Objective It was reported that p53 apoptotic peptide (N37) could inhibit p73 gene through being bound with iASPP, which could induce tumor cell apoptosis. To further explore the function of N37, we constructed the cloning plasmid of DNA fragment encoding p53 (N37) apoptotic peptide by using DNA synthesis and molecular biology methods. Methods According to human p53 sequence from the GenBank database, the primer of p53(N37) gene was designed using Primer V7.0 software. The DNA fragment encoding p53 (N37) apoptotic peptide was amplified by using self-complementation polymerase chain reaction (PCR) method and cloned into the pGEM-T Easy vector. The constructed plasmid was confirmed by endonuclease analysis and sequencing. Results The insertion of objective DNA fragment was confirmed by plasmid DNA enzyme spectrum analysis, p53 (N37) gene was successfully synthesized chemically in vitro. The sequencing result of positive clone was completely identical to the human p53(N37) sequence in GenBank using BLAST software (http://www. ncbi. him. nih. gov/cgi-bin /BLASTn). Conclusion The cloning of DNA fragment encoding p53(N37) apoptotic peptide was constructed by using DNA synthesis and pGEM-T Easy cloning methods. With the constructed plasmid, we could further investigate the function of N37 peptide.

ObjectiveTo study the cloning and sequencing of mature fragment of human bone morphogenetic protein-4 gene. Methods The template DNA was obtained from the human osteosarcoma cell line U2OS. By using RT- PCR method, the cDNA coding for the mature fragment of BMP-4 was amplified, cloned into the vector pUC19, and sequenced by Sanger Dideoxy-mediated Chain Termination method. Results The mature fragment of BMP4 cDNA was obtained by RT-PCR and determined by sequencing. Through the computer search on Genebank, the analysis showed that the homology of nucleotides and amino acids between cDNA of rhBMP4 mature fragment of this study and the published sequence was 99％. Sequence analysis showed that there were two differences, one was at base 1154 (201): G→C, which had no influence on the corresponding amino acids (Val). Another was at basel222 (269):C→T, the mutation at the base 1222 had the change of Ala to Val. Conclusion The mature fragment of BMP4 gene has been cloned. The results will be of great significance in treatment of skeletal injuries and diseases.

Objective:To construct a recombinant adenovirus vector harboring fusion gene NT4-p53(N15)-Ant,laying a foundation for gene therapy research of malignant tumors.Methods:The p53(N15)-Ant gene was obtained by T-vector method and was inserted in pBV220/NT4 vector after digested with restriction enzyme.The fusion gene of NT4-p53(N15)-Ant was subcloned into the shuttle plasmid of adenovirus;the products were cotransfered into HEK-293 cell line with helper plasmid PJM17.The recombinant adenovirus was produced by homologous recombination of above 2 plasmids in HEK-293 cells and its titer was measured by plaque-forming.The expression of Ad.NT4p53Ant in transfected 293 cells was confirmed by reverse transcription polymerase chain reaction(RT-PCR)procedure.The effect of Ad.NT4p53Ant on HepG2 cell line was measured by a colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT)assay.Results:The p53(N15)-Ant gene was confirmed by restriction enzyme digestion and DNA sequencing.High titer of recombinant adenovirus was obtained by homologous recombination in HEK-293 cells(1?10 11pfu/ml).The expression of NT4-p53(N15)-Ant gene in 293 cells was confirmed by RT-PCR.Ad.NT4p53Ant had strong killing effect on HepG2 cells.Compared with Ad.GFP,Ad.NT4p53Ant significantly decreased the survival rate of HepG2 cells.Conclusion:The recombinant adenovirus vector encoding gene NT4-p53(N15)-Ant has been successfully constructed in this experiment by molecular cloning and in vitro recombination techniques,laying a foundation for further research of gene therapy of cancer.

Objective To investigate the treatment of spinal cord injury (SCI) by recombinant adeno-associated virus (AAV) transducing the hNGF gene, and construct and produce the vector of hNGF recombinant AAV. Methods The resulting gene of hNGF was inserted into the KpnⅠ-BamHⅠ site of vector plasmid pSSHG-Neo to construct the vector of hNGF recombinant AAV. The recombinant AAV viral stock was packaged. Renal embryo 293 cell was co-transfected with the rAAV vector of plasmid pSSHG/hNGF, packaging plasmid pAAV/Ad and helper adenovirus pasmid pFG140 instead of adenovirus by calcium phosphate precipitation. Results The recombinant viral stock vector of plasmid pSSHG/hNGF was constructed successfully. The results of dot blot showed that we had obtained the rAAV stocks of high titre 1.46?10 12 PFU?mL -1. Conclusion We prepared the viral stock of rAAV-hNGF that can serve as the experimental study of gene therapy of SCI.

Objective To study the prokaryotic expression of fusion gene A?-HBcAg and analyze the immunoreactivity and immunogenicity of expression protein. Methods Recombinant plasmid pBV220/A?-HBcAg was transformed into E.coli DH5?, and expressed by temperature inducing. The bacteria were split by ultrasonic wave. The expression of the fusion protein was studied by SDS-PAGE and Coomassie brilliant blue staining. The immunoreactivity of the fusion protein was determined using ELISA. After immunized intraperitoneally with the fusion protein, 5 Balb/c mice's sera titers of anti- A? and anti-HBc were evaluated by ELISA. Results Fusion protein was in sediment of the split bacteria as inclusion bodies and its expression level was 5% of the total sediment protein. The fusion protein had both immunoreactivity of A? and HBcAg. The titers of anti-A? and anti-HBc were very low after 3 times of immunization. After immunization for 5 times, the titers reached 1∶800 and 1∶3 200 for anti-A? and anti-HBc, respectively. Conclusion Recombinant gene A?-HBcAg can be expressed in E.coli DH5? and the expression protein has certain immunoreactivity and immunogenicity. It indicates that further work should be done to enhance the expression level of fusion gene A?-HBcAg and improve the immunogenicity of the fusion protein.

Objective To prepare a hybridoma secreting stab le monoclonal antibodies against ?-amyloid peptide (A? 1-42) with high titer. Methods By genetic engineering technology, A ? gene was recombined with the MIR of HBcAg to get the A? and HBcAg fusi on protein. Spleen cells from BALB/c mice immunized with A? and HBcAg f usion protein were fused with mouse myeloma cells SP2/0. Results Two strains of hybridomas (1H 7 and 1F 3) secreting stable monoclonal antibodies raised against A? 1-42 were ob tained. The subtypes of A? 1-42 antibodies were IgG 3. C onclusion The A? 1-42 monoclonal antibodies obtained have high titers and specificity.

Objective To construct and expr es s a recombinant plasmid of nonstructural protein NS3-NS4 of hepatitis C virus ( HCV), and to identify the antigenicity of the expressed protein. Methods A gene region encompassing the nonstructural protei n NS3-NS4 of HCV was amplified by polymerase chain reaction (PCR) from the pUC1 9/HCV template. The recombinant expression plasmid containing the pBV220/NS3-NS 4 sequence was constructed, and the nonfused NS3-NS4 recombinant protein was ex pressed in E.coli DH5? efficiently. The recombinant protein was det ected by SDS-PAGE and ELISA. Results We successfully constructed and expressed the recom binant plasmid in prokaryote. Its antigenicity was detected with 50 standard ser a. Compared with the second-generation diagnostic Kit, the total detection rate was 96%. Conclusion The whole NS3-NS4 protein, a region of dominant immunogenicity, should be the effective component of the HCV diagnostic Kit and provide the clue for developing HCV DNA vaccine.

Objective To express the hNGF? gene segment encoding mature peptide (hNGF? ) in E.coli and determine its bioactivity. Methods The resulting gene of hNGF? was subclonedinto the hNGF? site of the expression vector plasmid pBV220. The ligation products were used to transform the competent E.coli DH 5?. The proteins of hNGF? were expressed by temperature induction. The expression products were dealed with solubilizing inclusion bodies and refolding protein. It was introduced into the expressed hNGF? tests of neurite growth of dorsal root knot of chicken embryo and tests of Brdu incorporation into PC12 cells was a biologically active protein. Results The recombinant plasmid pBV220/NGF? was successfully constructed. The NGF? was inserted to pBV220 plasma, a prokaryotic expression vector. Expression of NGF? in E.coli was induced by raising temperature to 42℃. SDS-PAGE electrophoresis showed that NGF? protein existed in inclusion. The solubility protein of NGF? was obtained through purification of inclusion by centrifugation and technique of protein repatriation. Recombinant NGF? protein was purified by affinity chromatography of heparin SepharoseCL-6B. The purity of NGF? was higher than 90% and yield of NGF? was 1.8～2.0mg/L expressing bacteria. The bioactivity of NGF? expressing prokaryotic cell was 1?10 5BU/g according to rule concerning examination of biological products in China. Conclusion The hNGF?gene with bioactivity can be expressed in E.coli.

Objective To construct the expression box of fusion gene NT4-p53(N15)-Ant.Methods The gene of p53(N15)-Ant was obtained by PCR of two primers templating with each other and T-vector cloning method.The positive clone was identified and analyzed by the restriction enzymes and sequencing respectively.After digested with restriction enzyme,the interest gene of p53(N15)-Ant was subcloned into the plasmid pBV220/NT4.Results The gene of p53(N15)-Ant was confirmed by the digestion of restriction enzyme and sequencing.The recombinant plasmid pBV220/NT4p53(N15)Ant was identified by the digestion of restriction enzyme and agarose gel electrophoresis and the results conformed theoretical values.Conclusion The plasmid pBV220 containing the expression box of NT4-p53(N15)-Ant was successfully constructed by molecular cloning and recombination techniques in vitro,which will guide further study on gene therapy of cancer.

Objective To investigate survivin as an anticancer therapeutic target by use of shepherdin [79-87],a novel peptide carrying the survivin sequence from Lys-79 through Leu-87,we constructed an recombinant vector containing fusion gene NT4-Ant-Shepherdin [79-87].Methods The gene of Ant-Shepherdin [79-87] was obtained by PCR and T-vector method.After cloned and digested with restricted enzyme,Ant-shepherdin [79-87] was inserted in PBV220NT4 vector.The recombinant vector was transformed into the competent cell,E.coli DH5?.The fusion gene of NT4-Ant-Shepherdin [79-87] was identified by agarose gel electrophoresis (AGE).Results DNA sequencing results verified that the sequence of Ant-Shepherdin [79-87] was consistent with what we had designed.After transformed E.coli DH5?,a fragment of 321 bp was confirmed.Conclusion The recombinant vector containing fusion gene NT4-Ant-Shepherdin [79-87] was successfully constructed in this experiment by molecular biology techniques,which provides the basis of further research of survivin for cancer gene therapy.