重组BPI23—Fcγ1融合蛋白在CHO细胞中的表达

The fusion gene of BPI23 and human Fc gamma 1 was obtained by PCR method, and the expression plasmid was constructed to express recombinant BPI23-Fc gamma 1 fusion protein in CHO cells. After transfection with the plasmid and selection by methotrexate, the cell lines expressing the fusion protein were obtained. The recombinant protein was purified using cation-exchange chromatography and its bioactivity was proved with bactericidal assays.     

CTLA4Ig融合蛋白在CHO细胞中的表达

CTLA4Ig是人CTLA4胞外区与人免疫球蛋白铰链区、CH2区、CH3区组成的融合蛋白,可以与B7结合,通过阻断B7与CD28的结合,从而阻断B7介导的T细胞活化必需的共刺激信号,可作为免疫抑制剂用于器官移植。将CTLA4Ig融合分子克隆到真核表达载体pCI-dhfr,并用脂质体方法转染到COS7和CHO-dhfr-细胞中,用氨甲喋呤筛选转染的CHO-dhfr-细胞。用RT-PCR、ELISA、细胞免疫荧光染色和Western-blot鉴定重组蛋白的表达。采用A蛋白纯化重组蛋白。     

炭疽毒素受体与人IgG1的Fc段融合蛋白ATR_Fc在CHO细胞中的表达

ATR_Fc是人炭疽毒素受体(ATR)的胞外区与人免疫球蛋白IgG1的铰链区、CH2区和CH3区组成的融合蛋白。表达该蛋白是为了获得结合PA的抗体样分子,通过阻断PA与细胞受体的结合,而阻止炭疽致死毒素和水肿因子进入细胞内,可作为预防和治疗炭疽感染的生物制品。将编码炭疽毒素受体N端1_227氨基酸的基因和编码Fc段的基因连接,插入到pcDNA3.1的HindⅢ和NotⅠ位点得到表达ATR_Fc融合蛋白的真核表达载体pcDNA3.1ATR_Fc,并用脂质体方法将该载体转染至CHO_K1细胞中,用G418筛选并获得ATR_Fc表达水平为10～15μg(106cells·d)的基因工程CHO细胞系ATR_Fc_1D5。采用蛋白A纯化重组蛋白,并用ELISA法鉴定ATR_Fc与PA的亲和性,表明ATR_Fc可与PA特异性结合。     

proUK-KGDW融合基因在CHO细胞中的高表达

利用常规分子生物学技术,构建了新型高效的proUK-KGDW融合基因的分泌型哺乳动物细胞表达载体。将该载体线性化后转染CHO/dhfr-细胞,经G418筛选获得阳性克隆,然后挑取表达水平较高的克隆进行MTX加压扩增,以提高proUK-KGDW杂合体的表达水平,经2～3轮MTX加压扩增,获得多株表达水平超过10μg/(106细胞·24h)的稳定的高表达细胞株,为proUK-KGDW杂合体的制备及功能研究奠定了基础。     

人肝细胞生长因子cDNA克隆在CHO细胞中的表达

肝细胞生长因子是一种由α、β链组成的杂合二聚体糖蛋白,能促进肝细胞、多种上皮细胞、内皮细胞和神经胶质细胞的有丝分裂,并对多种肿瘤细胞具有细胞毒性作用或者抑制其生长［１～４］,其作用无种属特异性,如人肝细胞生长因子能促进大鼠肝细胞增殖［５］。由于天然Ｈ...     

重组人尿激酶原在CHO细胞中的高效表达及其纯化

用鸡β- globin的MAR序列和人看家基因延伸因子1α(hEF-1α)的调控序列以及旱獭RNA稳定与输出序列,构建了重组人尿激酶原(recombinant human pro-urokinase,rhPro - UK)的高效表达载体,在CHO细胞中获得了rhPro - UK的高效稳定表达,rhPro - UK的表达水平达到1299 IU(以百万细胞1d的表达量计).采用阳离子交换层析、疏水层析和凝胶排阻层析的三步工艺纯化表达rhPro - UK的CHO细胞培养上清液,rhPro - UK的纯度达到98％、回收率为60％ ～70％.     

炭疽毒素受体与人IgG1的Fc段融合蛋白ATR_Fc在CHO细胞中的表达

ATR-Fc是人炭疽毒素受体（ATR）的胞外区与人免疫球蛋白IgG1的铰链区、CH2区和CH3区组成的融合蛋白。表达该蛋白是为了获得结合PA的抗体样分子,通过阻断PA与细胞受体的结合,而阻止炭疽致死毒素和水肿因子进入细胞内,可作为预防和治疗炭疽感染的生物制品。将编码炭疽毒素受体N端1-227氨基酸的基因和编码Fc段的基因连接,插入到pcDNA3-1的HindⅢ和NotⅠ位点得到表达ATR-Fc融合蛋白的真核表达载体pcDNA31/ATR9Fc,并用脂质体方法将该载体转染至CHO-K1细胞中,用G418筛选并获得ATR-Fc表达水平为10～15μg/(106cells·d)的基因工程CHO细胞系ATR-Fc-1D5。采用蛋白A纯化重组蛋白,并用ELISA法鉴定ATR-Fc与PA的亲和性,表明ATR-Fc可与PA特异性结合。     

hK1-L-Fc融合蛋白在CHO细胞中的表达及其活性研究

为进一步改造重组人激肽释放酶1(hK1),以期提高其生物活性,制备了通过柔性接头相连接的重组人激肽释放酶1-L-IgG1 Fc融合蛋白(hK1-L-Fc)。采用重叠延伸PCR技术构建hK1-L-Fc融合基因,克隆至表达载体pcDNA3.1,在中国仓鼠卵巢细胞(CHO-S)中表达。利用Protein A 亲和层析柱纯化融合蛋白,SDS-PAGE、Western blotting、飞行时间质谱(MALDI-TOF-MS)、HPLC检测表达产物,底物法检测融合蛋白的体外活性。结果显示:成功构建pcDNA3.1-hK1-L-Fc重组表达载体;获得稳定表达融合蛋白的细胞株;无血清悬浮批式培养的表达量在0.7 mg/L以上;纯化的蛋白其纯度在95%以上,分子量约60 kDa;活性检测显示其比活性在9.2 U/mg以上,较hK1-Fc蛋白提高了18%以上。     

人血小板生成素（hTPO）全长cDNA克隆及其在CHO细胞中的表达

以人胎肝ｍＲＮＡ为模板,采用ＲＴ－ＰＣＲ扩增了人ＴＰＯ编码区全长ｃＤＮＡ,全序列测定结果表明与国外报道序列一致;进而构建了ｐｃＤＮＡ３－ＴＰＯ永久表达载体,转染ＣＨＯ细胞后经Ｇ４１８加压筛选、ＴＰＯ表达稳定性等项指标测试,获得了稳定分泌ＴＰＯ的工程细胞株。     

Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.     

VPAC2在CHO细胞的表达及鉴定

PAC2是垂体腺苷酸环化酶激活多肽(Pituitary adenylate cyclase activating polypeptide,PACAP)和血管活性肠肽(vasoactive intestinal peptide,VIP)的共同受体,介导多种重要生物学功能。为获得稳定特异表达VPAC2的中国仓鼠卵巢(Chinesehamsterovary,CHO)细胞,将pcDNA-VPAC2表达载体转染CHO细胞,G418筛选转染阳性克隆,PACAP38标准品诱导阳性克隆细胞的胞内cAMP生成,筛选出对PACAP38最为敏感的阳性单克隆细胞株(VPAC2-CHO),运用RT-PCR、Westernblot和免疫荧光法检测VPAC2受体表达情况,利用VPAC2受体特异激动剂通过竞争性结合试验和促进胞内第二信使cAMP生成的活性检测实验证实,VPAC2-CHO特异表达有功能的VPAC2。Scatchard作图分析显示VPAC2-CHO的VPAC2受体密度为(1.1±0.2)pmol/mg膜蛋白,PACAP38与VPAC2的解离常数Kd值为(0.55±0.10)nmol/L。特异表达VPAC2受体细胞系的构建为深入研究该受体理化性质、生物学功能以及筛选、开发VPAC2受体新型特异激动剂和拮抗剂等研究奠定了基础。     

The role of the cell cycle in determining gene expression and productivity in CHO cells

Understanding the relationships between cell cycle and protein expression is critical to the optimisation of media and environmental conditions for successful commercial operation of animal cell culture processes. Using flow cytometry for the analysis of the early phases of synchronised batch cultures, the dependency of product expression on cell cycle related events has been evaluated in a recombinant CHO cell line. Although the production of recombinant protein is initially found to be cell cycle related, the maximum specific protein productivity is only achieved at a later stage of the exponential phase which also sees a maximum in the intracellular protein concentration. Subsequent work suggests that it is the batch phase/medium composition of cultures which is the major determinant of maximum specific productivity in this cell line. Furthermore the effect of the positive association between S phase and specific productivity is subordinate to the effect of batch phase/medium composition on the specific productivity of batch cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence of cytoskeleton organization on recombinant protein expression by CHO cells

In this study, we assessed the importance of cytoskeleton organization in the mammalian cells used to produce therapeutic proteins. Two cytoskeletal genes, Actin alpha cardiac muscle 1 (ACTC1) and a guanosine triphosphate GTPase-activating protein (TAGAP), were found to be upregulated in highly productive therapeutic protein-expressing Chinese hamster ovary (CHO) cells selected by the deprivation of vitamin B5. We report here that the overexpression of the ACTC1 protein was able to improve significantly recombinant therapeutic production, as well as to decrease the levels of toxic lactate metabolic by-products. ACTC1 overexpression was accompanied by altered as well as decreased polymerized actin, which was associated with high protein production by CHO cell cultured in suspension. We suggest that the depolymerization of actin and the possible modulation of integrin signaling, as well as changes in basal metabolism, may be driving the increase of protein secretion by CHO cells.     

促红细胞生成素基因表达载体的构建及其在CHO细胞中表达的研究

用从基因文库中所克隆的促红细胞生成素（ＥＰＯ）基因组基因,构建了３种由不同启动子调控的表达载体——ｐＯＰ１３／ＥＰＯ,ｐＲＳＶ／ＥＰＯ,ｐＣＭＶ／ＥＰＯ,在这３个表达载体的构建过程中对基因的转录起始效率、内含子的剪接、５′非翻译区和３′非翻译区对基因表达的影响等因素都加以考虑．用脂质体转染法分别将上述３个载体导入ＣＨＯ细胞,经瞬时表达,用ＥＬＩＳＡ方法检测,表达量分别为１９０ｍＩＵ／ｍｌ、１６０ｍＩＵ／ｍｌ、４４７ｍＩＵ／ｍｌ．用表达载体ｐＯＰ１３／ＥＰＯ转染ＣＨＯ－Ｋ１２细胞,在４００μｇ／ｍｌ的Ｇ４１８浓度下筛选稳定表达细胞克隆,获得了表达量约为１６０ＩＵ／１０６细胞（４８ｈ）的Ｃ１０细胞株．表达产物经Ｗｅｓｔｅｒｎｂｌｏｔ检测发现了ＥＰＯ阳性条带．用ＴＦ－１细胞对ＥＰＯ进行了生物活性检测,初步证实所表达的ＥＰＯ有生物活性．     

乙肝病毒S抗原和preS1抗原表位融合蛋白(S/preS1) 在CHO细胞中的稳定高效表达

含前S蛋白的重组乙型肝炎疫苗是第3代乙肝疫苗研发的重点,有望替代现在广泛使用的基因工程疫苗。构建表达乙肝病毒S抗原和preS1抗原表位 (21-47位氨基酸) 融合蛋白 (S/preS1) 的真核表达载体HMRCHEF53u/Neo-S/preS1并转染CHO-S细胞,经ELISA和有限稀释克隆筛选获得了S/preS1表达效率高、体外培养生物学性状好的CHO细胞系10G6。Western blotting分析证实10G6表达的S/preS1同时保留S和preS1的天然免疫原性。10G6细胞在以活细胞密度和preS1/S浓度为评价指标的连续批次培养过程中保持着稳定的目的产物表达效率和良好的生长特性。采用无血清流加培养工艺,10G6细胞的活细胞密度和preS1/S浓度分别达到7×106～10×106 cells/mL和17～20 mg/L。