Construction of Eukaryotic Expression Plasmid pcDNA3-FHIT and Its Transient Expression in COS-1 Cells

Objective: To construct eukaryotic expression plasmid pcDNA3-fragile histidine triad(FHIT) and obtain its transient expression in COS-1 cells. Methods: FHIT gene was cloned from normal human thyroid tissue by RT-PCR and then inserted into eukaryotic expression vector pcDNA3. After the sequence was confirmed, the recombinant plasmid pcDNA3-FHIT was transfected into COS-1 cells by cation liposome. The transient expression in the cells was measured by immunocytochemistry. Results: The sequence of FHIT in pcDNA3 was correct and high expression was obtained in COS-1 cells. Conclusion: The eukaryotic expression plasmid pcDNA3-FHIT was constructed successfully and could highly express FHIT protein in COS-1 cells. This will be potentially useful for the research on gene therapy.     

Construction and identification of eukaryotic eukaryotic expression plasmid pcdna3.1-bace and its transient expression in cells

Objective： To generate eukaryotic expression vector of pcDNA3.1-BACE and obtain its transient expression in COS-7 cells and high expression in the neuroblastoma SK-N-SH cells. Methods： A 1503 bp cDNA fragment was amplified from the total RNA of human neuroblastoma by RT-PCR method and cloned into plasmid pcDNA3.1. The vector was identified by digestion with restriction enzymes BamHI and XhoI and sequenced by Sanger-dideoxy：mediated chain termination. The expression of BACE gene was detected by immunocytochemistry method. Results： The results showed that the cDNA fragment included 1503 bp total coding region. The recombinant eukaryotic cell expression vector of pcDNA3.1-BACE was constructed successfully, and the sequence of insert was identical to the published sequence. The COS-7 cells and the neuroblastoma SK-N-SH cells transfected with the pcDNA3.1-BACE plasmid expressed high level of BACE protein in cytoplasm. Conclusion： The recombinant plasmid pcDNA3.1-BACE can provide very useful tool for researching the mason of Alzheimer＇s disease and lays the important foundation for preventing the AD laterly.     

Cloning of Integral Mature Peptide Gene of Human GDF-5

The integral mature peptide gene of human growth differentiation factor-5 (GDF-5) was出人物cloned to provide the essential foundation for study on the biological characteristics of GDF-5 at gene and protein levels. Two primers were chemosynthesized according to the hGDF-5 sequence reported in Genbank. The hGDF-5 gene was gained by RT-PCR methods from the total RNA extracted from human fetus cartilage tissue, and was cloned into vector pMD18-T. The sequence of recombinant plasmid pMD18-T-hGDF-5 was analyzed by sequence analysis. DNA agarose gel electrophoresis showed that the product of RT-PCR was about 380bp, and double enzyme digestion of the recombinant plasmid corresponded with it. The result of sequence assay was in agreement with the reported hGDF-5 sequence in Genbank. Our results showed that the integral mature peptide gene of human GDF-5 was cloned successfully from human fetal cartilage tissue, and totally identified with the seouence of human GDF-5 in Genhank.     

Construction of Eukaryotic Expression Vector of Human CC10 Gene and Expression of CC10 Protein in Lung Adenocarcinoma A549 Cell Line

Summary： A mammalian expression plasmid pcDNA3. 1-hCC10 was constructed and identified, then CC10 protein expression in A549 lung cancer cell line was detected. A 273 bp cDNA fragment was amplified from the total RNA of normal lung tissue by using RT-PCR and cloned into expression plasmid cDNA3. 1, and the recombinant plasmid was identified by employing double digestion restriction enzymes HindⅢ and BanH 1 and the cDNA sequence was assayed by the Sanger dideoxymediated chain termination method. The segment was then transfected into the A549 lung cancer cell line. The protein expression of CC10 was detected by immunofluorescence and Western blot. Our results showed that the cDNA fragment included the entire coding region （273 bp）. The re combinant eukaryotic cell expression vector of pcDNA3. 1-hCC10 was successfully constructed, and the sequence of the insert was identical to the published sequence. A549 cells line transfected with the pcDNA3. 1-hCC10 expressed high level of CC10 protein. The recombinant plasmid cDNA3. 1- hCC10 may serve as an effecnve tool for the study of tumorogenesis and tumor treatment.     

Cloning and Subcloning of cDNA Coding for Group Ⅱ Allergen of Dermatophagoides Farinae

Objective: To clone, sequence and subclone the cDNA coding for group Ⅱ allergen of Dermatophagoidesfarinae(Der f2). Methods: The cDNA gene fragment of Der f2 was amplified by RT-PCR. After being purified, the gene fragment was cloned into a vector pMD-18T. The recombinant plasmid pMD-18T-Der f2 was transformed into E. coli JM109. Positive clones were screened and identified by PCR and digested with restriction endonuclease, and the sequence of inserted Der f2 cDNA was also analyzed. Then Der f2 was subcloned into the vector of pET-32a( ). Results: The Der f2 cDNA was specifically amplified from RNA by RTPCR. The recombinant plasmid pMD-18T-Der f2 and pET-32a( ),Der f2 was constructed and digested by SacⅠ and NotⅠ, and the size of gene fragment was 455bp and in accordance with the expected one. Conclusion: The pET-32a( )-Der f2 subclone was constructed successfully.     

Cloning of NHE-1 gene fragment from human lung can.cer cells and construction of its antisense expression vector

Objective: To clone the partial sequence of Na^ /H^ exchanger- 1 (NHE- 1) gene of human lung cancer cells and insert it reversely into the multiclone site of pLXSN in order to construct an antisense expression vector for tumor gene therapy it~ vivo. Methods: With use of the upstream and downstream primers containing Barn H I and EcoR I in their 5‘ ends respectively, a partial sequence of the first exon of NHE-1 gene was cloned in a length of 454 bp from genomic DNA of human lung cancer cell A549 with PCR method. The product was then direetionally and reversely insert into the multiclone site of pLXSN. Finally, the constructed recombinant was identified with agarose gel electrophoresis and DNA sequencing. Results: The cloned fragment was 461 bp in length and successfully ligated to pLXSN with the identification by agarose gel etectrophoresis. DNA sequencing confirmed that the fragment cloned and inserted into the vector was identical with the targeted one. Conclusion: The targeted fragment is successfully cloned and reversely inserted into pLXSN in our experiment. The antisense expression vector of NHE-1, pNHE-1, was constructed successfully.     

Expression of the human era cDNA in E.coli

Objective: To amplify human era (Hera) gene, then express it in E.coli. Methods: Human era gene, after amplified by PCR and identified by sequencing, was inserted into the expression vector pGEX-4T3 in which exogenous gene was controlled by Ptac promoter. The recombinant plasmid pGEX-Hera was transformed into DH5 ( and induced with IPTG chemically. Results: The human era gene was amplified and the sequence was correct. When the bacteria with pGEX-Hera was induced, an anticipated 65 000 protein band appeared on SDS-PAGE gel and amounted to 23% of total bacterial protein. Conclusion: The human era gene has been successfully amplified and efficiently expressed in E.coli.     

Cloning of human melanoma antigen MAGE-A9 and its expression in hepatocellular carcinomas

Objective：To express the melanoma associated gene MAGE-A9 recombinant protein, obtain the anti-MAGE-A9 monoclonal antibody and to examine the expression of MAGE-A9 in hapatocellular carcinoma specimens. Methods：MAGE-A9 cDNA was cloned from human hepatocellular carcinoma tissue by using RT-PCR, and then subcloned into the plasmid pMD18-T. After sequencing, the MAGE-A9 was cloned into the prokaryotic expression vector pBAD/gⅢ to construct the recombinant expression vector pBAD/gⅢ - MAGE-A9, and was transformed into E. coli TOP10. The recombinant MAGE-A9 protein was expressed under induction of L-Arabinose, and was purified through Hitrap column. The anti-MAGE-A9 monoclonal antibody was generated. The expression of MAGE-A9 in hepatocellular carcinoma specimens was examined through ABC assay. Results：The cDNA sequence of the cloned MAGE-A9 gene was consistent with the reported sequence. By affinity column and SDS-PAGE, the purified MAGE-A9 fusion protein displayed a band of Mr 35,000, and subsequently the anti-MAGE-A9 monoclonal antibody was obtained. We found that MAGE-A9 expressed in the cytoplast of positive cells and MAGE-A9 antigen was detected in 8 cases out of 39 （21%） hepatocellular carcinoma specimens. Conclusion：MAGE-A9 antigen was expressed in a fair proportion of hepatocellular carcinoma specimens, these patients might be suitable candidates for immune involving antigen, encoded by the MAGE-A9 gene.     

Recombinant human heparin- binding neurite- promoting factor express ed with yeast stimulates neurites outgrowth .

Objectives Heparin- binding neurite- promoting factor (HBNF) is a heparin- binding protein primarily found in the brain, which can stimulate neurite outgrowth in vitro.We expressed recombinant human heparin- binding neurite- promoting factor (hrHBNF) using a yeast system, and observed its activity in stimulating neurite outgrowth in vitro.Methods cDNA encoding mature human HBNF was amplified from total RNA isolated from an 18- week aborted human fetal brain by RT- PCR method.After amplification, the HBNF cDNA gene was cloned into pPIC9K, a shuttle expression vector for yeast system.The positive clone of expression vector bearing HBNF cDNA gene was obtained by screening.Verified recombinant vector was then used to transform Pichia strain GS115 by electroporation.His+ transformants were selected on minimal dextrose medium (MD) plates which were histidine free.His+ yeast recombinants with multi- copy inserts were screened in vivo by their resistance to G418.PCR analysis was used to confirm the integration of the HBNF cDNA gene into the Pichia genome.Secreted expression of hrHBNF protein in culture medium was obtained when the positive clone containing the HBNF cDNA gene was induced by methanol. The hrHBNF product purified by gel chromatography was added to cultured rat pheochromocytoma (PC12) cells to observe its ability to stimulate neurite outgrowth.Results In the recombinant expression vector, the insert was sequenced to show exactly the sequence encoding human HBNF according to Genbank data.The HBNF cDNA gene was cloned downstream to the α- factor, and its open reading frame was in frame with the α- factor signal sequence in pPIC9K.SDS- PAGE showed that the molecular weight of the induced expression product was about 18 kDa, consistent with that of human HBNF reported in the literature.The protein product did promote neurite outgrowth in cultured rat pheochromocytoma (PC12) cells.Conclusion Recombinant human heparin- binding neurite- promoting factor can be expressed with a yeast system, and its product possesses the biological activity to promote neurite outgrowth.     

人组织激肽释放酶基因的克隆及测序分析

目的 克隆中国人组织激肽释放酶基因,为开展基因治疗高血压研究奠定基础。方法 提取人胰腺组织总RNA,逆转录后进行PCR扩增。将PCR产物回收、插入质粒KS,酶切鉴定后双向测序分析。结果 本实验克隆的激肽释放酶基因与GenBank报告的激肽释放酶基因相比,有一个碱基不同,同源性为99.8%。结论 克隆的中国人组织激肽释放酶基因可用于基因治疗等深入研究工作。     

重组人白细胞介素10基因的克隆及其在真核细胞中的表达

目的：克隆编码人白细胞介素10（hIL-10）基因的全长cDNA,构建真核表达载体,并在中国仓鼠卵巢（CHO）细胞中进行表达.方法：应用逆转录多聚酶链反应（RT-PCR）技术,从活化的正常人外周血单个核细胞中扩增出hIL-10 cDNA,将测序正确的hIL-10 cDNA克隆至真核表达载体pcDNA3构建重组表达载体.采用磷酸钙法转染CHO细胞,用ELISA法检测hIL-10基因的表达,用单四唑（MTT）检测hIL-10蛋白的活性.结果：RT-PCR产物插入Teasy载体,经序列测定证实hIL-10基因全序列克隆成功.在CHO细胞培养上清中有hIL-10的表达,该产物能抑制淋巴细胞转化.结论：成功构建了真核表达质粒pcDNA3-hIL-10,为进一步研究hIL-10在自身免疫、移植免疫及炎症性疾病中的作用打下了基础.     

人甲状腺刺激激素受体cDNA的克隆及真核表达载体的构建

目的克隆人甲状腺刺激激素受体(hTSHR)基因cDNA并构建其真核表达载体。方法以人甲状腺组织cDNA为模板,聚合酶链反应(PCR)扩增hTSHR基因编码区的全部序列,克隆入pGEM-T载体中,经限制性内切酶、DNA序列分析鉴定目的基因后,定向亚克隆到真核细胞表达载体pcDNA3.1中,并进行双酶切鉴定。结果PCR扩增的特异性片段长度为2 337 bp,以此构建的pGEM-T-hTSHR克隆载体,经限制性内切酶酶切及DNA序列分析证实载体中带有hTSHR基因编码区的目的片段,重组质粒pcDNA3.1-hTSHR经KpnⅠ和XbaⅠ双酶切后显示5 1000 bp和2 300 bp左右的2个条带,DNA序列分析显示与GenBank中的hTSHR基因cDNA序列一致,证明hTSHR基因已成功克隆入真核细胞表达载体pcDNA3.1中。结论成功构建了野生型pcDNA3.1-hTSHR重组真核表达载体。     

人透明带蛋白3真核表达载体的构建

目的 构建人ZP3蛋白(从第23位到348位氨基酸)真核表达载体,用于在毕赤酵母中表达重组人ZP3蛋白.方法 通过PCR方法扩增编码人ZP3蛋白的基因,并将其克隆入毕赤酵母表达载体pPICZ构建真核表达质粒pPICZα-hZP3.结果 经PCR扩增获得的hZP3基因大小为978 bp,其DNA测序结果分析表明:pPIC...     

小鼠血管抑素基因的克隆、测序与真核表达载体的构建

目的：克隆小鼠血管抑素（mAS)基因，测序并构建其真核表达载体。方法：从小鼠肝组织中提取总RNA，用RT－PCR方法将mAS的cDNA扩增出，克隆到pcDNA3真核表达载体中，测定其DNA序列。结果：测序结果表明我们克隆的血管抑素基因，其序列与GenBank中鼠的mAS基因序列有4个碱基不同，所编码的氨基酸有3个发生突变。结论：已成功构建鼠mAS的真核表达载体，为进一步应用AS进行肿瘤基因治疗研究打下基础。     

健康人APOBEC3G基因的克隆及真核表达载体的构建与鉴定

目的克隆人载脂蛋白B mRNA编辑酶催化多肽样3G（apolipoprotein B mRNA editing enzyme,catalyticpolypeptide 3G,APOBEC3G）基因cDNA,构建APOBEC3G基因的真核表达载体,并在体外进行表达和鉴定。方法采用RT-PCR技术从健康人外周血单个核细胞中克隆APOBEC3G基因,将该基因插入pcDNA3.1真核表达载体,构建pcDNA3.1-A3G真核表达质粒,并利用脂质体将重组质粒pcDNA3.1-A3G转染HepG2和HL7702细胞,经免疫细胞化学、Western blot等方法检测APOBEC3G真核表达情况。结果双酶切鉴定和测序结果表明,APOBEC3G真核表达载体构建成功。免疫细胞化学和Western blot结果均显示,转染pcDNA3.1-A3G的HepG2和HL7702细胞APOBEC3G蛋白均高表达,转染空质粒pcDNA3.1和未转染的HepG2和HL7702细胞APOBEC3G蛋白低表达或不表达。结论成功构建了人载脂蛋白B mRNA编辑酶催化多肽样3G真核表达载体pcDNA3.1-A3G,为进一步研究APOBEC3G抗HBV作用奠定实验基础。     

人血管内皮生长因子基因cDNA的克隆及其在COS-7细胞中的表达

目的：克隆和在COS－7细胞中表达人血管内皮生长因子165（VEGF165）。方法：利用RT－PCR方法，从新鲜卵巢癌组织中扩增到人VEGF165的全长cDNA，并测定其核酸序列；利用基因重组技术构建VEGF165的真核表达载体pcDNA3.1-VEGF165；应用脂质体介导的基因转移术将这一表达载体导入COS－7细胞后，免疫组化（SP法）检测瞬时表达的产物。结果：经酶切鉴定和基因测序证实，克隆的基因片段为人VEGF65cDNA，重组质粒pcDNA3．1－VEGF165转染COS－7细胞后，免疫组化检测有VEGF表达，结论：成功克隆人VEGF165基因，并在COS－7细胞获得表达。