小鼠Fancd2os基因真核表达质粒的构建及其对人胚肾上皮细胞增殖和凋亡的影响

目的构建小鼠Fancd2os基因的真核表达质粒,并分析Fancd2os基因对人胚肾上皮细胞(HEK293T)增殖和凋亡的影响。方法以小鼠睾丸组织DNA为模板扩增Fancd2os基因c DNA全长片段,并克隆至真核表达载体p3×FLAG-CMV-14,构建重组真核表达质粒p3×FLAG-CMV-14-Fancd2os。经Lipofectamine TM 2000将重组真核表达质粒瞬时转染HEK293T细胞,分别采用RT-PCR及Western blot法检测转染细胞中Fancd2os基因m RNA转录及蛋白表达情况;经CCK8法和流式细胞术分别检测过表达Fancd2os对HEK293T细胞增殖能力及紫外线诱导HEK293T细胞凋亡的影响。结果经双酶切及测序鉴定,重组真核表达质粒p3×FLAG-CMV-14-Fancd2os构建正确;转染p3×Flag-CMV-14-Fancd2os的HEK293T细胞可见Fancd2os基因m RNA转录及其蛋白表达;过表达Fancd2os的HEK293T细胞的增殖活性显著下降(P0.05);过表达Fancd2os可显著促进紫外线诱导的HEK293T细胞凋亡(P0.01)。结论成功构建了重组真核表达质粒p3×Flag-CMV-14-Fancd2os,过表达Fancd2os可抑制HEK293T细胞的增殖,促进其凋亡。     

小鼠截短型可溶性腺苷酸环化酶基因的克隆表达及活性鉴定

目的克隆表达小鼠截短型可溶性腺苷酸环化酶(mtsAC)基因,并检测其活性,为后续研究提供技术平台。方法Trizol法提取小鼠睾丸组织总RNA,RT-PCR法扩增mtsAC基因,经测序正确后,克隆至真核表达载体pcDNA4-V5,转染HEK293T细胞,Western blot及免疫荧光染色法检测目的蛋白的表达,并检测HEK293T细胞中cAMP的水平。结果所构建的重组表达质粒pcDNA4-V5-mtsAC经酶切和测序证明构建正确,插入的mtsAC基因长度为1549bp;表达的重组mtsAC蛋白相对分子质量为48000,在胞浆及胞核中呈均一性表达;转染质粒pcDNA4-V5-mtsAC的HEK293T细胞中cAMP的水平较未转染的HEK293T细胞升高约17倍,并可被NaHCO3和CaCl2激活。结论已成功克隆了mtsAC基因,并在HEK293T细胞中表达了具有活性的重组mtsAC蛋白,建立了可用于mtsAC特性研究及男性不育药物筛选的技术平台。     

瓣状内切核酸酶1基因重组真核表达质粒的构建及鉴定

目的构建瓣状内切核酸酶1(Flap endonuclease 1,FEN1)基因重组真核表达质粒,并进行鉴定。方法提取L02细胞总RNA,逆转录合成cDNA,经巢式PCR扩增FEN1基因,定向克隆至载体pcDNA3.1,构建重组真核表达质粒,经酶切及测序进行鉴定。将鉴定正确的真核表达质粒转染293T细胞,Western blot检测FEN1蛋白的表达。结果 FEN1基因重组真核表达质粒经酶切及测序鉴定证明构建正确;在相对分子质量约47 000处可见目的蛋白条带,转染细胞中FEN1蛋白表达量较空载体转染组及空白细胞对照组提高约3倍。结论已成功构建了FEN1基因重组真核表达质粒,并可在293T细胞中过表达FEN1。     

人源大麻素Ⅰ型受体基因真核表达质粒的构建及表达

目的构建人源大麻素Ⅰ型受体(human cannabinoid receptor 1,hCBⅠ)基因GV230真核表达质粒,并于HEK-293细胞中进行表达。方法以人脑皮质细胞的总RNA为模板,扩增获得hCBⅠ基因,克隆至真核表达载体GV230,构建重组表达质粒GV230-hCBⅠ,筛选阳性克隆,脂质体瞬时转染HEK293细胞。置倒置荧光显微镜下观察,并采用激光共聚焦显微镜(confocal laser scanning microscope,CLSM)和Western blot法检测hCBⅠ基因在HEK293细胞中的表达。结果重组表达质粒GV230-hCBⅠ经双酶切及测序鉴定,构建正确。转染48 h后,转染率约40%,CBⅠ蛋白主要于细胞膜分布及表达,且于相对分子质量约50 000处可见特异性条带。结论成功构建了重组真核表达质粒GV230-hCBⅠ,并于HEK293细胞中表达,为进一步研究CBⅠ的生物学功能奠定了基础。     

小鼠丝氨酸/精氨酸蛋白特异激酶2基因真核重组表达质粒的构建及其对微管蛋白聚合的影响

目的构建小鼠丝氨酸/精氨酸蛋白特异激酶2(serine/arginine-rich protein specific kinase 2,SRPK2)基因(srpk2)的真核重组表达质粒,并分析其对微管蛋白α-Tubulin聚合的影响。方法利用RT-PCR法扩增srpk2的全长cDNA序列,通过分子克隆技术构建真核重组表达质粒pLV-EGFP(2A)Puro-srpk2;在脂质体Lipofectamine 2000的介导下,将真核重组表达质粒及空载体pLV-EGFP2(A)Puro分别转染人胚肾HEK293T细胞,同时设空白对照组(未转染)。采用RT-PCR及Western blot法分别检测HEK293T细胞中srpk2基因mRNA转录及SRPK2蛋白的表达情况;Western blot法分析HEK293T细胞中聚合态和游离态α-Tubulin的含量。结果双酶切及DNA序列鉴定证明真核重组表达质粒pLV-EGFP2(A)Puro-srpk2构建正确,且在HEK293T细胞中实现了srpk2基因过表达。真核重组表达质粒转染组HEK293T细胞中游离态α-Tubulin含量显著低于空载体转染组及空白对照组(P0.05),而聚合态α-Tubulin水平明显高于空载体转染组及空白对照组(P0.05)。结论成功构建了srpk2基因的真核重组表达质粒,SRPK2可促进α-Tubulin微管蛋白聚合。     

人Makorin环指蛋白1基因shRNA真核表达质粒的构建及鉴定

目的构建人Makorin环指蛋白1(MKRN1)基因shRNA真核表达质粒,并检测其对HEK293细胞MKRN1基因表达的影响。方法设计并合成特异性针对人MKRN1基因的小干扰片段,定向克隆至带有卡那霉素抗性基因和绿色荧光蛋白基因的真核表达载体pGenesil-1中,对重组质粒进行酶切鉴定及DNA序列分析,并用脂质体将其转染到HEK293细胞中,观察其对细胞MKRN1基因mRNA转录和蛋白表达水平的影响。结果经酶切鉴定和序列分析证明,3个人MKRN1基因shRNA真核表达质粒及其阴性对照质粒构建正确,转染HEK293细胞后,3个shRNA真核表达质粒在mRNA水平对细胞MKRN1基因的抑制率分别为52.4%、26.2%和42.9%,在蛋白水平对细胞MKRN1基因的抑制率分别为69.1%、27.9%和50.0%。结论已成功构建了人MKRN1基因的shRNA真核表达质粒,为进一步研究MKRN1基因的功能及其与人端粒酶逆转录酶的关系奠定了基础。     

人Makorin环指蛋白1基因沉默对HEK293细胞的影响

目的探讨特异性抑制人Makorin环指蛋白1(MKRN1)基因的表达对HEK293细胞的影响。方法用脂质体将前期筛选出的含最有效干扰序列的人MKRN1基因shRNA真核表达质粒,转染至HEK293细胞中,观察其对人端粒酶逆转录酶(hTERT)基因mRNA转录水平、蛋白表达水平及细胞增殖的影响。结果人MKRN1基因shRNA真核表达质粒转染HEK293细胞后,在mRNA和蛋白水平均能明显上调细胞hTERT基因的表达,且细胞明显增殖。结论抑制HEK293细胞中人MKRN1基因的表达,可导致hTERT基因mRNA转录水平和蛋白表达水平上调,并促进细胞增殖。     

双启动子双报告基因真核表达质粒的构建及表达

目的构建双启动子双报告基因真核表达质粒,并在293T细胞内进行表达。方法分别以pEGFP-N1和pDsRed-N1为模板,PCR扩增EGFP和DsRed基因,插入表达载体pCI中,分别构建重组表达质粒pCI-EGFP和pCI-DsRed,利用载体上BglⅡ和BamHⅠ为同尾酶的特点,将两个表达质粒酶切后拼接,构建双启动子双报告基因重组真核表达质粒pCI-cmvDsRed-cmvEGFP,转染293T细胞,荧光显微镜观察EGFP和DsRed的表达,Western blot和流式细胞术检测EGFP和DsRed蛋白的表达。结果重组真核表达质粒pCI-cmvDsRed-cmvEGFP经酶切鉴定和测序证实构建正确,两启动子为顺向相连;EGFP和DsRed蛋白在293T细胞中可同时正确表达。结论成功构建了双启动子双报告基因真核表达质粒,并在293T细胞中表达,为实现两种外源基因在真核细胞内的同步表达及示踪提供了有效的分子工具。     

高表达转录因子ZNF191基因的慢病毒颗粒的构建及鉴定

目的构建转录因子ZNF191基因的慢病毒颗粒,为进一步研究ZNF191的生物学功能及其在肿瘤基因治疗方面的应用奠定基础。方法从HEK293细胞中提取总RNA,RT-PCR扩增ZNF191基因,亚克隆至pLVX-AcGFP-N1质粒中,经酶切及DNA测序鉴定正确后,将含有ZNF191基因的重组质粒pLVX-ZNF191结合辅助质粒转染HEK293T细胞,包装重组慢病毒,Western blot检测感染的293T细胞内ZNF191蛋白的表达。结果酶切及测序鉴定pLVX-ZNF191质粒构建正确,Western blot检测重组慢病毒能显著提高293T细胞的ZNF191蛋白表达水平。结论已成功构建了ZNF191基因重组慢病毒表达载体pLVX-ZNF191,为进一步研究ZNF191的生物学功能及肿瘤的基因治疗奠定了基础。     

Lipocalin-2基因表达产物对人胚肾细胞增殖的影响

目的构建Lipocalin-2(Lcn-2)基因真核表达质粒PL-2,并检测其及前期原核表达的重组Lcn-2蛋白对人胚肾细胞HEK293增殖的影响。方法利用RT-PCR从鼠巨噬细胞RAW264.7中扩增Lcn-2基因,克隆入真核表达质粒pEGFP-C1中,构建重组质粒PL-2。转染HEK293细胞,通过荧光观察和RT-PCR鉴定Lcn-2基因的表达。将重组质粒PL-2和前期原核表达的重组Lcn-2蛋白作用于HEK293细胞,通过MTT法检测细胞增殖情况,Westernblot法检测细胞增殖核抗原(PCNA)的表达。结果重组真核表达质粒PL-2经酶切和测序鉴定,证明构建正确。经荧光观察和RT-PCR鉴定,转染重组质粒PL-2的HEK293细胞中有Lcn-2基因的表达。加入重组Lcn-2蛋白后,HEK293细胞较对照组明显增殖,细胞中PCNA的表达也较对照组明显升高,而重组质粒PL-2对HEK293细胞增殖以及细胞中PCNA的表达均无影响。结论已成功构建了Lcn-2基因真核表达质粒,其对HEK293细胞的增殖无影响,而原核表达的重组Lcn-2蛋白对HEK293细胞的增殖有一定的促进作用,推测Lcn-2基因的表达产物可能通过与细胞膜受体结合促进HEK293细胞的增殖。     

High-level expression of recombinant IgG1 by CHO K1 platform

The Chinese Hamster Ovary (CHO K1) cell was used to express a targeted anti-cancer monoclonal antibody by optimizing the platform of the construction of production cell line in this study. The adherent CHO K1 was first adapted to suspension culture in chemical defined medium. Then the glutamine synthetase (GS) vector was applied to construct a single plasmid to overexpress a monoclonal antibody IgG1. Post transfection, the production of cell pool was optimized by glutamine-free selection and amplification using various concentrations of methionine sulfoximine. The best cell pool of CHO K1/IgG1 was used to screen the top single clone using the limiting dilution cloning. Finally, a high IgG1 production of 780 mg/L was obtained from a batch culture. This study demonstrated that the construction of high producing cell line, from gene to clone, could be completed within six month and the gene amplification improved protein production greatly.     

Platelet-activating factor stimulates expression of IL-1 beta mRNA in THP-1 cells

A human monocytic cell line (THP-1) was used to study the effects of PAF (platelet-activating factor) on the expression of IL-1 beta mRNA. THP-1 cells were incubated with 10 pM PAF in the presence or absence of 0.1 μg/mL endotoxin for 4 hr, after which cytoplasmic RNA was extracted and subjected to Northern hybridizations. PAF, alone and in combination with endotoxin, caused an increase in mRNA levels for IL-1 beta. The magnitude of the effects of PAF on IL-1 beta mRNA levels matched closely the effects seen at the level of protein synthesis, suggesting that the effects of PAF on IL-1 beta release may result largely from its effects on IL-1 beta mRNA levels.     

Ultrafine carbon particles down-regulate CYP1B1 expression in human monocytes

Background  

Cytochrome P450 monoxygenases play an important role in the defence against inhaled toxic compounds and in metabolizing a wide range of xenobiotics and environmental contaminants. In ambient aerosol the ultrafine particle fraction which penetrates deeply into the lungs is considered to be a major factor for adverse health effects. The cells mainly affected by inhaled particles are lung epithelial cells and cells of the monocyte/macrophage lineage.     

细胞因子信号抑制因子1基因真核高表达质粒的构建及其在NOK细胞中的表达

目的构建细胞因子信号抑制因子1(suppressors of cytokine signaling 1,SOCS1)基因真核高表达质粒,并在口腔上皮细胞系NOK细胞中进行表达。方法提取健康人外周静脉血基因组DNA,PCR扩增SOCS1基因,与p EGFPN1载体连接,构建重组真核表达质粒p EGFP-N1-SOCS1,用Eco RⅠ和Bam HⅠ双酶切鉴定及测序后,转染NOK细胞,采用荧光显微镜及Western blot法检测转染细胞中SOCS1蛋白的表达。结果重组真核表达质粒p EGFP-N1-SOCS1经双酶切及测序鉴定证实构建正确。p EGFP-N1-SOCS1转染NOK细胞72 h后获得表达,SOCS1蛋白的表达量为(134.67±9.07)%,较转染空质粒组约升高4倍,二者差异有统计学意义(P=0.001)。结论成功构建了SOCS1基因真核高表达质粒,为进一步研究SOCS1的生物学功能奠定了基础。     

Chemical synthesis and expression of the HIV-1 Rev protein

The HIV-1 Rev protein is responsible for shuttling partially spliced and unspliced viral mRNA out of the nucleus. This is a crucial step in the HIV-1 lifecycle, thus making Rev an attractive target for the design of anti-HIV drugs. Despite its importance, there is a lack of structural, biophysical, and quantitative information about Rev. This is mainly because of its tendency to undergo self-assembly and aggregation; this makes it very difficult to express and handle. To address this knowledge gap, we have developed two new highly efficient and reproducible methods to prepare Rev in large quantities for biochemical and structural studies: 1) Chemical synthesis by using native chemical ligation coupled with desulfurization. Notably, we have optimized our synthesis to allow for a one-pot approach for the ligation and desulfurization steps; this reduced the number of purification steps and enabled the obtaining of desired protein in excellent yield. Several challenges emerged during the design of this Rev synthesis, such as racemization, reduced solubility, formylation during thioester synthesis, and the necessity for using orthogonal protection during desulfurization; solutions to these problems were found. 2) A new method for expression and purification by using a vector that contained an HLT tag, followed by purification with a Ni column, a cation exchange column, and gel filtration. Both methods yielded highly pure and folded Rev. The CD spectra of the synthetic and recombinant Rev proteins were identical, and consistent with a predominantly helical structure. These advances should facilitate future studies that aim at a better understanding of the structure and function of the protein.     

白细胞介素-7基因慢病毒表达载体的构建及在CHO-K1细胞中的稳定表达

目的构建白细胞介素-7(interleukin-7,IL-7)基因慢病毒表达载体,并在CHO-K1细胞中稳定表达,为生产试剂级的IL-7奠定基础。方法参照Gen Bank中公布的人IL-7基因核苷酸序列(J04156.1),人工合成该条基因,亚克隆至表达载体pLV-CMV-EF1-GP上,构建重组慢病毒表达质粒pLV-CMV-EF1-GP-r IL-7,在HEK293T细胞中进行病毒包装,检测病毒滴度后,收获细胞毒液,感染CHO-K1细胞,经嘌呤霉素筛选,获得稳定表达IL-7的CHO-K1-r IL-7细胞。荧光显微镜观察感染细胞绿色荧光蛋白的表达;RT-PCR法检测IL-7基因m RNA的转录;ELISA法检测IL-7的含量;Western blot法检测IL-7的表达。结果重组慢病毒表达质粒pLV-CMV-EF1-GP-r IL-7经双酶切及测序结果证明构建正确。慢病毒的滴度为3×10~8 TU/ml。慢病毒感染CHO-K1细胞24、48和72 h后,均可见特异性绿色荧光蛋白表达,且随着培养时间的延长,荧光强度明显增强;CHO-K1-r IL-7细胞可扩增出约540 bp的IL-7基因片段;经ELISA检测,样品中IL-7含量为0.6 ng/ml;表达产物主要存在于CHO-K1-r IL-7细胞上清中,且具有良好的反应原性。结论成功构建了IL-7基因慢病毒重组表达质粒,并实现了IL-7在CHO-K1细胞上的稳定表达。     

人胰岛素样生长因子-1的融合表达及纯化

目的利用原核表达系统融合表达人胰岛素样生长因子-1(human insulin-like growth factor-1,hIGF-1),并进行纯化及鉴定。方法根据大肠埃希菌密码子偏好性对hIGF-1天然基因序列进行同义突变,并人工合成,PCR扩增后,克隆至pET48b(+)载体,构建重组原核表达质粒,转化大肠埃希菌BL21(DE3),IPTG诱导表达。表达的融合蛋白Trx A-hIGF-1经Ni2+亲和层析进行纯化,纯化产物经肠激酶酶切后,进行Western blot鉴定。结果重组表达质粒pET48b-Trx A-hIGF-1经菌落PCR及测序证实构建正确;表达融合蛋白相对分子质量约为26 000,表达量约为菌体总蛋白的40%,主要以可溶形式存在于菌体裂解上清中,可溶性蛋白占总目的蛋白的比例不低于90%,纯化后纯度不低于90%,蛋白浓度为0.5 mg/ml;纯化的Trx A-hIGF-1融合蛋白可被肠激酶切割为相对分子质量约为8 000的hIGF-1蛋白和18 000的Trx A蛋白,hIGF-1蛋白可与兔抗hIGF-1多克隆抗体特异性结合。结论成功表达了hIGF-1融合蛋白,为其生物学活性的研究及规模化生产奠定了基础。