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1.
利用5L生物反应器悬浮培养HEK-293 N3S细胞生产携带绿色荧光蛋白基因的重组腺病毒(recombinant adenovirus-green fluorescent protein,Ad-GFP),为规模化生产腺病毒基因药物建立一种稳定可行的生产工艺。复苏的种子细胞进行逐级放大最后接入5L搅拌式生物反应器中,采用含5%胎牛血清(FBS)的DMEM/F12培基灌流培养293 N3S细胞,当细胞密度达到(2~4)×106个/mL时感染Ad-GFP,48h后收获细胞,经两步氯化铯超速离心获得纯化的Ad-GFP。采用紫外分光光度计比色法和高压液相色谱法(HPLC)测定病毒颗粒数和纯度,采用组织培养半数感染剂量(TCID50)法检测腺病毒的感染滴度。连续培养10~12d,细胞密度可达到(2~4)×1066个/mL左右,纯化的Ad-GFP感染滴度和颗粒数分别为1.0×1011IU/mL和1.68×1012VP/mL,比活性为6.0%,A260A280比值为1.33,产品纯度达到99.2%。建立了5L生物反应器悬浮培养293 N3S细胞生产重组腺病毒Ad-GFP的生产工艺,对携带其他基因的重组腺病毒药物生产具有一定的指导意义。  相似文献   

2.
目的:用色谱法纯化制备携带绿色荧光蛋白基因的重组腺病毒。方法:采用5L生物反应器悬浮培养HEK-293N3S细胞进行病毒的扩增,冻融法裂解细胞,通过超滤、Source15Q离子交换层析和Sepharose4Fast Flow凝胶过滤层析进行分离纯化,采用分光光度法和高效液相色谱法分析重组腺病毒产品的纯度,采用组织培养半数感染剂量方法测定病毒的感染滴度。结果:通过两步色谱层析得到产品,经分光光度法分析,其D260nm/D280nm比值为1.21,高效液相色谱法分析其纯度达96.5%,产品滴度为1.0×1011U/mL,病毒颗粒数为1.76×1012/mL,比活性为5.68%。结论:确定了稳定可行的重组腺病毒色谱分离工艺,得到的产品在纯度、滴度和比活性等方面均符合国家食品药品监督管理局的标准。  相似文献   

3.
吴全德  黄文林 《生物工程学报》2014,30(11):1786-1790
腺病毒载体是极具发展前景的基因治疗载体之一,为获得一条新型腺病毒规模化生产工艺,研究采用5 L振荡激流式一次性生物反应器悬浮培养HEK293细胞来生产重组腺病毒载体。细胞经种子链逐步扩增后,接种至AP10生物反应器,采用CD293无血清培养基流加培养悬浮HEK293细胞,细胞密度达约2.0×106个/m L时,以30 MOI(Multiplicity of infection)感染重组Ad-IFNγ(Recombinant adenovirus-interferon gamma),48 h后收获细胞,3次冻融裂解离心收获上清病毒粗产品。采用壳蛋白免疫法快速测定粗产品滴度。结果表明,采用振荡激流式一次性生物反应器,流加HEK293细胞悬浮培养6 d,密度可达2.0×106个/m L,Ad-IFNγ粗产量达1.49×1013 IFU(Infectious unit),单细胞包装量达3 800 IFU/cell。采用阴离子交换层析法纯化重组腺病毒,回收率35.9%。建立了利用5 L激流式一次性生物反应器悬浮培养HEK293细胞生产重组腺病毒载体Ad-IFNγ的初步工艺。  相似文献   

4.
目的采用体外连接技术构建含红色荧光蛋白(RFP)报告基因的重组腺病毒载体,为基因治疗与基因疫苗研究提供重要工具。方法将pTurbo RFP-N上的含红色荧光蛋白基因片段,经酶切连接定向克隆至转移载体pShuttle上,构成重组质粒pShuttle-TurboRFP-N。用I-CeuI/PI-SceI双酶切重组质粒pShuttle-TurboRFP-N和骨架质粒pH5′040 pkGFP-II,回收目的片段,连接,获得重组腺病毒载体pH5.′040.CMV.RFP-N。将其线性化后,经脂质体介导转染至AD293细胞内包装出重组腺病毒颗粒。通过荧光显微镜观察其在AD293细胞内的包装效率和RFP表达情况。扩增并再次感染AD293细胞检测病毒颗粒感染能力,并测定其生物活性及滴度。结果经酶切鉴定,证明已正确构建重组腺病毒载体AdH5.′040.CMV.RFP-N;经AD293细胞包装成具有感染性的病毒颗粒,并能在真核细胞中高效表达目的基因;扩增纯化后获得重组腺病毒颗粒数为3.6×1012vp/mL,滴度达1×1010pfu/mL。结论成功构建了携带红色荧光蛋白报告基因的重组腺病毒载体,并能够在AD293细胞中高效地表达,为基因治疗与基因疫苗研究提供重要工具。  相似文献   

5.
本文研究在生物反应器中用微载体连续灌注培养Vero细胞生产狂犬病毒制备技术。在5L体积的生物反应器中,加入含10g/L微载体的199培养基,接种Vero细胞至细胞浓度达到1×105/mL,培养7d后细胞可生长至6~7×106/mL,然后以感染复数(MOI)为0.01接种狂犬病毒VaG株,接毒后24h开始收获,连续收获12d左右,收获的病毒滴度范围在6.0~8.5logLD50/mL,收获的病毒原液经浓缩、灭活和纯化等步骤制备成疫苗,各项质量指标均达到《中国生物制品规程》2000年版要求。实验表明,用生物反应器微载体灌注培养制备人用Vero细胞狂犬病疫苗小试工艺可行。  相似文献   

6.
表达PRRSV M蛋白重组腺病毒的构建及其免疫特性研究   总被引:4,自引:0,他引:4  
本研究通过RT-PCR方法扩增猪繁殖与呼吸综合征病毒(PRRSV)S1株的M蛋白基因,将其克隆重组到人血清5型腺病毒载体中,转染293细胞,制备重组腺病毒rAd-M.RT-PCR和IFA方法鉴定,结果表明rAd-M可表达M基因的mRNA和M蛋白.纯化的rAd-M重组腺病毒经293细胞连续传25代,滴度稳定为107.8TCID50/mL.动物免疫试验结果表明,该重组腺病毒rAd M能够刺激机体产生PRRSV的特异性抗体免疫和细胞免疫应答反应,从而为PRRSV结构蛋白功能及其基因工程疫苗研究奠定了基础.  相似文献   

7.
Wang Y  Zhang SF  Liu Y  Zhang F  Zhang JX  Hu RL 《病毒学报》2011,27(5):442-446
构建表达狂犬病病毒弱毒SRV9糖蛋白(GP)的重组人5型腺病毒,检测其对小鼠的免疫效果。将狂犬病病毒SRV9株GP基因的完整开放阅读框克隆到腺病毒表达系统中的穿梭质粒多克隆位点,构建重组穿梭质粒pac-Ad5CMV-Gs9,以罗氏转染液介导线性化骨架质粒和重组穿梭质粒共转染293AD细胞,细胞病变后取培养物进行PCR鉴定并电镜观察,在293AD细胞上测定病毒滴度。以106 TCID50重组腺病毒腹腔接种昆明小鼠,免疫后不同时段采尾静脉血通过荧光抗体病毒中和试验(FAVN)检测小鼠血清狂犬病中和抗体效价。正确构建重组穿梭质粒pacAd5CMV-Gs9;获得表达狂犬病病毒SRV9株GP蛋白的缺陷型重组人5型腺病毒;病毒滴度达到106 CFU/mL以上;腹腔接种小鼠14d后均产生了抗狂犬病中和抗体,有效保护率达90%。成功获得了表达狂犬病病毒GP基因的重组腺病毒,该腺病毒免疫小鼠可产生保护性中和抗体,为进一步开发新型兽用狂犬病疫苗奠定了物质基础。  相似文献   

8.
目的:构建含小鼠细胞因子信号抑制因子-1基因(SOCS1)的重组腺病毒载体(Ad5F35-SOCS1),探讨其介导SOCS1基因在小鼠树突状细胞中的表达。方法:设计含AgeI和NheI酶切位点的SOCS1基因上下游引物,以质粒pEF-FLAG-1/mSOCS1为模版,通过PCR扩增获得SOCS1全部序列,片段回收后经AgeI和NheI酶切,再定向插入到经AgeI和NheI酶切的质粒pDc316-LacZa中,获得重组穿梭质粒pDC316-SOCS1,经AgeI和NheI酶切、PCR及测序等鉴定后,用脂质体将穿梭质粒pDC316-SOCS1与腺病毒骨架质粒pBHGF35共转染293细胞,经位点特异性重组获得重组腺病毒Ad5F35-SOCS1,行PCR鉴定,经293细胞扩增、纯化制备高滴度病毒液,TCID50法测定病毒滴度。用获得的重组腺病毒感染小鼠树突状细胞,以免疫组化检测SOVD1的表达。结果:成功构建了含小鼠SOCS1基因的重组腺病毒载体,病毒感染滴度为1.4×10~9IU/ml,该载体能有效介导SOCS1基因在小鼠树突状细胞中的表达。结论:重组腺病毒载体能将SOCS1基因转入小鼠树突状细胞并有效表达,为基因转染制备耐受性树突状细胞奠定了基础。  相似文献   

9.
本研究通过RT-PCR方法扩增猪繁殖与呼吸综合征病毒(PRRSV)S1株的M蛋白基因,将其克隆重组到人 血清5型腺病毒载体中,转染293细胞,制备重组腺病毒rAd-M。RT-PCR和IFA方法鉴定,结果表明rAd-M可表 达M基因的mRNA和M蛋白。纯化的rAd-M重组腺病毒经293细胞连续传25代,滴度稳定为107.8 TCID50/ mL。动物免疫试验结果表明,该重组腺病毒rAd-M能够刺激机体产生PRRSV的特异性抗体免疫和细胞免疫应 答反应,从而为PRRSV结构蛋白功能及其基因工程疫苗研究奠定了基础。  相似文献   

10.
以我国分离的首株人H5N1亚型禽流感病毒(A/Anhui/1/2005)作为研究对象,通过引入内部核糖体进入位点序列(IRES),构建共表达H5N1亚型禽流感病毒膜蛋白基因M1和HA的重组腺病毒。PCR扩增禽流感病毒H5N1亚型M1和HA基因的全长可读框片段,先后亚克隆入pStar载体,然后扩增M1-IRES-HA片段并将其插入穿梭载体pShuttle-CMV,再与pAd-Easy载体在BJ5183菌中通过同源重组产生重组腺病毒载体,转化293细胞,包装出重组腺病毒Ad-M1/HA。将Ad-M1/HA感染293细胞,可观察到明显细胞病变效应,用免疫荧光及Western-blot方法均检测到M1和HA基因的表达。共表达M1和HA双基因的重组腺病毒的成功构建为开发新型重组腺病毒流感疫苗奠定了基础。  相似文献   

11.
 以RT PCR法从大鼠脑组织中克隆bcl XL 基因 ,将其定向插入带rep cap基因和neu基因的三功能腺相关病毒 (AAV)载体 ,转染HeLa细胞并以G4 18筛选 ,然后用腺病毒感染筛选后的细胞克隆 ,包装成重组腺相关病毒 .用带rep cap基因的C12细胞筛选和滴定产生重组腺相关病毒的细胞克隆 ,粗制细胞裂解物中病毒滴度最高只有 3× 10 5IU ml.从产病毒量较高的克隆大量制备重组病毒 ,经肝素柱高压液相亲和层析法纯化、浓缩病毒后获得了高达 4× 10 11IU ml的重组病毒 .为研究脑缺血动物模型中BCL XL 的抗脑细胞凋亡作用打下了基础  相似文献   

12.
Recombinant adenoviruses became one of the vectors of choice for delivery and expression of foreign proteins for gene therapy and vaccination purposes. Nevertheless, the production of adenovirus is currently limited by the so-called "cell density effect", i.e., a drop in cell specific productivity concomitant with increased cell concentration at infection (CCI). This work describes the characterisation and optimisation of the infection process in order to improve recombinant adenovirus type 5 yields at high cell densities. For that purpose, 293 cells adapted to suspension were grown in 2l bioreactors and infected at different cell concentrations, using different re-feed strategies, while evaluating cell metabolism. The consumption of amino acids is enhanced during infection, although no amino acid limitation was detected for cells infected at concentrations in the range of 2 x 10(6)cell/ml, for which the highest volumetric productivity was obtained in batch mode. Conversely, infecting at cell concentrations in the range of 3 x10(6)cell/ml led to complete depletion of glucose, glutamine and threonine before the optimal harvesting time, a significant decrease in volumetric productivity being observed; the effect of amino acids and glucose addition at infection time on cell specific and volumetric productivity of adenovirus was assessed, no improvement on adenovirus production being achieved. The effect of ammonia, present in high concentrations at 3 x10(6)cell/ml, was evaluated and seem to be detrimental; an 1.8-fold increase on adenovirus volumetric productivity was obtained for infections performed at 3 x10(6)cell/ml when non-ammoniagenic medium was used.  相似文献   

13.
High-titer adenovirus vector production in 293S cell perfusion culture   总被引:1,自引:0,他引:1  
Human 293S cells culture for recombinant adenovirus production is traditionally carried out in batch at a maximum of 6 x 10(5) cells/mL. A previous report demonstrated that fed-batch, applied to the adenovirus/293S cells system, improves the volumetric production of viral proteins by increasing the cell density at which cells can be infected, up to 2 x 10(6) cells/mL, without reducing the per-cell yield of product. To increase this cell density limit, the adenovirus production was performed in a perfusion system where the cells were separated by means of a tangential flow filtration device. 293S cell growth to 14 x 10(6) cells/mL was achieved in 10 days, at a medium renewal rate of 1 volume of medium per reactor volume and day (VVD). For adenovirus production, three 293S cell cultures were perfused at 1 VVD in parallel and infected at an average density of 8 x 10(6) cells/mL. One of the cultures was set at 37 degrees C and the two others at 35 degrees C. After a rapid initial cell loss, the average cell density stabilized at 5.75 x 10(6) cells/mL, 12 h postinfection, which was 8 times higher than the cell density in the batch control. This allowed the production of 3.2 x 10(9) infectious viral particles/mL (IVP/mL) at 37 degrees C and 7.8 x 10(9) IVP/mL at 35 degrees C, this last result being 5.5 times higher than the control. To our knowledge, this nonconcentrated titer is the highest value that has ever been published for adenovirus vector production. These observations lead to the conclusion that perfusion is an efficient tool to maintain, at high cell density, a specific production rate level sufficient to increase significantly the adenovirus volumetric production. Furthermore, it shows that perfusion at 35 degrees C can improve viral titer by 2.4-fold compared to 37 degrees C, in accordance with a previous study on adenovirus batch production.  相似文献   

14.
15.
应用AdMax腺病毒载体系统构建携带siSPK1基因的重组腺病毒,进一步研究SPK1基因对N2a细胞凋亡的影响。设计可形成小发夹结构的SPK1-siRNA模板cDNA序列,克隆至质粒pDC316-siRNA,构建SPK1-siRNA穿梭质粒pDC316-SPK1,经鉴定正确后,将pDC316-SPK1与辅助包装质粒(pBHG loxΔE1,3 Cre)共转染至HEK293细胞,包装纯化并扩增重组腺病毒颗粒,终点稀释法测定病毒滴度;病毒感染N2a细胞,Western blot法检测SPK1蛋白的表达;Hoechst33258染色检测SPK1基因对N2a细胞凋亡的影响。酶切后PCR分析、测序鉴定表明,pDC316-SPK1构建成功,病毒纯化后滴度为2.50E+10 PFU/mL;重组病毒可在蛋白水平抑制SPK1的表达;SPK1基因抑制后,N2a细胞凋亡增加(P〈0.001)。上述结果表明,含有SPK1-siRNA的重组腺病毒构建成功,且具有抑制SPK1蛋白表达的功能,该基因沉默后,N2a细胞凋亡增加。  相似文献   

16.
Recombinant retroviruses are now an established tool for gene delivery. Presently they are mainly produced using adherent cells. However, due to the restrictive nature of adherent cell culture, this mode of production is hampered by low cell-specific productivity and small production units. The large-scale production of retroviral vectors could benefit from the adaptation of retrovirus packaging cell lines to suspension culture. Here, we describe the ability of a 293 packaging cell line to produce retroviral vectors in suspension culture at high titer. Adherent 293GPG cells, producing a Moloney Murine Leukemia Virus (MoMLV) retrovirus vector pseudotyped with the vesicular stomatitis virus G (VSVG) envelope protein and expressing a TK-GFP fusion protein, were adapted to suspension culture in calcium-free DMEM. At a cell density similar to adherent cell culture, the suspension culture produced retroviral vector consistently in the range of 1 x 10(7) infectious viral particles/mL (IVP/mL), with a specific productivity threefold higher than adherent culture. Furthermore, at the same medium replacement frequency, the suspension producer cells could be cultured at higher density than their adherent counterparts, which resulted in virus titer of 3-4 x 10(7) IVP/mL at 11.0 x 10(6) cells/mL. This corresponds to a 10-fold increase in viral concentration compared to adherent cells. The capacity to up scale the retroviral vector production was also demonstrated by performing a 2 VVD perfusion culture for 9 days in a 3L Chemap bioreactor. The combination of suspension and perfusion led to a 20-fold increase in maximum virus productivity compared to the adherent culture.  相似文献   

17.
In recent years, CRISPR/Cas9 has rapidly become one of the most promising genome editing tools because it is simple and easy to use and cost effective. However, the large size of Cas9 sequences limits its application in clinically promising vectors and it also impacts non-viral transfection. In this study, CRISPR/Cas9 adenovirus vectors that target the buffalo 18s rDNA gene were constructed, transfected into 293 cells for adenovirus packaging, and the adenovirus was used to knockout the 18s rDNA gene in buffalo mammary epithelial cells. The results demonstrated that the CRISPR/Cas9 adenovirus vectors for the buffalo 18s rDNA gene could efficiently target the sites as revealed by the fluorescence reporter system. After amplification, the adenovirus titer of Sn458-18s1 and Sn458-18s2 reached 1.03 × 109PFU/mL and 1.05 × 109 PFU/mL, respectively. For buffalo mammary epithelial cell infection, the efficiency was 100% when the multiplicity of infection (MOI) ≥ 100 PFU/mL. There were 9 mutational clones found in the 20 clones, and the gene mutagenesis rate reached 45%. Of these, 2 clones were 35-bp deleted and 7 clones were 12-bp deleted. These results suggested that the adenovirus system overcame the low transfection efficiency of the buffalo mammary epithelial cells associated with using lipid-based methods or electroporation. Moreover, we preliminary developed an efficient technique for multiple-locus gene targeting at repeated sequences of the buffalo genome.  相似文献   

18.
薛玲  李航  张静  王净  吴雅岚  姬秋和  叶菁 《生物磁学》2011,(6):1083-1086
目的:利用AdEasy腺病毒表达系统构建含有小鼠脂肪储存小滴蛋白5(LSDP5)基因的重组腺病毒。方法:从小鼠肝脏cDNA克隆出LSDP5基因全长,克隆至pMD18-T载体中,酶切测序。回收酶切产物,连接到腺病毒穿梭载体pShuttle-CMV,构建pShuttle-CMV-LSDP5重组质粒,经PmeI酶切线性化后转化至含有腺病毒骨架质粒pAdEasy-1的BJ5183中。筛选阳性克隆,提取重组质粒,PacI酶切线性化并转染AD293细胞进行包装,提取病毒DNA,鉴定重组病毒并检测病毒滴度。结果:LSDP5基因克隆经测序证实与Genebank公布一致,双酶切重组pMD18-T载体得到1400 bp左右的片段。重组穿梭载体经Kpn I和Sal I双酶切后得到预期片段。PacI酶切得到30 Kb大片段和4.5 Kb小片段。转染AD293细胞后收集病毒,经PCR鉴定,获得理想的目的片段。取病毒上清反复感染AD293细胞以扩增病毒,最后所得病毒滴度为2.5×109pfu/ml。结论:成功构建了携带脂肪储存小滴蛋白5基因的重组腺病毒载体,为进一步研究LSDP5基因功能奠定基础。  相似文献   

19.
TNF-a是一种具有广泛生物学活性的前炎症细胞因子, 参与哮喘整个病理生理过程。可溶性肿瘤坏死因子受体(sTNFR)可以拮抗肿瘤坏死因子活性, 已被用来治疗与TNF相关的炎性疾病。将sTNFRI-IgGFc基因插入腺病毒穿梭质粒pDC316, 与辅助质粒pBHGloxΔE1, 3Cre共转染HEK293细胞, 重组产生Ad-sTNFRI-IgGFc, PCR鉴定毒种正确后, 进行扩增、纯化和滴度测定, 转染人气道平滑肌细胞, 利用RT-PCR、免疫组化方法, 流式细胞仪, ELISA检测转染后细胞中sTNFRI-IgGFc的转录和表达。实验结果证明成功构建了Ad-sTNFRI-IgGFc腺病毒载体, 感染性滴度达3×1010 TCID50/mL, 200 moi转染气道平滑肌细胞阳性率达99.32%, 转染后气道平滑肌细胞在mRNA和蛋白水平均有sTNFRI-IgGFc表达。转染上清稀释64倍后仍对TNF有拮抗活性。为将表达sTNFRI-IgGFc腺病毒基因治疗哮喘的实验研究提供了基础。  相似文献   

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