On the practice of cultivating scientific thinking in the teaching of Gene Engineering curriculum / 中华医学教育探索杂志

Gene Engineering is a compulsory course for students majoring in biotechniques. Therefore, we have made innovative reforms to strengthen the "scientific thinking" elements in the course in all aspects of the course, such as content design, teaching strategies, inspection and evaluation methods on the teaching. Briefly, the contents are designed to reflect the scientific rules and thinking manners, and milestone findings and stories are mined thoroughly. During the teaching process, students are inspired and enlightened based on the history by scene reappearance to form the scientifc thinking. During the evaluation process, students are advocated to share their stories in gene engineering field and the underlying scientific thinking manners are taken into the assessment. With the teaching reform in the Gene Engineering course, the "scientific thinking" ability of the students has been significantly improved.

Strain engineering and fermentation technology for production of long-chain dicarboxylic acid: a review / 生物工程学报

Long-chain dicarboxylic acid (DCA), a building block for synthesizing a variety of high value-added chemicals, has been widely used in agriculture, chemical, and pharmaceutical industries. The global demand for DCA is increasing in recent years. Compared with chemical synthesis which requires harsh conditions and complicated processes, fermentative production of DCA has many unparalleled advantages, such as low cost and mild reaction conditions. In this review, we summarized the chemical and microbial synthesis methods for DCA and the commercialization status of the fermentation process. Moreover, the advances of using molecular and metabolic engineering to create high-yielding strains for efficient production of DCA were highlighted. Furthermore, the challenges remaining in the microbial fermentation process were also discussed. Finally, the perspectives for developing high titer DCA producing strains by synthetic biology were proposed.

Study on choroidal neovascularization genetically engineered mice model / 国际眼科杂志(Guoji Yanke Zazhi)

@#With the maturity of genetic engineering technology, a variety of genetic engineering mouse models for the development of key factors and processes of choroidal neovascularization(CNV)have been adapted to meet the needs of different research points in the CNV process. For example, VEGF164 RPE65 transgene, Tet/VMD2/VEGF, <i>etc.</i> which are key factors in the process of CNV. ApoE overexpression rats are an important model of spontaneous CNV formation in AMD-like lesions; Ccl2/Cx3cr1-deficient mice associated with changes in retinal pigment epithelial(RPE); choroidal neovascularization and retinal neovascularization can be seen in SOD1-/-aging, Vldlr-/-directed mutation, <i>etc</i>; retinal neovascularization secondary to choroidal neovascularization can be found in Cp-/-Heph-/Y knockout mice, <i>etc</i>. The main advantages of the CNV genetic engineering mouse model are rapid induction and short time of occurrence; strong correlation with CNV pathophysiology, which can compare various biological components of CNV and facilitate the study of its mechanism; closely relating to human CNV, and providing research methods for human CNV treatment evaluation. However, there are also limitations, such as low induction rate, low percentage and small area of CNV; frenquent occurrence of retinal angiomatous hyperplasia,which interferences CNV research. Researchers might select the appropriate model according to his own needs and modify the corresponding experimental parameters as needed.

Progress in interleukin 24 gene engineering protein / 军事医学

Interleukin 24 ( IL-24) has a good prospect in tumor therapy because it can specifically inhibit proliferation in a variety of tumor cells in vitro and in vivo and induce apoptosis of tumor cells without affecting normal cells .Gene therapies which use recombinant adenovirus as a vector have some limitations that restrict the clinical application of IL-24. In comparison, protein drugs have tremendous advantages .In this paper, the progress in research on IL-24 gene engineering protein is elaborated .

To constructe,package and identificate the lentiviral vector with overexpression gene Grp78 / 重庆医学

Objective To constructe ,package and identificate the lentiviral vector with overexpression gene Grp78 .Methods We used lentiviral vector and genetic engineering technology to obtain the aim gene fragment and to constructe recombinant plas‐mid .we prepared competent cells and transform the cells .Through positive clone sequencing ,lentivirus was packaged and virus titer was tested .Results Positive cloning sequence comparison results show that the test was passed .Melt curve did not appear mixed peak ,also did not appear abnormal peak broadening .It means that does not appear pollution ,primer dimers and nonspecific amplifi‐cation in the experiments .Conclusion The construction ,packaging and identification of lentiviral vector with over expression gene Grp78 are sucessful .

Enhanced proliferation and differentiation of Oct4- and Sox2-overexpressing human adipose tissue mesenchymal stem cells

Mesenchymal stem cells (MSCs) are attractive candidates for clinical repair or regeneration of damaged tissues. Oct4 and Sox2, which are essential transcription factors for pluripotency and self-renewal, are naturally expressed in MSCs at low levels in early passages, and their levels gradually decrease as the passage number increases. Therefore, to improve MSC proliferation and stemness, we introduced human Oct4 and Sox2 for conferring higher expansion and differentiation capabilities. The Oct4-IRES-Sox2 vector was transfected into human adipose tissue MSCs (ATMSCs) by liposomal transfection and used directly. Oct4 and Sox2 were successfully transfected into ATMSCs, and we confirmed maintenance of MSC surface markers without alterations in both red fluorescent protein (RFP) (control) and Oct4/Sox2-ATMSCs. Enhanced proliferative activity of Oct4/Sox2-ATMSCs was shown by WST-1 assay, and this result was further confirmed by cell counting using trypan blue exclusion for a long period. In addition, FACs cell cycle analysis showed that there was a reduction in the fraction of Oct4/Sox2-ATMSCs in G1 with a concomitant increase in the fraction of cells in S, compared with RFP-ATMSCs. Increased levels of cyclin D1 were also seen in Oct4/Sox2-ATMSCs, indicating acceleration in the transition of cells from G1 to S phase. Furthermore, Oct4/Sox2-overexpressing ATMSCs showed higher differentiation abilities for adipocytes or osteoblasts than controls. The markers of adipogenic or osteogenic differentiation were also upregulated by Oct4/Sox2 overexpression. The improvement in cell proliferation and differentiation using Oct4/Sox2 expression in ATMSCs may be a useful method for expanding the population and increasing the stemness of ATMSCs.

Deepen Teaching Reform for Course of Gene Engineering and Improve Teaching Quality / 微生物学通报

Gene engineering is the main course of biological engineering. It should be adapted to the demand of innovation spirit, practice ability and comprehensive quality of students. Educational reform of gene engineering conducted by constructing system of theory and practice, optimizing course teaching content, strengthening practice teaching content, using modern teaching technology, strengthening web course construction and improving teaching methods. We pay attention to impart specialty knowledge and learning methods to students. Its aim was to increase teaching effects and meet the demands of bioengineering specialty and qualified personal training in 21 century.

Advances in Molecular Modification of Microbial Enzymes / 中国生物工程杂志

In recent years,more and more of the enzyme proteins have been carried out using recombinant microorganism bioreactor for large scale production.For reasons of improved the catalytic capability and environmental suitability,or enhanced expression level of the protein,a variety of genetic engineering technology according to protein molecule modification have been applied extensively.Major strategies and achievements of molecular modification for microbial enzyme,such as site-directed mutagenesis,error-prone PCR,DNA shuffling and optimum codon design were reviewed.

Enhancement of CEA specific RNAi on gene engineering adenovirus H101 for treatment of tumor tissue of nude mice transplanted with human esophageal cancer cells / 基础医学与临床

Objective To study the effect of gene engineering adenovirus H101 for treating esophagus carcinoma EC9706 induced by CEA gene specific silencing and to explore internal influential factor of H101 sensitivity. Methods To construct the siRNA expression vector of CEA and to inhibit the expression of CEA through RNA interference and gene transfection in EC9706 cell,stable CEA gene silencing system was set up,compared with empty vector group and non-transfected EC9706 cell,the model of athymic mouse subcutaneous transplantation tumor of human esophagus carcinoma EC9706 cell was established followed by injection with H101. The mRNA and protein expressions of CEA were detected by real time PCR and immunohistochemistry,the tumor size was measured. Results Silencing CEA gene by applying RNAi can inhibit CEA mRNA and protein expression in nude mice model with transplanted human esophageal cancer cells,there was no evident influence on tumor growth and mass oftumor. After using H101,the tumor size of interfering group was much smaller than that of empty vector group and normal control group(P

MAR increased transgene expression in stable transfected CHO cells / 基础医学与临床

Objective To study the effect of human ?-globin matrix attachment region(MAR) on transgene expression in stably transfected CHO cells.Methods Expression vector was constructed,which contained the ?-globin MAR in both sides of Chloramphenicol acetyltransferase(CAT) reporter gene expression cassette in cis,then transfected into CHO cells.The CAT reporter gene expression was analyzed by ELISA method.Results The ?-globin MAR enhanced the CAT gene expression 5.5-fold in stably transformed CHO cells,while the transgene expression variation among individuals of transformants was decreased.Conclusion MAR increase transgene expression in stably transfected CHO cells.

The Study on Gene Engineering Strains Producing Lycopene / 中国生物工程杂志

Lycopene—a kind of important active compound of caroteinoids, is greatly beneficial to human health with its diverse biological functions. With the elucidation of lycopene biosynthetic pathway and cloning genes of relative enzymes from microorganisms, it is possible to regulate lycopene biosynthesis via genetic engineering. The biosynthesis pathways of lycopene and gene cloning of lycopene biosynthetic enzymes in microorganisms were reviewed, and gene engineering strains documented in previous works including: E.coli and yeast constructed by genetic recombination, mold strains enhanced the ability of producing lycopene by gene manipulation were summarized. At last, compared with the present methods, the problems existed in the process of construction were pointed out.

Antimicrobial Activity of Recombinant Human ?-Defensin 3 on Clinically Isolated Multidrug-resistant Strains / 中华医院感染学杂志

OBJECTIVE To analyze the antimicrobial activity of recombinant human ?-defensin 3(rhBD-3) on clinically isolated multidrug-resistant bacterial strains.METHODS The antimicrobial activity of rhBD-3 on clinically isolated multidrug-resistant Staphylococcus aureus,Enterococcus faecium,Acinetobacter baumannii and Pseudomonas aeruginosa from the wards of burns department was measured by turbidity method.RESULTS rhBD-3 Demonstrated antimicrobial activity against all the strains in a dose-dependent manner.The minimal inhibitory concentration(MIC) to Gram-positive strains and Gram-negative strains was 4 ?g/ml and 8 ?g/ml,respectively.CONCLUSIONS rhBD-3 Has significant antimicrobial activity against clinically isolated multidrug-resistant strains and thus implies therapeutic potential as an effective substitute for the present drug-resistant bacteria.

Construction,expression and identification of human B7-H1-Fc chimeric molecule in mammalian CHO cell / 第三军医大学学报

Objective To construct eukaryotic expression plasmid of human B7-H1 extracellular region-hIgG1Fc-pCI-neo eukaryotic expression vector,and express the functional fusion protein in mammalian CHO cell.Methods Full-length human B7-H1 encoding sequence was amplified from human activated T cell cDNA library by PCR,fused with hIgG1Fc,then transformed into pCI-neo expression vector and verified by sequencing.The validated recombinant was transfected into mammalian CHO cell by lipofectamine reagent.The supernatant of the cultured cell was collected and analyzed by the sandwhich ELISA to detect if there was the fusion protein,and the fusion protein was purified by HiTrap recombination protein Protein A affinity chromatography.The concentrated supernatant or purified fusion protein were used for Western blotting after SDS-PAGE to identify the molecular weight and immune activity of the fusion protein of B7-H1.Results The extracellular region of hB7-H1 about 727 bp was cloned from human T cell cDNA library and was inserted with hIgG1Fc into the eukaryotic expression vector pCI-neo.After the transfection of recombinant into mammalian CHO cell by lipofectamine reagent,the expression of B7-H1 fusion protein was detected in the cultured CHO cell supernatant by the sandwhich ELISA.The immune activity of the fusion protein was verified by Western blotting,and its molecular weight was about 51.76?10~(3),very close to the expected value.Conclusion The hB7-H1-Fc chimeric molecule was successfully constructed and the expression of its functional fusion protein in mammalian CHO cells lays a foundation for further research on the role of B7-H1 in immune tolerance,autoimmune diseases.

Recent advance in recombinant calcitonin / 医学研究生学报

By taking advantage of gene engineering,recombinant calcitonin analogue can be successfully obtained.This method can greatly improve the biological activity of the analogue of calcitonin and lower its antigenicity.In this review we discuss the recent progress in the production of recombinant human calcitonin.

Construction and expression of single chain variable Fv of anti-idiotype monoclonal antibody against Vibrio alginolyticus / 中国免疫学杂志

Objective:To clone the single chain variable Fv of anti-idiotype monoclonal antibody against vibrio alginolyticus.Methods:Total RNA was extracted from hybridoma cell 2F4 secreting MAb against vibrio alginolyticus and cDNA was amplified by retropolymerase chain reaction(RT-PCR),the expression vector pTAT-AL1 was constructed for the recombinant V_H-V_L expression.The transformed E.coli BL21 cells were propagated and induced by IPTG.Results:The V_H gene contained 369 base pairs and encoded 123 amine acid residues;The V_L gene contained 339 base pairs and encoded 113 amine acid residues;There were four FRs three CDRs and two characteristic cysteine residues in the V_H and V_L gene,respectively.ELISA results showed the ScFv retained almost the same antigen affinity and specificity as its parent monoclonal anitbody.Conclusion:The single chain variable Fv of anti-idiotype monoclonal antibody against vibrio alginolyticus was constructed successfully and expression products was found in the periplasmic space and inclusion bodies.This ScFv might be a new generation of gene engineering vaccine of the anti-idiotype monoclonal antibody against vibrio alginolyticus in fishery.

Expression and activity of the chermic F(ab')_2 fragment of anti-CD20 antibody / 中国免疫学杂志

Objective:To highly express the secretory gene engineering anti CD20F(ab') 2 in the E.coli,simplify working processes and enhance the bioactivity of the antibody fragment.Methods:Using the single factor analysis to optimize the ferment parameters.A culture method which most suit for E.coli secreting anti CD20F(ab') 2 was selected.The anti CD20F(ab') 2 was extracted from periplasm then purified it using the strptococal protein G affinity colume and the S200 HR size exclusion chromatography.The activity of anti CD20F(ab') 2 inhibiting the growth of cell Daudi in vitro was eamined using MTT.Results:Using the optimized culture method,the yield of anti CD20F(ab') 2 was distinctly enhanced from 1.9～2.2 mg/L to 3.7～4.3 mg/L and the proportion of anti CD20F(ab') 2 in all the extract also was improved from 9.7～13.2% to 38.1%～46.8%.After the S200 HR size exclusion chromatography,the purity of anti CD20F(ab') 2 could exceed 85%.The outcome of MTT exmiination showed the IC 50 of anti CD20F(ab') 2 and Fab' were 14.6 ?g/ml and 39.5 ?g/ml,respectively.Conclusion:The gene engineering anti CD20F(ab') 2 could highly express in the E.coli by using the optimized culture method.The anti CD20F(ab') 2 inhibited the growth of Daudi cells in vitro more strong than anti CD20Fab'.

Construction and expression of anti-CD3?anti-CD19 bispecific diabody and analyzing its specific binding activity / 中国癌症杂志

Background and purpose:Bispecific antibody is a special pattern of genetic engineering antibody.It possesses 2 antigen binding sites, one directed at the effecter cells and the other at the target cells which are always the tumor cells.This enables the effecter cells to recruit to the targets efficiently and exert cytotoxic effect.The experiment was aimed to construct the expression vector of anti-CD3?anti-CD19 bispecific antibody, purification and analyzing the activity of the diabody.Methods:Applyed PCR and overlap PCR to construct the expression vector of anti-CD3?anti-CD19 bispecific antibodies, transformed it into E.coli 16C9 for expression.Purified the diabody by His-tag affinity chromatography and confirmed by 12% SDS-PAGE and Western blot.Antigen binding activity of the diabody was analyzed by FACS.Results:The DNA sequence of the expression vector containing anti-CD3?anti-CD19 fragment was comfirmed.The yield of purified diabody was up to 5 mg/L.The molecular weight of diabody was correctly indicated by SDS-PAGE and Western blot.The diabody could specifically bind to CD19+ Raji cells and CD3+ Jurkat cells while competitively inhibit binding of HIT3a and HIT19a to the cells.Conclusion:Successfully constructed the expression vector of anti-CD3?anti-CD19 bispecific antibodies, the diabody acquired by the method could be produced with high level expression and also had high specific affinity with CD19+ Raji cells and CD3+ Jurkat cells.

Detection of Anti-Spirometra erinaceieuropaei Antibody Using ELISA / 中国寄生虫学与寄生虫病杂志

Objective To study specific diagnosis of Spirometra erinaceieuropaei . Methods An enzyme linked immunosorbent assay (ELISA) was studied using highly pure gene engineering antigen expressed by the recombination of the cloned cysteine proteinase gene of Spirametra erinaceieuropaei with expression vector pMAL TM c2. Six sera from patient infected with Spirometra erinaceieuropaei were detected using this method. Results and Conclusion The results showed that the gene engineering antigen reacted strongly with the sera from Spirometra erinaceieuropaei infected patients, but did not with the sera from Cysticercus cellulosae infected patients.

STUDY ON RESISTANCE GENE KNOCK OUT FROM INTEGRATED ALKALINE PROTEASE GENE ENGINEERING STRAIN / 微生物学通报

The knock out vector pHK was constructed with E coli vector pET 28a and shuttle vector pHY300PL, by using denatured DNA and homologous recombination technique, the kanamycin resistance gene ( Kan r) from integrated alkaline protease gene engineering strain BP071 was knocked out successfully, and the 11 positive clones were obtained The yield of the best positive clone BP0715 was stable as same as BP071 The methods provided the good experience for the industrial microbiology research, and it was foundation for studying on the safety of genetically modified organisms

Construction of recombinant adenovirus plasmid containing GST-? gene / 重庆医科大学学报

Objective: To constuct recombinant adenovirus vector carrying the human glutathione transferase ?(GST-?)gene and amplify the adenovirus vector in HEK293cells.Methods: GST-? gene with the suitable enzyme site are amplified from the original plasmid through PCR,then subcloned into the shuttle plsmid pAdtrackCMV after digested by the same enzyme.The recombinant plasmid pAdtrack-GST-? 1inearized by PmeI,plasmid pAdtrack-GST-? was electroporated into E.coli BJ5183 cells that had been electroporated adenovirus backbone plasmid pAdEasy-1.Picking up the homologous recombant plasmid AdEasy-GST-?,then it was digested with PacI and transfected into HEK293 cells to package the adenovirus,followed by identification of the recombinant adenovirus by means of observation of green fluorescence protein expression under fluorescent microscope.Recombinant virus supernatant infected HEK293 cells repeatedly,and the green fluorescence protein expression Was detected.Results: Through the restriction enzyme digestion and expression of GFP,recombinant adenoviral vector GST-? gene was constructed successfully.Conclusions: The recombinant adenoviral vector containing GST-? gene was successfuHy constructed,and based for the gene therapy of GST-? transferring the hematopoietic stem cells.