鼠OAZ1基因的真核表达质粒的构建及其在COS7细胞中的表达

构建鼠OAZ1基因真核表达质粒,观察OAZ1-GFP融合蛋白在绿猴肾细胞COS7中的表达。利用RT-PCR的方法获得鼠OAZ1基因,再利用重叠延伸PCR缺失掉OAZ1序列中的205位碱基T,由此获得无需阅读框移码即可编码全长OAZ1的突变基因。将此突变基因克隆入真核表达载体pEGFP-N1获得重组质粒pEGFP-N1-OAZ1-T。将此质粒瞬时转染COS7后,采用RT-PCR,Western blotting,免疫荧光技术观察检测目的基因的表达情况。结果显示,酶切和测序证明质粒pEG-FP-N1-OAZ1-T构建正确,转染COS7细胞后,OAZ1-GFP融合蛋白能在细胞中高效表达。成功构建了pEGFP-N1-OAZ1-T真核表达质粒,在COS7细胞内,该质粒能成功指导OAZ1-GFP融合蛋白合成。     

人P2X7真核表达载体的构建及稳定转染细胞株的建立

目的：构建人P2X7基因的真核表达载体,并通过转染获得稳定表达P2X7分子的HEK293细胞株。方法：以人脑组织P2X7cDNA为模板扩增出P2X7基因,插入到真核表达载体pEGFP-N1中,构建重组质粒pEGFP-N1/P2X7。用X-fect试剂盒将重组质粒转染HEK293细胞,通过G418辅助荧光筛选建立稳定表达P2X7-EGFP细胞株。经流式细胞仪、Western blot和激光共聚焦显微镜检测,了解人P2X7在HEK293细胞中的表达水平及细胞内定位。结果：重组质粒pEGFP-N1/P2X7构建正确,建立了稳定表达人P2X7的HEK293细胞系。Western blot和流式细胞仪检测证实,P2X7在HEK293细胞系中成功表达,激光共聚焦显微镜检测显示P2X7-EGFP定位在细胞膜上。结论：重组载体pEGFP-N1/P2X7构建成功并建立了稳定表达人P2X7的HEK293细胞系,为进一步研究P2X7离子通道结构和功能奠定基础。     

HCMV IE86对ARPE-19细胞周期的影响

目的:构建真核表达质粒pEGFP-N3-IE2,瞬时转染ARPE-19细胞,探讨其时视网膜色素上皮细胞周期的影响.方法:采用PCR方法从质粒pIEP86AD169扩增出IE2片段,将其定向克隆于pEGFP-N3,构建真核表达质粒pEGFP-N3-IE2,酶切、测序鉴定,脂质体介导瞬时转染ARPE-19细胞,RT-PCR、免疫荧光法分析IE2基因在mRNA和蛋白水平的表达.流式细胞仪检测转染前后细胞周期的变化.结果:成功构建重组质粒pEGFP-N3-IE2,并在被转染细胞中检测到IE2基因的表达;瞬时转染后细胞周期表现为S期细胞比例显著增高.结论:成功构建pEGFP-N3-IE2真核表达质粒,重组质粒能在ARPE-19细胞中顺利表达IE2,IE2基因可以引起ARPE-19细胞的细胞周期S期阻滞.     

广西巴马小型猪ABCA1基因真核表达载体的构建与鉴定

本研究目的是克隆广西巴马小型猪ABCA1基因CDs序列并构建真核表达载体。利用RT-PCR技术从广西巴马小型猪肝脏组织中扩增出ABCA1基因编码序列,直接插入至pEGFP-N1真核表达载体的KpnⅠ酶切位点,构建出重组质粒。之后将重组质粒pEGFP-N1-ABCA1转染PK15细胞,并于24 h、48 h后观察细胞荧光表达情况。收集转染48 h后的PK15细胞通过qRT-PCR检测ABCA1 mRNA相对表达量。结果表明:本实验成功构建了广西巴马小型猪ABCA1基因真核表达载体,重组质粒pEGFP-N1-ABCA1转染PK15细胞后,有绿色荧光蛋白表达,且与转染pEGFP-N1和空白组相比,pEGFP-N1-ABCA1转染组的ABCA1 mRNA表达量显著提高。本研究为下一步研究ABCA1基因的功能和调控及生产转ABCA1基因广西巴马小型猪奠定基础。     

广西巴马小型猪TRβ1基因真核表达载体的构建及鉴定

本试验为了克隆广西巴马小型猪甲状腺激素受体β1(TRβ1)基因编码序列并构建该基因的真核表达载体,取广西巴马小型猪肝脏组织作为材料,采用RT-PCR技术扩增出TRβ1基因编码序列,与pMD19-T-Simple载体连接,测序正确的重组质粒双酶切后,连接pEGFP-N1载体,构建pEGFP-N1-TRβ1真核表达载体。经双酶切和测序鉴定后,重组质粒pEGFP-N1-TRβ1转染293T细胞,培养48 h后,在荧光显微镜下观察细胞荧光蛋白的表达情况。结果表明,本试验成功克隆了广西巴马小型猪的TRβ1基因cDNA序列,序列长度为1 368 bp,编码455个氨基酸,与参考序列的同源性为99.6%,有5处位点突变,且全为同义突变。阳性重组质粒pEGFP-N1-TRβ1转染293T细胞48 h后,在荧光显微镜下观测出绿色荧光表达。     

黑曲霉Z6植酸酶基因phyA的克隆及表达

目的:克隆植酸酶基因phyA,构建毕赤酵母表达载体,转化毕赤酵母,并对重组工程菌的表达产物进行初步酶学性质研究.方法:以植酸酶高产菌株-黑曲霉Z6染色体DNA为模板,PCR扩增得到植酸酶基因phyA,序列鉴定后连接到毕赤酵母穿梭载体pPIC9K上,构建重组质粒pPIC9K-phyA,电击转化毕赤酵母KM71,筛选得到重组转化子.对重组工程菌表达产物进行SDS-PAGE分析和酶活性研究.结果:phyA序列分析表明该基因具有典型的植酸酶活性位点保守序列ArgHisGlyAlaArgTyrPro,与NC-BI已发表的植酸酶基因同源性较高,达到94%以上.该序列已提交GenBank,序列号为DQ318022.重组工程菌KM71-phyA7的PCR扩增证实了植酸酶基因已整合到酵母基因组中,植酸酶能有效分泌和表达,粗酶液酶活可达875U/mL.结论:植酸酶基因phyA在毕赤酵母中成功表达,为今后的定向改组奠定了基础.     

艾滋病患者APOBEC3G 基因的克隆和真核表达

目的 建立艾滋病( AIDS) 患者载脂蛋白B mRNA 编辑酶催化多肽样蛋白3G( APOBEC3G) 的真核表达体系。方法 采用反转录-聚合酶链反应( RT-PCR) 技术从AIDS 患者外周血单个核细胞( PBMC) 中获取APOBEC3G 基因编码区, 将其克隆到pMD18-T载体上, 测序验证正确后再将其转接入真核表达载体pEGFP-N1 中, 然后将重组质粒pEGFP-N1-A3G 转染HEK293T细胞, 分别用RT-PCR 法和蛋白印迹法( Western 印迹法) 验证APOBEC3G 在mRNA 和蛋白水平的表达。结果 从AIDS 患者体内克隆的APOBEC3G 基因编码区长度为1 154 bp, 测序结果与GenBank 中APOBEC3G 参考序列( NM021822) 比对发现存在2 处差异, 分别位于mRNA 第588 位和746 位碱基处。重组质粒pEGFP-N1-A3G转染HEK293T 细胞, 在荧光显微镜下观察到融合蛋白A3G-EGFP的表达, RT-PCR 法和Western blot 法分别验证了蛋白在mRNA 和蛋白水平的表达。结论 成功构建了AIDS 患者APOBEC3G 蛋白的真核表达体系, 为进一步研究APOBEC3G 在HIV-1 感染中的作用奠定了基础。     

重组pEGFP—C2-p100-TSN点突变质粒构建及表达

目的将6个不同的p100-TSN．Mutants基因片段分别定向连入PEGFP—C2质粒中,使P100-TSN突变蛋白能够与绿色荧光蛋白在COS7细胞中融合表达,从而为进一步研究p100蛋白TSN结构域的功能奠定实验基础。方法利用EcoR I和Xho I双酶切方法从6个pcDNA3．1（＋）-p100-TSN．Mutants重组质粒中分别获得p100-TSN．Mutants的cDNA片段,将其连人pEGFP—C2质粒载体中,再将成功构建的6个pEGFP—C2-p100-TSN．Mutants质粒分别转染COS7细胞中,荧光显微镜下观察绿色荧光蛋白表达。结果①将重组质粒进行双酶切鉴定可见p100-TSN．Mutants的cDNA片段;②转染重组质粒后可观察到绿色荧光蛋白的表达。结论①6个pEGFP—C2-p100-TSN．Mutants重组质粒构建成功;②p100-TSN突变蛋白可与绿色荧光蛋白在COS7细胞中融合表达。     

小鼠GITRL基因真核表达质粒的构建转染及其在Kupffer细胞中的表达

目的构建含小鼠GITRL基因的真核表达质粒pEGFP-N1-GITRL,体外转入小鼠Kupffer细胞。方法利用PCR方法扩增GITRL基因,克隆至pEGFP-N1载体,选择阳性克隆并进行序列测定。以脂质体化学法转染至Kupffer细胞中。结果构建了真核表达质粒pEGFP-N1-GITRL,基因测序与GenBank中发表的序列完全一致,体外瞬时转染Kupffer细胞,RT-PCR及WB检测该Kupffer细胞表达GITRL。结论成功构建了真核表达质粒pEGFP-N1-GITRL,并在小鼠Kupffer细胞成功表达,为进一步研究GITRL在Kupffer细胞中的的生物学功能提供研究基础。     

异种化人肾细胞癌特异性抗原G250真核表达载体的构建及表达

目的：构建含有人肾细胞癌特异性抗原G250（CAⅨ）主要T细胞表位区域、猴和鼠CAⅨ部分片段区域融合基因tG250的真核表达质粒,并在猴肾COS7细胞中表达。方法：通过基因合成和PCR技术构建人、猴和鼠G250区域融合基因tG250,将其插入含有人Igκ链前导信号肽（sig）、人IgG-Fc和糖基磷脂酰肌醇（GPI）锚定信号肽融合基因序列的细胞膜锚定修饰真核表达载体pCI-Fc-GPI中,继而又将酶切后的sig-tG250-Fc-GPI融合基因导入含有细小病毒内部核糖体结合位点（IRES）基因且可以共表达人GM-CSF和B7.1融合基因的真核表达载体pVAX1-IRES-GM/B7中;将构建的重组质粒pVAX1-sig-tG250-Fc-GPI-GM/B7转染COS7细胞,利用流式细胞仪和免疫荧光检测其表达。结果：测序结果表明tG250融合基因序列正确,PCR和酶切鉴定证明已将其连入真核表达载体pVAX1-IRES-GM/B7中;流式细胞仪和免疫荧光的检测结果显示,重组质粒pVAX1-sig-tG250-Fc-GPI-GM/B7在COS7细胞中得到很好的表达。结论：构建了重组质粒pVAX1-sig-t G250-Fc-GPI-GM/B7,且在COS7细胞中有效表达,为以G250为靶点的抗肾细胞癌基因疫苗的构建与功能研究奠定了基础。     

人TIMP-1 cDNA克隆、真核表达载体构建及在COS-7细胞中的表达

根据 Gen Bank中 TIMP- 1基因的碱基序列 ,用 RT- PCR方法从人的正常肾组织中克隆出包含信号肽在内的 TIMP- 1全长 c DNA序列 .采用 T- A克隆的方法将之插入 p CRR2 .1中间载体 ,DNA测序证实该片段序列与文献报告的完全一致 .利用亚克隆的方法将 TIMP- 1 c DNA片段克隆到 pc DNA3载体上 ,构建出 pc DNA3/ TIMP- 1的真核表达载体 ,通过脂质体 DOTAP转染至 COS-7细胞 ,Northern印迹及原位杂交证实在 COS- 7细胞上获得人 TIMP- 1的高效表达 ,细胞增殖实验表明 TIMP- 1的高产表达可促进 COS- 7细胞的增殖 ,证实了所转染人 TIMP- 1的生物活性     

Expression of a gene encoding a rabbit sperm membrane protein in mammalian cells.

A general mammalian expression vector designated pSV2-EP was reconstructed by inserting an oligonucleotide fragment into pSV2-dhfr. This vector allowed insertion of cDNAs with EcoRI cohesive ends. The pSV2-EP contains a simian virus 40 (SV40) early promoter, origin for DNA replication, SV40 poly-A site, splicing site, an initiator ATG downstream from the promoter and an EcoRI site for the insertion of cDNA fragment screened from lambda gt11 expression libraries. A recombinant plasmid (pS-VRS-1) was constructed by inserting RSD-1, a cDNA encoding a rabbit sperm tail protein, into the EcoRI site of the pSV2-EP vector. Chinese hamster ovarian (CHO) dhfr-negative cells were cotransformed with pSV2-dhfr and pSVRS-1 by the calcium phosphate method. In selective culture medium without thymidine and hypoxanthine, several cell lines were obtained containing mRNA and DNA that hybridized with RSD-1. One of these transformed cell lines stained intensely with anti-rSMP-B antibodies, demonstrating that the RSD-1 was expressed in the transformed CHO cells.     

Antibodies generated in response to plasmid DNA encoding zona pellucida glycoprotein-B inhibit in vitro human sperm-egg binding

To investigate the immunogenicity of plasmid DNA encoding bonnet monkey (Macaca radiata) zona pellucida (ZP) glycoprotein-B (bmZPB), the cDNA corresponding to bmZPB, excluding the N-terminal signal sequence and C-terminus transmembrane-like domain, was cloned in mammalian expression vector VR1020 downstream of tissue plasminogen activator signal sequence under cytomegalovirus promoter (VRbmZPB). In vitro transfection of COS-1, COS-7, CHO, HEK-293, and UM-449 mammalian cells with VRbmZPB plasmid DNA led to the expression of bmZPB. Expression of bmZPB in transfected cells was cytosolic. Flow cytometry analysis of COS-1 cells transfected with VRbmZPB revealed that approximately 15% cells expressed bmZPB. The expressed bmZPB has an apparent molecular weight of 57 kDa. Immunization of male BALB/cJ mice with VRbmZPB plasmid DNA in saline as compared to VR1020 immunized group, elicited significant antibodies against E. coli expressed recombinant bmZPB as evaluated in ELISA. The antibodies generated by VRbmZPB plasmid DNA recognized bonnet monkey as well as human ZP. The immune sera obtained from mice immunized with VRbmZPB plasmid DNA also inhibited, in vitro, the binding of spermatozoa to the ZP in the hemizona assay. These studies, for the first time, demonstrate the feasibility of DNA vaccine to generate antibodies against ZP that recognize native protein and inhibit human sperm-oocyte binding.     

Molecular cloning of human immune interferon cDNA and its expression in eukaryotic cells.

Starting with mRNA derived from Staphylococcal enterotoxin A induced human splenocytes, dsDNA was synthesized and inserted into unique BamHI site of the eukaryotic expression vector pSV529 (1). A recombinant plasmid containing human immune interferon (IFN-gamma) cDNA was identified by hybridization of plasmid inserted DNA bound onto nitrocellulose filters with mRNA derived from SEA-induced splenocytes, translation of the eluted RNA in Xenopus laevis oocytes and assaying for IFN activity. Plasmids containing the entire human IFN-gamma cDNA sequence were identified by colony hybridization and were sequenced. A unique coding region was identified which predicted a protein of 166 amino acids, the 20 N-terminal amino acids of which presumably represent a signal peptide. After transfection of monkey cells with plasmid DNA isolated from one of the recombinant clones (pHIIF-SV-gamma 1), IFN was excreted into the culture medium. This IFN was not distinguishable from human IFN-gamma by serological criteria or by cell target species specificity.     

Electroporation-mediated transfection of mammalian cells with crude plasmid DNA preparations

We designed a simple and reproducible electroporation-mediated transfection procedure with which to screen mammalian expression vector-constructed cDNA libraries. Using a specific chamber composed of five parallel electrodes, the recipient cells can be electroporated separately with 40 plasmid DNA preparations in a single experiment. Over 300 crude plasmids prepared from E. coli (DH-5) carrying a pcD2neo-vector-derived cDNA library were tested. The efficiency of stable transfection by electroporation with crude plasmid DNA preparations was 10-times higher than with the CsCl-purified plasmid DNA. When the crude plasmids were digested with RNase, the efficiency of stable transfection markedly decreased, indicating that the contaminating bacterial RNA in the crude plasmid preparations has a strong carrier effect during the electroporation. Even when salmon sperm DNA or genomic DNA from the recipient cells was used as the carrier of the purified plasmid, the efficiency was not higher than that using the crude preparations. This procedure is useful not only for screening a number of cDNAs but also for routinely introducing biologically active foreign genes into cultured mammalian cells.     

人ABCB6表达载体的构建及稳定表达细胞株A375的筛选

目的构建表达载体pIRES2-ZsGreen1-ABCB6,在转染的人黑素瘤细胞系株A375中筛选其稳定表达的细胞株。方法抽取健康人外周血,分离外周血单个核细胞,提取总RNA,逆转录获取cDNA序列,加入特异性引物经PCR扩增获得ABCB6cDNA双链,再经过BglII、EcoRI双酶切PCR产物及质粒载体pIRES2-ZsGreen1,酶切产物经回收、T4DNA连接酶连接,产物转化到大肠杆菌DH5α,挑取阳性克隆经菌落PCR鉴定、酶切鉴定和测序分析,以确定构建质粒正确。转染人黑素瘤细胞株A375,G418筛选稳定表达ABCB6的单克隆细胞株,应用荧光显微镜鉴定ABCB6蛋白的表达情况。结果 pIRES2-ZsGreen1-ABCB6质粒经菌落PCR、酶切、测序鉴定正确,经过G418筛选后获得稳定细胞株,在荧光显微镜下可观察到绿色荧光蛋白在A375细胞中的表达。结论表达载体pIRES2-ZsGreen1-ABCB6构建正确,并成功筛选出稳定表达ABCB6的A375细胞株,为进一步研究ABCB6的生物学功能奠定了良好基础。     

Construction and characterization of a recombinant plasmid encoding the gene for the thymidine kinase of Herpes simplex type 1 virus.

We have constructed a hybrid plasmid by insertion of the thymidine kinase (TK) gene of Herpes simplex virus (HSV) type I at the BamHI site on Escherichia coli plasmid pBR322. The restriction endonuclease cleavage site map for the viral DNA fragment was determined for ten nucleases, and the insert in the recombinant plasmid has the same restriction nuclease digestion pattern as bona fide viral DNA. This result indicates that the plasmid contains an accurate copy of the viral DNA. The viral TK gene carried on the plasmid can be introduced into mammalian cells where it is expressed. This source of DNA with a selectable marker should be of considerable practical use in gene-transfer experiments in mammalian cells.     

人工合成的黑曲霉NRRL3135菌株植酸酶基因在毕酵母系统中的高效表达

本文以黑曲霉（Aspergillus niger)NRRL3135菌株植酸酶基因为对象,通过基因人工合成的方法去除了该基因的内含子与信号肽编码序列,换用在毕赤酵母（Pichia pastoris）中使用频率较高的密码子以优化其表达。该人工合成植酸酶基因（PhyA-as)以N端融合的方式正确插入到毕赤酵母表达载体pPICZαA。通过电击将重组表达载体整合人酵母染色体DNA中得到重组转化子。SDS－PAGE结果与表达产物酶这性质研究表明植酸酶得到分泌表达,且与天然产物性质基本一致。筛选得若干株高产基因工程菌,其中SPAN－Ⅲ菌株达到了在摇庆培养条件下,每毫升发酵液产生165000u植酸酶的水平,基本满足工业化生产的要求。