pEGFP-BLCAP真核表达载体的构建和鉴定

目的：构建真核表达载体pEGFP—BLCAP并转染骨肉瘤细胞SOSP—M。方法：从培养的人骨肉瘤细胞系SOSP—M细胞中提取总RNA，经RT—PCR获得BLCAP基因的cDNA，测序正确后插入真核表达载体pEGFP—C2中。构建的重组质粒经脂质体介导转染SOSP—M细胞，经观察荧光蛋白表达和Western blot鉴定目的蛋白在转染后的SOSP—M细胞中的表达情况。结果：RT—PCR成功地扩增出一条约280bp的片段，经限制性内切酶分析和DNA序列测定证实目的基因cDNA已插入重组质粒；荧光显微镜下观察到转染后的SOSP—M细胞发出较强绿色荧光，Western blot证明BLCAP能以融合蛋白的形式在SOSP—M细胞中获得表达。结论：构建了真核表达载体pEGFP—BLCAP并成功表达目的蛋白。     

TSLC1真核表达载体的构建及在HepG2肝癌细胞中的表达

目的构建TSLC1真核表达载体,并转染到肝癌细胞HepG2中使之表达,为研究TSLC1的抗肝癌作用奠定基础。方法利用RT-PCR技术扩增TSLC1基因全长,并与真核表达载体pRe-ceiver-M29进行连接;应用双酶切、PCR以及测序鉴定此连接载体;脂质体Lipofectamine 2000介导重组载体转染到HepG2细胞中,经G418筛选建立稳定转染细胞株,分别采用RT-PCR和免疫组化法检测其表达。结果RT-PCR获得了约1 329 bp大小的TSLC1基因片段;经过双酶切、PCR以及测序鉴定证实TSLC1基因片段正确插入真核表达载体pReceiver-M29中;与对照组比较,RT-PCR方法可见显示转染组细胞TSLC1mRNA表达明显增多,免疫组化法可见显示转染组细胞TSLC1蛋白表达明显增高。结论成功构建了真核表达载体pReceiver-M29-TSLC1,建立了稳定转染TSLC1的HepG2细胞株。     

真核表达载体pEGFP-N1-Ubc9的构建及表达

目的：克隆人类Ubc9基因cDNA全长,构建Ubc9的真核表达载体并表达。方法：采用RT-PCR技术从人小细胞肺癌NCI-H446细胞总RNA中扩增Ubc9 cDNA基因片段,经过酶切鉴定后,克隆至pUCM-T载体,测序证实碱基序列无误后,再克隆至真核绿色荧光蛋白表达载体（pEGFP-N1）上,并转染到真核细胞,观察其在真核细胞中的表达。结果：测序证实克隆的Ubc9全长cDNA阅读框正确完整,酶切和序列测定证实Ubc9正确插入pEGFP-N1载体中,该重组载体能够在真核细胞中表达。结论：成功构建了真核表达载体pEGFP-N1-Ubc9。     

人Elf5基因真核表达质粒的构建及其亚细胞定位

目的：构建人Elf5真核细胞表达质粒,并研究其在乳腺癌MCF7细胞中的亚细胞定位。方法：PCR扩增人Elf5基因,将其克隆入真核表达载体pEGFP-C1中,构建含人Elf5基因的真核表达质粒pEGFP-hElf5。利用酶切鉴定,DNA测序,免疫印迹及免疫荧光方法鉴定Elf5真核表达质粒及Elf5蛋白的亚细胞定位。结果：经酶切鉴定、DNA测序、免疫印迹方法确认Elf5基因成功导入真核表达载体pEGFP-C1。瞬时转染MCF7细胞,Elf5蛋白表达在细胞核中。结论：pEGFP-Elf5真核表达质粒构建成功,Elf5定位在细胞核中。     

人细胞周期素D1和B1真核表达载体的构建及对HeLa细胞的转染

目的：构建人细胞周期素cyclin D1和cyclin B1的真核表达载体,并将其瞬时转染到HeLa细胞株中。方法：以HeLa细胞总RNA为模板,通过RT-PCR扩增cyclin D1和cyclin B1基因编码的cDNA,并将扩增的cDNA片段插入p3XFLAG-CMV~（TM）-14真核表达载体,分别构建p3XFLAG-cyclin D1和p3XFLAG-cyclin B1重组质粒,重组子经酶切分析和测序鉴定后,用脂质体介导的基因瞬时转染法,将重组正确的表达载体转染HeLa细胞,用Western-blot技术检测细胞中FLAG融合蛋白的表达。结果：经酶切鉴定和Western印迹分析证实人cyclin D1和cyclin B1的真核表达载体构建成功,并能在瞬时转染的HeLa细胞中表达分子量大小相符的重组蛋白。结论：成功构建了人cyclin D1和cyclin B1的真核表达载体,为两种细胞周期素及其相关蛋白的功能研究奠定了基础。     

AIF基因真核表达载体的构建及其在MGC-803中的表达

[目的]检测胃癌标本中AIF基因是否存在碱基突变;构建AIF基因真核表达载体AIF—GFP;并检测其在胃腺癌细胞株MGC-803中的表达。[方法]应用RT—PCR的方法分别从5例胃癌组织标本中提取总RNA,进一步扩增出全长的AIF基因;将其克隆到DMD19-T载体进行测序,并亚克隆至真核表达载体pEGFP—N2,重组载体AIF—GFP采用脂质体法瞬时转染MGC-803细胞,荧光显微镜观察AIF—GFP在细胞中的表达：[结果]由5例胃癌组织标本提取的mRNA所扩增出的AIF基因的序列与GeneBank中AIF（AF100928）序列完全一致．胃癌组织中均未发现碱基突变;构建了真核表达载体AIF—GFP;并将其转染MGC-803进行表达。[结论]胃癌中AIF基因未发生碱基突变;AIF-GFP真核表达载体,在MGC-803细胞中获得了表达,为进一步探讨AIF在胃癌诊治中的应用奠定了基础。     

真核表达载体pEGFP-N1-Ubc9的构建及表达

目的:克隆人类Ubc9基因cDNA全长,构建Ubc9的真核表达载体并表达。方法:采用RT-PCR技术从人小细胞肺癌NCI-H446细胞总RNA中扩增Ubc9 cDNA基因片段,经过酶切鉴定后,克隆至pUCM-T载体,测序证实碱基序列无误后,再克隆至真核绿色荧光蛋白表达载体(pEGFP-N1)上,并转染到真核细胞,观察其在真核细胞中的表达。结果:测序证实克隆的Ubc9全长cDNA阅读框正确完整,酶切和序列测定证实Ubc9正确插入pEGFP-N1载体中,该重组载体能够在真核细胞中表达。结论:成功构建了真核表达载体pEGFP-N1-Ubc9。     

4ARE强化的双荧光蛋白报告基因载体的构建及鉴定

目的:构建4ARE强化的红、绿双荧光蛋白报告基因载体。方法:人工合成3对ARE序列,经退火和磷酸化后插入pARE-TK-GFP载体,构建成p4ARE-TK-GFP载体。以质粒pDsRed2-N1为模板,PCR扩增红色荧光蛋白及其启动子序列与pMD18-T载体连接,构建成pMD-DsRed载体。用Ade I酶和BspT I酶对载体pMD-DsRed和p4ARE-TK-GFP进行双酶切,连接产物转化大肠埃希菌DH5α 感受态细胞后,挑取阳性克隆子进行质粒抽提酶切及测序鉴定。结果:经酶切及DNA测序证实,目的片段ARE及DsRed的序列完全正确,重组双荧光蛋白报告基因载体成功转入DH5α。结论:成功构建4ARE强化的双荧光蛋白报告基因载体并在DH5α内表达,为进一步研究ARE的调控作用奠定了基础。     

p53蛋白及其突变体对RhoE基因转录调控的影响

目的:探索p53蛋白及其突变体对RhoE基因转录调控的影响。方法:构建pEGFP-wt-p53质粒,利用基因定点突变PCR技术构建p53单点和双点突变体质粒,构建pGL3-RhoE-promotor-Luc质粒。以P21基因的启动子序列(P21-promoter-luc)为阳性对照,EGFP-N1空载体为阴性对照,将野生型和突变型p53质粒瞬时转染PC3(p53-null)细胞,利用双荧光素酶报告基因和Westernblot方法检测p53蛋白及其突变体蛋白在转录水平和蛋白水平对RhoE基因表达的影响。结果:电泳及测序表明以上质粒均构建成功。双荧光素酶报告基因检测显示野生型p53蛋白可调控RhoE基因转录,但其突变体丧失对RhoE的转录调控作用(P0.05),且不同位点的p53突变体蛋白之间对RhoE转录调控作用的差别无统计学意义(P0.05)。Western blot结果与基因转录结果一致。结论:RhoE是受p53蛋白调控的基因之一,而p53突变体失去了在转录水平调控RhoE表达的作用。     

MDR1启动子/荧光素酶质粒的构建及其在朊蛋白高表达胃癌细胞中的活性检测

目的：构建含MDR1基因启动子的荧光素酶报告基因质粒,并检测其在朊蛋白高表达胃癌细胞系中的活性表达。方法：PCR克隆人MDR1基因启动子片段,通过亚克隆将启动子分别插入到pMD18-T载体和荧光素酶报告基因pGL3-Enhancer载体中,建立含MDR1启动子的荧光素酶报告基因质粒pGL-MDR1,并经测序及酶切确定扩增序列;脂质体基因转染法将pGL-MDR1转染入朊蛋白高表达胃癌细胞系SGC7901-PrP,并测定其荧光素酶活性。结果：PCR克隆出MDR1启动子经DNA测序证实序列正确,pGL-MDR1转染入朊蛋白高表达胃癌细胞系的荧光素酶活性,较转染入pcDNA3.1空载体细胞系相比升高3-5倍。结论：成功构建含MDR1启动子的荧光素酶报告基因质粒;上调朊蛋白表达可激活MDR1的转录活性。     

p53 mutation arising in Arg72 allele in the tumorigenesis and development of carcinoma of the urinary tract.

PURPOSE: It is known that a common p53 polymorphism, encodingeither proline (Pro) or arginine (Arg) at residue 72, produces marked change in the structure of p53. Furthermore, the Arg72-containing allele is preferentially mutated and retained in various human tumors, suggesting that polymorphic residue within p53 modifies mutant behavior. We studied to determine whether Arg72 could be a risk factor for p53 mutations in human transitional cell carcinomas (TCCs). In addition, the relationship between the status of p53 codon 72 polymorphism and clinicopathological factors of this tumor were also analyzed. EXPERIMENTAL DESIGN: We analyzed the correlation between the p53 mutations and genotypes of its codon 72 using genomic DNAs from the TCCs by direct DNA sequencing. Loss of heterozygosity was determined using a p53 microsatellite marker (TP53) amplified by PCR. RESULTS: There was a bias to mutate and express the Arg allele in the p53 -mutated TCCs arising in individuals with heterozygosity (Pro/Arg). The Arg72-containing allele was preferentially retained in these tumors. The prevalence of cases with p53 mutations within Arg72-containing allele was higher for advanced-stage TCCs (chi(2) = 5.320, P = 0.021) than for TCCs with those arising in Pro72-containing allele. CONCLUSIONS: Our in vivo findings suggested that p53 mutation alleles containing Arg72 are preferentially selected during tumorigenesis and affect mutant behavior in TCCs, and revealed that TCCs with p53 mutation arising in Arg72-containing allele became progressively more abundant with increase in tumor stage.     

人ＳＴＡＴ３基因ｓｉＲＮＡ真核表达质粒的构建及鉴定

目的：构建针对人ＳＴＡＴ３基因的ｓｉＲＮＡ真核表达质粒,检测其在细胞水平对ＳＴＡＴ３基因表达的抑制效果。方法：用ＤＮＡ重组技术将针对人ＳＴＡＴ３基因ｍＲＮＡ序列不同位点设计的３个ｓｉＲＮＡ序列克隆到真核表达质粒ｐＲＮＡＴ-Ｕ６.１／ｎｅｏ中构建重组体ｐＲＮＡＴ-Ｕ６.１-ｓｉＲＮＡ,重组质粒经ＰＣＲ检测及测序分析,用脂质体转染重组质粒至人食管癌Ｅｃａ-１０９细胞,Ｇ４１８筛选获得阳性克隆,ＲＴ-ＰＣＲ和Ｗｅｓｔｅｒｎｂｌｏｔ检测ＳＴＡＴ３基因ｍＲＮＡ和蛋白的表达,筛选最佳沉默效率的ｓｉＲＮＡ。结果：ＰＣＲ检测及测序分析结果均提示重组质粒构建正确。ＲＴ-ＰＣＲ和Ｗｅｓｔｅｒｎｂｌｏｔ检测证实ｐＲＮＡＴ-Ｕ６.１-ｓｉＲＮＡ３具有最佳的沉默效率。结论：成功构建人ＳＴＡＴ３基因ｓｉＲＮＡ真核表达质粒,并证实其能够从ｍＲＮＡ和蛋白水平抑制ＳＴＡＴ３基因的表达。     

Common tumour p53 mutations in immortalized cells from Hupki mice heterozygous at codon 72

Codon 72 of human p53 gene is polymorphic, encoding arginine or proline. Here we report construction of a human p53 knock-in (Hupki) mouse encoding the codon 72(pro) variant. The new strain was crossed with the original Hupki mice (codon 72(arg/arg)) to obtain primary embryonic fibroblasts polymorphic at codon 72 or homozygous for codon 72(pro). The fibroblasts, cultured under standard conditions, immortalized within 12 weeks and acquired p53 mutations similarly to Hupki codon 72(arg/arg) cells investigated previously. Sequencing of human p53 exons 4-9 in immortalized cultures revealed missense mutations found repeatedly in human tumours. In cell lines ensuing from benzo(a)pyrene-treated cultures the combined p53 mutation pattern from experiments with the 3 codon 72 genotypes showed a predominance of strand-biased G to T transversions (18 of 36 mutations), and mutations recurring at smokers' lung tumour hotspot codons 157 and 273, supporting involvement of tobacco carcinogens in shaping the mutation signature in lung cancers of smokers. Mutations in cell lines from unexposed cultures did not cluster at these codons and G to T transversions were uncommon (2 of 52 mutations) (Fisher's exact test P<0.0001). Most mutations (13/16) in cell lines derived from cells polymorphic at codon 72 were found on the proline allele, with loss of the arginine allele.     

干扰iASPP基因对转染MCF-7细胞凋亡变化的观察

目的:观察iASPP基因干扰RNA转染乳腺癌细胞MCF-7后的干扰效果及细胞凋亡变化。方法:设计特异性siRNA序列,将序列克隆至PGCsilencerTM H1/Neo/GFP质粒中,用脂质体将重组子转染至MCF-7细胞中,用RT-PCR方法检测i ASPP的表达,蛋白质印迹法检测蛋白表达的变化,流式细胞仪检测细胞凋亡的情况。结果:iASPP干扰质粒转染MCF-7细胞后,iASPP的mRNA表达和蛋白表达减少40%～50%;p53蛋白相对表达量由转染阴性质粒的0.37增加到转染干扰质粒的0.64;细胞凋亡率由原来的17.8%和16.2%分别增加到53.5%和51.3%。结论:抑制内源性i ASPP能有效地恢复乳腺癌细胞MCF-7中p53的抑癌功能,为乳腺癌的治疗提供新的思路。     

Construction of a fusion expression plasmid containing the G250 gene and human granulocyte-macrophage colony stimulating factor and its significance in renal cell carcinoma

This study aimed to construct a eukaryotic expression plasmid containing the G250/MN/CA IX (G250) and human granulocyte-macrophage colony stimulating factor (hGM-CSF) genes, and to detect the expression of these proteins in vitro by recombinant plasmids in eukaryotic cells. pORF-hGM-CSF and pcDNA3.0-G250 were used as the template to amplify G250 and hGM-CSF by routine polymerase chain reaction (PCR). The two PCR products were cloned into the eukaryotic vector pVAX1, in order to construct a recombinant plasmid pVAX1-G250-hGM, and the plasmid was transfected into human embryonic kidney 293 cells. The protein expression was then determined by immunocytochemistry, atomic force microscopy, ELISA and Western blotting. DNA sequencing showed that the cloned G250 and hGM-CSF sequences were consistent with the reported Gene Bank ones. Moreover, a high expression was noted following recombinant plasmid transfection of the G250 and hGM-CSF proteins. Thus, the eukaryotic expression vector pVAX1-G250-hGM containing G250 and hGM-CSF was constructed, allowing for the investigation of the anti-G250 antigen vaccine and immune response mechanisms of biological immunotherapy in renal cell carcinoma.     

Effect of RNA interference of iASPP on the apoptosis in MCF-7 breast cancer cells

ASPP family is proved to be apoptotic specific regulators of p53. Among them, iASPP acts as an inhibitor of p53. To investigate the effect of the iASPP RNAi on the apoptosis of breast cancer cell MCF-7, we transfected the recombinant plasmid PGCsilencer H1/Neo/GFP/RNAi into MCF-7. The iASPP expression was analyzed by RT-PCR and Western blot. The cell apoptosis was detected by FCM. The results show that the expression of iASPP is descended and the apoptosis rate is increased after transfection. Therefore, we conclude that inhibition of expression of iASPP may resume the ability of p53 to induce apoptosis in MCF-7 cells.     

真核表达p27构建胃癌顺铂耐药细胞模型的研究

目的:构建p27真核表达载体并导入胃癌SGC-7901细胞获得稳定表达p27的稳定细胞株,以研究p27在胃癌SGC-7901细胞顺铂耐药中所发挥的功能。方法:以乳腺文库为模板,PCR扩增出p27编码区,并将其连接到pCDNA 3.0-Flag载体上,转染293T细胞后分别用定量PCR和Western blot检测其表达情况,并通过Western blot检测SGC-7901细胞过表达Flag-p27稳定细胞株是否构建成功。通过CCK-8药物敏感性实验检测p27在胃癌细胞顺铂耐药中所发挥的功能。结果:双酶切和测序结果表明,pCDNA 3.0-Flag-p27构建成功,并在293T中成功表达,胃癌SGC-7901细胞过表达Flag-p27细胞株建立成功,通过耐药曲线表明过表达p27可以引起SGC-7901细胞顺铂耐药。结论:成功构建了带Flag标签的p27真核表达载体,为进一步研究p27在胃癌耐药中的功能奠定了基础。     

The N-terminus of a novel isoform of human iASPP is required for its cytoplasmic localization

ASPP1 and ASPP2 are both proteins that interact with p53 and enhance its ability to induce apoptosis by selectively elevating the expression of proapoptotic p53-responsive genes. iASPP(RAI) is a third member of the family that is the most conserved inhibitor of p53-mediated apoptosis. Here, we have described iASPP, a longer form of iASPP(RAI), which at 828 amino acids is more than twice the size of iASPP(RAI). Using two antibodies that recognize both iASPP and iASPP(RAI), we report that this longer form of iASPP is the predominant form of the molecule expressed in cells. Like iASPP(RAI), iASPP also binds to p53 and inhibits apoptosis induced by p53 overexpression. However, whereas iASPP(RAI) is predominantly nuclear, the N-terminus of iASPP is entirely cytoplasmic, and the longer iASPP is located in both the cytoplasm and the nucleus. The effect upon subcellular localization of the longer N-terminus of iASPP means that this new, longer form of the molecule may be subject to greater regulation and provides another layer in the control of p53-induced apoptosis.