms/10(6) cells, as compared with the calibration curve of mIL-12.     

杆状病毒-昆虫细胞表达系统在复合体重组表达应用中的研究进展

真核细胞大部分生命活动是通过复合体催化的。因此,系统了解这些复合体的生物学功能,将在健康和疾病方面具有重要意义。由于内源性复合体存在低丰度和异质性特点,因而直接提取复合体存在一定的困难。杆状病毒-昆虫细胞表达系统可成功表达复合体,为研究复合体的结构和功能提供新的手段。综述近年来利用杆状病毒-昆虫细胞表达系统进行蛋白质复合体结构和功能研究的进展。     

人白细胞介素12在昆虫细胞中的表达

人白细胞介素12(hIL-12)是一种异源二聚体细胞因子,由p40和p35两个亚基通过二硫键连接而成。首先构建了两个表达载体pVL1392-hp40和pVL1393-hp35,并分别与线性化多角体病毒基因组DNA共转染昆虫细胞株Sf9,用目视法筛选出重组病毒AcNPV-hp40和AcNPV-hp35。利用PHA激活的人淋巴细胞增殖试验,在条件培液中检测到重组hIL-12的生物活性,表达量约为1.5～2μg/10⁶细胞。经实时BIA和Northern blot分析,hp35和hp40两亚基在昆虫细胞中获得表达。非还原条件下的Westernblot结果显示,重组hIL-12的表观分子量为76kDa,hp40同源二聚体的表观分子量为92kDa。重组ph40具有明显抑制hIL-12生物活性的作用。     

人白细胞介素18在大肠杆菌中的表达,纯化和复性

用ＲＴ－ＰＣＲ从ＰＨＡ刺激的人外周血单个核细胞（ＰＢＭＣ）中扩增出人白细胞介素１８（ＩＬ－１８）的ｃＤＮＡ,克隆到表达载体ｐＪＷ２中,经热诱导后在大脾性杆菌中得到高效表达。表达的重组ＩＬ－１８占菌体总局旧白质的２０％,表达的蛋白南分子量约为１８ＫＤ,与预期分子量相符。产物主要以包涵体的形式存在。包涵体经超声破碎、洗涤后以８ｍｏｌ／Ｌ尿素溶解,经Ｓｅｐｈａｄｅｘ Ｇ－１００柱纯化后,纯度可达９０％以     

猪白细胞介素18基因的克隆、表达及生物学活性检测

通过RT-PCR方法直接从猪脾脏淋巴细胞中扩增出猪白细胞介素18(pIL-18)成熟蛋白基因的cDNA, 克隆到pGEM-T载体, 构建重组质粒pGEM-T-IL18, 转化E.coli JM109感受态细胞, 取PCR和酶切鉴定为阳性的重组质粒进行序列测定。测序结果表明, pIL-18成熟蛋白基因核苷酸长度为474 bp, 编码157个氨基酸。将其克隆到表达载体pGEX6P-1中, 构建重组质粒pGEX-IL18, 转化E.coli BL21感受态细胞, 用IPTG诱导表达。重组菌菌体裂解物SDS-PAGE可检测到相对分子质量为45 kD的重组蛋白, 占菌体总蛋白的28%左右, 以包涵体形式存在。对包涵体进行洗涤, 用MTT法测定表明, 重组蛋白能明显刺激猪脾脏T淋巴细胞增殖反应的活性。     

猪白细胞介素18基因的克隆、表达及生物学活性检测

通过RT-PCR方法直接从猪脾脏淋巴细胞中扩增出猪白细胞介素18(pIL-18)成熟蛋白基因的cDNA, 克隆到pGEM-T载体, 构建重组质粒pGEM-T-IL18, 转化E.coli JM109感受态细胞, 取PCR和酶切鉴定为阳性的重组质粒进行序列测定。测序结果表明, pIL-18成熟蛋白基因核苷酸长度为474 bp, 编码157个氨基酸。将其克隆到表达载体pGEX6P-1中, 构建重组质粒pGEX-IL18, 转化E.coli BL21感受态细胞, 用IPTG诱导表达。重组菌菌体裂解物SDS-PAGE可检测到相对分子质量为45 kD的重组蛋白, 占菌体总蛋白的28%左右, 以包涵体形式存在。对包涵体进行洗涤, 用MTT法测定表明, 重组蛋白能明显刺激猪脾脏T淋巴细胞增殖反应的活性。     

猪细小病毒VP2蛋白在昆虫细胞中的表达及其特性

将猪细小病毒(Porcine Parvovirus, PPV)vp2基因重组到杆状病毒BacToBac表达系统的pFastBacⅠ质粒中,构建了pFastvp2质粒。在DH10Bac大肠杆菌中,pFastvp2与改造过的苜蓿夜蛾核型多角体病毒(AcNPV)基因组(Bacmid)发生同源重组,从而获得重组穿梭载体Bacmidvp2,转染Sf9细胞得到重组病毒AcNPVvp2。SDSPAGE和Westernblotting分析可见大小约为64kD的特异性带,表明AcNPVvp2在Sf9细胞中成功地表达了PPV VP2蛋白。红细胞凝集试验和间接ELISA进一步证实,表达的VP2蛋白具有与全病毒相同的血凝活性和相似的抗原性。电镜观察VP2蛋白的粗提物,发现VP2蛋白可自行装配成许多病毒样粒子(VLPs)。     

IL—2重组杆状病毒载体的构建及表达

将人白细胞介素２（ＩＬ－２）ｃＤＮＡ插入苜蓿银纹夜蛾核型多角体病毒（ＡｃＮ－ＰＶ）的转移载体ｐＶＬ１３９２中。经限制性酶切和ＤＮＡ杂交筛选、鉴定出重组转移载体ｐＶＬ１３９２－ＩＬ２。重组载体通过在昆虫细胞内共转染将ＩＬ－２ｃＤＮＡ转移到野生型ＡｃＮＰＶＤＮＡ中。经３２Ｐ标记探针鉴定出重组病毒Ａｃ１３９２－ＩＬ２。用重组病毒感染昆虫细胞,结果表达产物有明显刺激ＣＴＬＬ细胞生长,［３Ｈ］－ＴｄＲ掺入法检测ＩＬ－２活性为１５００ＩＵ／ｍ１。     

猪IL-18在乳酸乳球菌中的表达及其生物活性的检测

为了在乳酸乳球菌中分泌表达具有生物活性的猪IL-18蛋白,并检测其生物活性,故通过分离猪外周血单核淋巴细胞(PBMC),以其为模板,采用RT-PCR方法扩增猪白细胞介素18(pIL-18)基因,将目的基因与乳酸乳球菌表达载体pAMJ399进行连接,并电转化至乳酸乳球菌MG1363中,通过SDS-PAGE和Western blotting分析检测目的蛋白的表达,并通过脾淋巴细胞增殖试验和细胞病变抑制法对pIL-18的生物活性进行检测。Western blotting分析检测结果与生物活性检测结果显示,在重组菌pAMJ399-pIL18/MG1363的上清和菌体沉淀中19 kDa处均出现pIL-18的特异蛋白反应带,且分泌表达的pIL-18蛋白能明显促进猪脾淋巴细胞的增殖,并对病毒增殖有明显的抑制作用。以上结果表明pIL-18可在乳酸乳球菌分泌表达,且表达产物具有良好的生物活性。     

Modelling baculovirus infection of insect cells in culture

Conclusions Infection of insect cells with baculovirus is a potentially attractive means for producing both viral insecticides and recombinant proteins. The continuation of mathematical modelling studies such as those reviewed in this paper are essential in order to realise the full potential of the system. Through mathematical models it is possible to predict complex behaviours such as those observed when infecting cells at low MOI or when propagating virus in a continuous culture system. A purely empirical analysis of the same phenomena is very difficult if not impossible.The present three models are — despite their complexity and the effort that has gone into developing them — all first generation models. They summarise, to a large extent, our present quantitative understanding of the interaction between baculovirus and insect cells, when looked upon as a black box system. The binding and initial infection processes are still quantitatively poorly understood and further work in this area is much needed. On the longer term, a second generation of models is likely to consider interior processes such as viral DNA and RNA accumulation in much more detail using a structured model of the infection cycle.     

Improved baculovirus system for transferring genes into insect and mammalian cells

A new set of eukaryotic expression vectors was constructed on the basis of baculoviruses. EcoRI fragments S, J, and P with the genes for late viral proteins p35 (polyhedrin), p39, and p10 were cloned from genomic DNA of the nuclear polyhedrosis virus. The promoter regions of these genes were used to construct double-and triple-promoter expression vectors. Baculovirus vectors containing an expression cassette with the cytomegalovirus promoter and the green fluorescent protein reporter gene were designed to express the cloned genes in cultured mammalian cells.     

白细胞介素-18及其抗肿瘤作用研究进展

白细胞介素18(IL-18)具有多向免疫调节功能。它对T细胞向Th1或Th2分化起有独特的调节能力。最近的研究表明了IL-18的结构、活化形式和调节树突状细胞、NK细胞等方面的重要功能。研究也发现许多肿瘤患者血清IL-18水平显著升高及其在肿瘤发生发展中的作用。本文主要介绍了IL-18的结构、产生与调节、生物学作用及其在肿瘤方面的研究进展。     

Expression and purification of the matrix protein of Nipah virus in baculovirus insect cell system

Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N‐terminally His‐tagged NiV M protein in insect cells. A time‐course study demonstrated that the highest yield of recombinant M protein (400–500 μg) was expressed from infected cells 3 days after infection. A single‐step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme‐linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:171–177, 2016     

Glial Expression of Interleukin-18 and its Receptor After Excitotoxic Damage in the Mouse Hippocampus

Interleukin (IL)-18, a member of the IL-1 cytokine family, is an important mediator of peripheral inflammation and host defence responses. However, although IL-1 is a key proinflammatory cytokine in the brain, little is known about IL-18 changes in glial cells under excitotoxic neurodegeneration. In this study, we characterized the expressions of IL-18 and IL-18 receptor (IL-18R) in kainic acid (KA)-induced excitotoxicity in mouse hippocampus by immunohistochemistry and Western blotting. IL-18 immunoreactivity was found in microglia whereas IL-18R immunoreactivity was observed in astrocytes. Levels of IL-18 and IL-18R in hippocampus homogenates increased progressively from day 1 post-KA and peaked at 3 days. This study demonstrates the cellular sources of IL-18 and IL-18R, and their temporal correlations after KA-insult, and suggests roles for IL-18 and IL-18R in glial cells in response to excitotoxic damage in the hippocampus.     

层粘连蛋白基因家族在猪多能干细胞中的表达谱分析

层粘连蛋白(Laminin)是细胞外基质的重要成分,对细胞生长、分化、运动、组织修复和再生等发挥重要调节作用。Laminin包括有A1-5、B1-4和C1-3共12个编码基因,以不同的表达模式发挥功能。其中Laminin A5作为可以支持多能细胞生长的重要基因,得到广泛研究。但是,在所有猪相关数据库中,均未能查到Laminin A5的信息。文中通过生物信息学分析,首次确认了猪Laminin A5的存在,并进行了克隆和测序验证。为揭示Laminin基因家族在猪诱导多能干细胞(i PSCs)中的表达特性,检测了Laminin在猪各组织、体细胞和i PS细胞中的不同表达模式。发现Laminin B1基因在猪多能干细胞中存在特异性可变剪接体(LAMB1-a),且该可变剪切体的表达量与猪多能干细胞的重编程程度呈正相关。为进一步揭示和利用Laminin作为细胞外基质,用于猪多能干细胞的获取和培养优化奠定了基础。     

Recombinant protein production in insect cell cultures infected with a temperature-sensitive baculovirus

Spodoptera frugiperda (IPLB-SF-21) insect cells were grown in shake-flasks and infected with a temperature-sensitive baculovirus to express the gene of chloramphenicol acetyl transferase (CAT) in serum-free medium (SF-900) and two serum-supplemented media (IPL-41 and Grace's). In temperature-shift experiments (cell growth at 33°C followed by virus replication at 27°C 3–4 days later), virus and CAT production were much poorer in the serum-free medium than in serum-supplemented media, though cell growth was virtually the same in the different media tested. In all the three media, highest virus and CAT titers were obtained at the lowest MOI (multiplicity of infection 0.02). This result is contrary to that obtained in constant-temperature culture (27°C for both cell growth and virus replication). Virus and CAT production was greatly improved when the entire culture was run at constant temperature. It appeared that infected cells were severely damaged at 33°C (6°C above the optimal 27°C), resulting in little or no virus and protein production. As a result of these temperature-shift experiments, a larger-scale (141 air-lift bioreactor) serum-free culture of Sf-9 insect cells was conducted at constant temperature (27°C) to produce recombinant protein (β-galactosidase). A cell density as high as 1×10⁷ cells.ml⁻¹, and a β-gal concentration of up to 104,000 unit.ml⁻¹ were achieved.